Dynamic visualization and genetic determinants of Sonic hedgehog

Dynamic visualization and genetic determinants of
Sonic hedgehog protein distribution during zebrafish
embryonic development
DISSERTATION
zur Erlangung des akademischen Grades
Doctor rerum naturalium
(Dr. rer. nat.)
vorgelegt
der Fakultät Mathematik und Naturwissenschaften
der Technischen Universität Dresden
von
Dipl.-Biol. Arndt Friedrich Siekmann
geboren am 12.02.1975 in Bad Oeynhausen
Gutachter:
Prof. Dr. Michael Brand
Prof. Dr. Bernard Hoflack
Prof. Dr. Uwe Strähle
Eingereicht am:
Tag der Verteidigung:
30.09.2004
2
Summary
The correct patterning of embryos requires the exchange of information between cells.
This is in part achieved by the proper distribution of signaling molecules, many of
which exert their function by establishing gradients of concentration. Because of this
property they were named “morphogens”, or “form giving” substances. Among these,
proteins belonging to the Hedgehog (Hh) family have received much attention, owing
to their unusual double lipid modification and their involvement in human disease,
causing congenital birth defects and cancer. Great efforts have been made in order to
elucidate the mechanisms by which Hh molecules are propagated in the embryo.
However, no conclusive evidence exists to date to which structures these molecules
localize and how they, despite their membrane association, establish a gradient of
concentration. Therefore, I decided to study the distribution of the vertebrate Hh
homolog, Sonic Hedgehog (Shh) in developing zebrafish embryos. By fluorescently
tagging Shh proteins, I found that these localize to discrete punctate structures at the
membranes of expressing cells. These were often regions from which filopodial
protrusions emanated from the cells. Punctate deposits of Shh were also located
outside of expressing cells. In dividing cells, Shh accumulated at the cleavage plane.
Furthermore, by making use of confocal microscopy and time lapse analysis, I
visualized Shh proteins moving in filopodial extensions present between cells. This
suggests a novel mechanism of Shh distribution, which relies on the direct contact of
cells by filopodia for the exchange of signaling proteins.
In a second part of my thesis, I characterized genes potentially implicated in
regulating Shh protein distribution and signaling function. I cloned three zebrafish
genes belonging to the Ext1 (exostosin) family of glycosyltransferases required for the
synthesis of Heparan Sulfate Proteoglycans and established a tentative link of these
genes to somitic Hh signaling. In addition, I characterized the developmental
expression and function of zebrafish Rab23, a small GTPase, which acts as a negative
regulator of the Shh signaling pathway. Performing knock-down experiments of
zebrafish Rab23, I found that Rab23 functions in left-right axis specification, a
process previously shown to depend on proper Shh signaling.
3
Table of contents
SUMMARY
2
TABLE OF CONTENTS
3
ACKNOWLEDGEMENTS
8
INTRODUCTION
9
THE ZEBRAFISH AS A MODEL SYSTEM
9
EMBRYONIC DEVELOPMENT OF ZEBRAFISH
10
GENERAL DEVELOPMENTAL CONCEPTS
11
THE SPEMANN-MANGOLD ORGANIZER
12
MECHANISMS OF INTERCELLULAR COMMUNICATION
13
THE MORPHOGEN CONCEPT
14
MORPHOGEN GRADIENT FORMATION
15
DIFFUSION
15
PLANAR TRANSCYTOSIS
16
ARGOSOMES
17
CYTONEMES
18
ALTERNATIVE WAYS OF GRADIENT FORMATION
19
THE HEDGEHOG SIGNALING PATHWAY
20
THE HH SIGNAL TRANSDUCTION PATHWAY
21
CYTOPLASMIC COMPONENTS OF THE HH SIGNALING PATHWAY
24
DISTRIBUTION OF HH PROTEINS
25
DEFECTS IN HH SIGNALING AND HUMAN DISEASE
26
ZEBRAFISH MUTATIONS IN HH PATHWAY COMPONENTS
27
TABLE OF CONTENTS
4
RELEVANCE OF THE EXTRACELLULAR MATRIX IN MORPHOGEN
SIGNALING
28
SYNTHESIS OF HSPGS
28
EXT FAMILY OF GLYCOSYLTRANSFERASES
29
DEVELOPMENTAL FUNCTIONS OF HSPGS
29
ABBREVIATIONS
31
MATERIALS AND METHODS
33
CHEMICALS AND SOLUTIONS
33
FISH HUSBANDRY AND CARE
34
IN VITRO RNA TRANSCRIPTION
34
RNA INJECTIONS
34
MORPHOLINO INJECTIONS
35
CELL TRANSPLANTATION
36
SYNTHESIS OF DIG OR FLUORESCEIN LABELED PROBES
36
WHOLE MOUNT IN SITU HYBRIDIZATION
37
DOUBLE WHOLE MOUNT IN SITU HYBRIDIZATION
37
IMMUNOHISTOCHEMISTRY
38
BIOTIN DETECTION OF TRANSPLANTED CELLS
38
CONFOCAL MICROSCOPY
39
HEAT SHOCK TRANSFORMATION OF BACTERIA
39
PLASMID PREPARATIONS
39
AGAROSE GEL ELECTROPHORESIS
40
EXTRACTION OF DNA FROM AGAROSE GELS
40
RESTRICTION DIGEST
40
DEPHOSPHORYLATION AND LIGATION OF DNA FRAGMENTS
40
TOPO CLONING
41
PREPARATION OF CDNA
41
DATABASE SEARCHES
42
PCR
42
RACE PCR
44
SITE DIRECTED MUTAGENESIS
44
GENERATION OF SHH-GFP FUSION PROTEINS
45
CLONING OF DN-EZRIN
46
TABLE OF CONTENTS
5
WESTERN BLOTTING
46
LIST OF PLASMIDS USED IN THIS STUDY
47
RESULTS
48
EXPRESSION OF SHH AND PTC1 AT GASTRULA STAGES
49
EXPRESSION OF PTC1 AT GASTRULA STAGES DEPENDS ON SHH SIGNALING
50
SHH CAN INDUCE TARGET GENE ACTIVATION IN ECTOPIC POSITIONS IN THE EMBRYO
51
ANALYSIS OF FLUORESCENT SHH FUSION CONSTRUCTS
53
CLONING OF SHH-NGFP AND SHH-VENUS
54
SIGNALING STRENGTH OF SHH-VENUS WAS COMPARABLE TO WT SHH
55
UBIQUITOUS OVEREXPRESSION OF SHH-VENUS OR SHH CAUSED SIMILAR
MORPHOLOGICAL CHANGES
56
TABLE 1. OVEREXPRESSION OF SHH, SHH-VENUS OR SHH-NGFP ALTERED EYE MORPHOLOGY.
56
ALTERATIONS IN MARKER GENE EXPRESSION INDUCED BY EITHER SHH OR SHH-VENUS
WERE COMPARABLE
58
TABLE 2. OVEREXPRESSION OF EITHER SHH OR SHH-VENUS INDUCED ECTOPIC EXPRESSION OF
PAX2.1.
58
TABLE 3. OVEREXPRESSION OF EITHER SHH OR SHH-VENUS REPRESSED PAX6.
59
TABLE 4. OVEREXPRESSION OF EITHER SHH OR SHH-VENUS INDUCES ECTOPIC EXPRESSION OF
FOXA2 IN THE DIENCEPHALON.
60
AUTOPROTEOLYTIC CLEAVAGE OCCURRED NORMALLY IN SHH-VENUS
61
TRANSPLANTED CELLS OVEREXPRESSING SHH-VENUS COULD INDUCE PTC1
62
EXPRESSION OF SHH-VENUS IN ITS ENDOGENOUS DOMAIN PARTIALLY RESCUED LOSS OF
EXT1C EXPRESSION IN SYU EMBRYOS
63
SUBCELLULAR LOCALIZATION OF SHH-VENUS
64
SHH-VENUS LOCALIZED TO PUNCTATE STRUCTURES AND FILOPODIA
65
TIME LAPSE MOVIES REVEALED TRAVELING OF SHH-VENUS ALONG FILOPODIA
68
TABLE 6. CONFOCAL MOVIES REVEAL SHH-VENUS TO BE PRESENT IN FILOPODIA
68
TABLE OF CONTENTS
6
CHARACTERIZATION OF FILOPODIAL EXTENSIONS IN GASTRULATING
EMBRYOS
70
FILOPODIA CONTAINED GFP-GPI BUT NOT CYTOSOLIC GFP
70
THE DISTRIBUTION OF SHH-VENUS DIFFERED FROM THAT OF GPI-YFP
72
FILOPODIA CONTAINED ACTIN
73
FILOPODIA EXTENDED IN THE EXTRACELLULAR SPACE
74
OVEREXPRESSION OF DN-EZRIN DIMINISHED SHH SIGNALING RANGE
74
IDENTIFICATION OF ZEBRAFISH GENES POSSIBLY INVOLVED IN SHH
SIGNALING
76
CLONING AND EXPRESSION OF ZEBRAFISH EXT1 GENES
76
EXPRESSION OF EXT1A
77
EXPRESSION OF EXT1B
81
EXPRESSION OF EXT1C
83
DEPENDENCE OF EXT1A AND EXT1C ON HEDGEHOG SIGNALING
84
KNOCK-DOWN OF EXT1A LEADS TO CURLY-DOWN BODY SHAPE
86
KNOCKDOWN OF EXT1B AND EXT1C
88
ANALYSIS OF ZEBRAFISH RAB23
89
CLONING OF ZEBRAFISH RAB23
89
TABLE 7. ALIGNMENT OF RAB23 PROTEINS FROM DIFFERENT SPECIES
90
EXPRESSION OF RAB23 DURING ZEBRAFISH DEVELOPMENT
91
KNOCK-DOWN OF RAB23 LEADS TO LATERALITY DEFECTS
91
TABLE 8. INJECTION OF RAB23 MORPHOLINO CAUSES RANDOMIZATION OF HEART SITUS
93
TABLE 9. GUT SITUS IS RANDOMIZED IN EMBRYOS INJECTED WITH RAB23 MORPHOLINO.
94
DISCUSSION
95
SIGNALING RANGE OF SHH
95
CONTROL OF SHH SIGNALING RANGE
96
SIGNALING STRENGTH OF FLUORESCENT SHH FUSION PROTEINS
97
SUBCELLULAR LOCALIZATION OF SHH-VENUS
98
LOCALIZATION OF SHH TO RAFT LIPID MICRODOMAINS
99
SHH-VENUS ACCUMULATION AT CELL JUNCTIONS
99
TABLE OF CONTENTS
7
CHARACTERIZATION OF SHH-VENUS LOCALIZATION IN FILOPODIA
100
MECHANISMS OF TARGETING SHH TO DISTINCT SUBCELLULAR COMPARTMENTS
101
INTERFERING WITH FILOPODIAL FORMATION DIMINISHES SHH SIGNALING RANGE
102
COMPARISON OF EXT1 EXPRESSION DOMAINS WITH OTHER SPECIES
103
RELATIONSHIP OF EXT GENES TO SITES OF SHH EXPRESSION
103
ZEBRAFISH EXT GENES RESPOND TO SHH SIGNALING
104
RELATIONSHIP OF EXT1 GENE EXPRESSION TO FGF SIGNALING
105
MORPHOLINO KNOCK-DOWN OF ZEBRAFISH EXT1 GENES
105
INTERFERING WITH RAB23 FUNCTION ALTERS ORGAN SITUS
106
REFERENCES
107
LIST OF PUBLICATIONS
123
8
Acknowledgements
I would like to thank Michael Brand, in whose laboratory this work was carried out,
for his support. Furthermore, I am grateful to all lab members for the great working
atmosphere and unforgettable lunch discussions. I would like to thank Florian Raible
and Gerlinde Reim for sharing several of their voluptuous dinners (if it would not
have been for the tomatoes), Muriel Rhinn for advice on scientific projects, great
vacations in America’s dairyland and for all the unmentionable little things. I would
also like to thank Irinka Castanon-Ortega for being a great friend.
I am grateful to Claudia Lohs, Muriel and Marta Luz for great evenings out in the
Neustadt and for the yet to come trip to the Erzgebirge.
Thanks to Tobias Langenberg for reading this thesis and for many valuable comments
on the manuscript.
Finally, I would like to thank my parents, my grandmother and my brother for their
continuous support during all of my life, making this work possible.
9
Introduction
During the course of development, a single cell will after many divisions ultimately
give rise to a complex organism consisting of various different cell types. These will
be organized into the tissues and organs of the body. This remarkable process poses a
multitude of questions. How do the cells know when to divide? How do they know
into which cell type they should differentiate and where they are in respect to the
other cells of the developing embryo? How is the cellular behavior controlled?
Finally, all cells contain the same set of genetic information encoded in their DNA,
while only a subset of this information will be used. For instance, the building blocks
for a muscle cell are much different from those needed to make a cell in the liver.
How then does a cell achieve the silencing of certain regions of the DNA while others
become selectively expressed? These are the questions developmental biology asks
and a growing understanding of the processes underlying the control of tissue
differentiation has spurred interest in this multi-disciplinary field of biology.
However, this understanding is far from completion and many questions remain.
In the introduction of my thesis, I will first provide a brief presentation of the
zebrafish model system in which this work was carried out. Then I will turn to general
concepts of how cells acquire their positional information and how this information is
translated into different cellular fates. Finally, I will introduce the components of the
Sonic Hedgehog signaling pathway and its relevance as a means of intercellular
communication to zebrafish development.
The zebrafish as a model system
The zebrafish (Danio rerio) has received much attention as a model organism to study
the processes underlying development because of several advantages. The embryos
develop outside the mother and are therefore amenable for experimental
manipulations and microscopic observations. These are facilitated by the translucency
of the embryos. The cloning and characterization of several promoters over the recent
years, which allow the specific expression of proteins, e.g. fluorescent ones, has
provided an additional tool for microscopical analysis.
INTRODUCTION
10
Zebrafish have a short generation cycle of approximately 12 weeks and the
development of the embryo is fast: within 48 hours a free swimming larvae has grown
from the fertilized egg, facilitating genetic analysis. Furthermore, forward genetic
screens have provided several thousand mutations affecting genes involved in many
developmental processes (Driever et al., 1996; Haffter et al., 1996). With the
“TILLING” technique, which is based on the PCR screening of a large number of
mutated alleles, researchers can now obtain mutants for known genes (Wienholds et
al., 2003). In addition, knock down techniques using morpholino oligonucleotides
have become available, allowing the specific inactivation of the studied genes
(Nasevicius and Ekker, 2000). The identification of the gene affected by a certain
mutation will be furthermore facilitated by the near completion of the zebrafish
genome sequencing.
Embryonic development of zebrafish
Development proceeds upon fertilization, after which the extra-embryonic chorion
lifts from the zygote and yolk-free cytoplasm streams to the animal pole of the cell to
form the blastodisc (Fig. 1). This blastodisc sits on top of a yolk cell. The subsequent
cleavages are called meroblastic, because they only take place at the animal pole of
the embryo. These first cleavages are synchronous, producing about 512 cells within 2
3/4 hours, forming a blastula. Upon mid-blastula transition, zygotic transcription is
activated and cell divisions lose their synchrony.
Subsequently, gastrulation starts at about 4 hpf with epiboly when cells start to
migrate around the yolk cell. At about 6 hpf, involution takes place, a process by
which cells at the future dorsal side of the embryo, or shield, start to migrate
underneath the overlying cells. In addition, cells converge towards the midline and
extend anteriorly. The gastrulation process will finally give rise to the 3 different
germ layers of the embryo and is finished by reaching the tailbud stage at 10 hpf.
During this gastrulation period the main body axes are specified.
After the tailbud stage, segmentation starts with the formation of the somites,
mesodermal blocks of tissue, which will give, rise to several organs, such as muscle,
spleen and blood. Furthermore, the process of neurulation subdivides the ectodermal
neural plate into a regionalized neural tube. At the end of the segmentation period, at
INTRODUCTION
11
about 24 hpf, most of the organ primordia are specified and as the first functioning
organ, the beating heart, is visible. The embryo now enters the pharyngula period and
hatches at about 48 hpf.
Fig.1.
Developmental
stages of the
zebrafish (Danio
rerio).
Stages are shown
with the time of
development after
fertilization
at
2 8 . 5 °C. Embryos
are oriented animal
to the top, dorsal to
the right (one cell to
tailbud stages).
From
25hpf,
embryos
are
oriented anterior to
the left, dorsal up.
Top views are
included for germ
ring and shield
stages.
Upon fertilization,
the zygote divides
and by 3.5 hpf, 8
cells are visible.
These continue to
divide
and
gastrulation starts at
about 6 hpf. Cells
involute around the
germ ring and
converge
and
extend toward the
dorsal side of the
embryo. At 10 hpf,
the tailbud stage,
this process is
complete
and
somitogenesis and
neurulation start. At
25 hpf, larvae reach
the
pharyngula
period. At 3 months
of development,
zebrafish reach
sexual maturity.
Modified
from
(Kimmel et al.,
1995)
and
F.
Raible, M. Brand
(pers. comm.)
General developmental concepts
The establishment of the embryonic body plan depends on a multitude of different
cellular and molecular processes, which will be ultimately translated into fate
INTRODUCTION
12
changes, and morphological alterations of tissues. I will introduce intercellular
communication and differential gene expression as two of the developmental concepts
important for these processes. They also directly impinge on my studies.
In order for an embryo to be properly patterned, genes must be activated in
certain regions of the embryo, while they need to remain silent in others. For instance,
the early neural plate is subdivided into forebrain, midbrain and hindbrain. The
forebrain and midbrain tissue expresses otx2, a transcription factor that controls the
expression of forebrain and midbrain specific genes. In contrast, the hindbrain
expresses another transcription factor, gbx1 (Rhinn and Brand, 2001; Rhinn et al.,
2003). If this transcription factor is now overexpressed, the extend of the otx2
expression domain becomes reduced and the embryo will develop excess hindbrain
tissue at the expense of fore-midbrain tissue (M. Rhinn, unpublished). This illustrates
how differential gene expression influences the development of embryonic tissues and
demonstrates the importance of regionally controlling gene expression.
This can be either achieved by an intrinsic program that tells the cells at which
time point of development which genes need to be switched on or off. This program
would thereby commit the cell to a certain fate. Alternatively, extrinsic factors can
instruct a cell to obtain this fate. In reality, these two possibilities do not exclude each
other, but are interdependent. During a given time window a cell is competent to
respond to a certain extrinsic or inductive cue, whose absence or presence is
subsequently translated into a specific differentiation program. Therefore, intercellular
communication sets the basis for differential gene expression. Further processes, such
as regulative development and plasticity, by which the embryo accommodates
alterations of its normal developmental program, are mediated through
communication between cells.
The Spemann-Mangold organizer
Early grafting experiments by Hilde Mangold and Hans Spemann in newt embryos
provided evidence for inductive interactions between different embryonic tissues.
When transplanted to the ventral side of a newt gastrula, the dorsal blastopore lip of
another embryo was able to induce the formation of a secondary axis in the host
embryo (Spemann, 1938). Most importantly, the resulting axis did not solely form
INTRODUCTION
13
from transplanted cells, but also instructed cells of the host embryo to differentiate
into tissues which they normally would not have formed. Conversely, removal of this
structure lead to the loss of dorsal tissue and therefore to a disorganized neural tube.
Because of these properties, the dorsal blastopore lip was referred to as an organizer.
The organizer concept was later expanded to any cell population that is able to
pattern surrounding tissue (Capdevila and Izpisua Belmonte, 2001; Niehrs, 2004).
Several organizers were subsequently identified in the embryo. These include the
already mentioned Spemann-organizer, or shield in zebrafish (Shih and Fraser, 1996),
the row1 cells in the vertebrate forebrain (Houart et al., 1998; Shimamura and
Rubenstein, 1997), the midbrain-hindbrain-boundary organizer (Raible and Brand,
2004; Rhinn and Brand, 2001; Wurst and Bally-Cuif, 2001), the zone of polarizing
activity in the limb buds (Capdevila and Izpisua Belmonte, 2001) and a recently
identified tail organizer in zebrafish (Agathon et al., 2003). In invertebrates, such as
the fruitfly Drosophila melanogaster, the anterior-posterior compartment boundary in
the wing imaginal disc comprises a region with organizing properties (Tabata and
Takei, 2004).
Powerful molecular approaches developed in the last decade have made it
possible to identify the molecules responsible for the observed inductive interactions.
However, much less is known how they exert their function on the neighboring tissue
and how they can elicit a multitude of different cellular outcomes.
Mechanisms of intercellular communication
Much work has focused on the mechanisms by which cells can communicate with one
another. Two different means of signaling are usually distinguished. These are
juxtacrine and paracrine signaling (Gilbert, 2003). In juxtacrine signaling the
exchange of information is direct, for instance via gap junctions, which connect
individual cells. Alternatively, receptors bound to the cell surface on one cell can
directly contact ligands on the surface of a neighboring cell. This mechanism is well
established for the Notch signaling pathway, which is important for processes such as
neural development by lateral inhibition (Portin, 2002).
In paracrine signaling, one cell secretes a signaling molecule, which travels over
a distance to neighboring cells and binds to receptors on these cells, eliciting a
14
INTRODUCTION
downstream signaling cascade. The existence of paracrine signaling was first
demonstrated by a study that addressed the inductive interactions needed to produce
kidney tissue (Grobstein and Dalton, 1957). By placing a filter between responding
and inducing tissues, the authors could show that induction still took place, even in
the absence of physical contact between the two tissues.
The morphogen concept
Many of the molecules shown to mediate the inductive properties of organizers were
subsequently demonstrated to work in a concentration dependent manner. These
findings were in agreement with previous theoretical concepts of pattern formation,
which postulated the existence of form giving substances, or “morphogens” (Turing,
1952; Wolpert, 1969). These morphogens are thought to be produced by a localized
source of cells and then establish a gradient of concentration over a field of cells. The
local concentration of the morphogen then directs the fate of nearby cells.
Fig.2. Morphogen gradient formation.
A morphogen (green) produced from a localized
source of cells (producing cells) travels over a
distance and generates a gradient of
concentration. The receiving cells interpret this
graded distribution of the morphogen by
differentially activating target gene transcription
(target gene x and target gene y) according to
different concentration thresholds (C1 and C2 ,
respectively) (Entchev and Gonzalez-Gaitan,
2002).
Cells receiving a low concentration of the signaling molecule respond by
expressing low threshold genes, while others, receiving a higher concentration
respond by expressing high threshold genes (Fig. 2) (Entchev and Gonzalez-Gaitan,
2002). Another feature of morphogens is their direct effect on cells at a distance,
which must not involve relay mechanisms. This concept provides an attractive
explanation of how cells in the embryo acquire positional information and how the
INTRODUCTION
15
multitude of cellular differences is coordinated by a much lower number of signaling
molecules.
Several molecules have been proven to function as morphogens during fly
development, which was also shown for some of their vertebrate cognates. These
molecules include Hh with its vertebrate homolog Shh, which I will discuss in detail
later, Wnt family members, like Wg, and members of the TGF family, such as Dpp
(Tabata and Takei, 2004). Furthermore, Squint has been shown to act as a morphogen
during early zebrafish development (Chen and Schier, 2001), while Activin is
implicated in a dose dependent patterning of Xenopus mesodermal tissue (McDowell
et al., 2001).
Morphogen gradient formation
While the signaling outcomes of different morphogen concentrations are well
established, considerable debate exists on how these molecules move through the
tissue and how stable morphogen gradients are established in the developing embryo.
Several models have been put forward to explain these processes and evidence
accumulates that they might not be mutually exclusive. Furthermore, different
molecules might use different means of gradient formation.
Diffusion
The simplest model predicts that morphogen molecules secreted from their source
diffuse through the extracellular space and thereby create a gradient of concentration,
with the highest concentration closest to the source. Experimental evidence for this
model was obtained in Xenopus animal cap experiments, where the establishment of a
gradient of Activin over a field of cells was monitored (McDowell et al., 2001). This
gradient was established even when the tissue was incubated at 4°C, a temperature in
which energy dependent processes, such as endocytosis, do not occur any more.
Therefore, the authors concluded that a passive process formed the Activin gradient.
Furthermore, a recent study in zebrafish suggests that Fgf8 protein spreads
extracellularly by a diffusion-based mechanism (Scholpp and Brand, 2004).
INTRODUCTION
16
However, in Drosophila, a recent study showed that a secreted GFP protein could
not establish a gradient of concentration in the wing imaginal disc (Entchev et al.,
2000). In addition, theoretical models have demonstrated that receptor binding of the
morphogen alone would prevent the formation of a concentration gradient (Kerszberg
and Wolpert, 1998). Therefore, interactions with receptor molecules and molecules in
the extracellular matrix, to which many morphogens have been shown to bind (Tabata
and Takei, 2004), have to be taken into consideration.
Planar transcytosis
The implications that other factors besides diffusion most probably influence the
generation of stable morphogen gradients lead to the development of alternative
models. One way of explaining gradient formation is that morphogens are transported
through a field of cells by repeated cycles of endocytosis and resecretion, known as
planar transcytosis. It has been initially proposed as a mechanism of Wg distribution
(Bejsovec and Wieschaus, 1995). The requirement for endocytosis was directly
addressed in the case of Dpp signaling in the Drosophila wing imaginal disc. A DppGFP fusion protein was used to visualize the distribution of Dpp and it was found to
colocalize with endocytic structures (Entchev et al., 2000). Furthermore, expression of
a dominant negative form of the small GTPase Rab5, which is thought to be rate
limiting in the early endocytic pathway, restricted the expression of Dpp target genes
to cells close to the Dpp source. In contrast, overexpression of Rab5 broadened the
expression domain of Dpp target genes.
Furthermore, the authors generated clones of cells mutant for shibire (shi),
encoding for dynamin, a GTPase that controls clathrin mediated endocytosis.
Generation of these clones shortly after a burst of Dpp-GFP prevented the formation
of Dpp-GFP positive endosomes in cells behind the shi clone, causing the formation
of “shadows” (Entchev et al., 2000). Thus, endocytosis seems to determine the
signaling range of Dpp in the Drosophila wing imaginal disc.
However, the results obtained for Dpp-GFP rely on the overexpression of DppGFP, which might cause ectopic accumulation of the protein in the endocytic
pathway. Other experiments showed by digestion of extracellular proteins that most of
the Dpp is actually present in the extracellular space (Teleman and Cohen, 2000).
INTRODUCTION
17
Therefore, the authors conclude that transport in the extracellular space accounts for
the observed Dpp gradient. In addition, theoretical modeling suggested that the
formation of the observed shadows behind shi clones could be a result of surface
receptor accumulation due to the inhibition of endocytosis (Lander et al., 2002). This
finding, however, could not be confirmed by antibody staining for the Dpp receptor
thickveins (Kruse et al., 2004).
In contrast to the results for Dpp and despite earlier findings (Bejsovec and
Wieschaus, 1995), recent data obtained for Wg showed that planar transcytosis most
probably does not play a role in Wg gradient formation (Strigini and Cohen, 2000).
Most of the Wg protein could be detected in a shallow extracellular gradient and was
internalized by wt cells behind a shi clone without a shadow forming. These results
obtained for Wg protein distribution lead to the hypothesis that endocytosis might
shape gradients of diffusible morphogens by mediating ligand degradation. This was
shown for Wg by using an HRP fusion construct, which resists lysosomal degradation
(Dubois et al., 2001). Wg protein was more efficiently degraded in the posterior part
of each wg transcribing stripe, leading to an asymmetric response of the target tissue
to Wg signaling.
The same mechanism of targeted degradation or ‘restrictive clearance’ was
described to control propagation of Fgf8 protein in gastrulating zebrafish embryos
(Scholpp and Brand, 2004). Furthermore, a recent study in Drosophila suggests that
endocytosis is required for Ptc mediated Hh degradation (Torroja et al., 2004).
Therefore, endocytosis most probably plays a role in shaping morphogen gradients by
either controlling ligand degradation or mediating planar transcytosis. An important
step towards disentangling these two functions of endocytosis will be the proof that a
morphogen is resecreted from a receiving cell.
Argosomes
The fact that some of the molecules that exhibit morphogen characteristics are lipid
modified and therefore thought to be bound to membranes has always been hard to
reconcile with them being secreted and diffusing through the extracellular space.
However, it was shown that upon labeling of membranes in certain regions of the
Drosophila wing imaginal disc by making use of a GFP linked to GPI, membranous
INTRODUCTION
18
particles could also be found in nearby cells (Greco et al., 2001). This suggests that
membrane of wing imaginal disc cells can vesiculate and travel through the disc
epithelium. Furthermore, these membrane pieces were found to colocalize with Wg
protein, providing evidence that Wg might move in association with membrane
fragments. Interestingly, argosomes were produced by many different regions of the
wing imaginal disc, implying that they might also play a role in the transport of other
morphogen molecules, such as Hh (Greco et al., 2001).
Cytonemes
Cells at the periphery of wing imaginal discs were found to extend long, actin
containing filopodia towards the anterior-posterior compartment boundary of the disc,
a known signaling center at which molecules such as Hh and Dpp are expressed
(Ramirez-Weber and Kornberg, 1999). Therefore, cells could make direct contact to
the source of a morphogen over a long distance. This is an attractive hypothesis,
because cellular protrusions were found to be formed by many different cell types.
For instance, the peripodial membrane of the wing imaginal disc is connected to the
underlying epithelium via cellular protrusions (Gibson and Schubiger, 2000).
Furthermore, in sea urchin embryos, migrating mesodermal cells send out long
extensions to overlying ectodermal cells (Vacquier, 1968). The same phenomenon can
also be observed in gastrulating zebrafish embryos (P. Herbomel, unpublished). In
addition, nerve cells extent long axons, and muscle cells contact these via myopodia,
cellular protrusions necessary for proper formation of neuro-muscular junctions
(Ramirez-Weber and Kornberg, 2000).
Despite these observations, functional evidence for the importance of these
structures in embryonic signaling is lacking so far. However, a recent study addressed
the contribution of filopodia to Fgf dependent tracheal formation in Drosophila (Sato
and Kornberg, 2002). Furthermore, filopodia were shown to mediate lateral inhibition
by the Delta/Notch pathway during the formation of Drosophila mechanosensory
bristles (De Joussineau et al., 2003). The authors showed that disturbing the formation
of filopodia in sensory organ precursor cells interfered with lateral inhibition and lead
to the formation of supernumerary mechanosensory bristles. Other studies in cultured
cells reported the existence of filopodial connections between individual cells
INTRODUCTION
19
(Rustom et al., 2004). Strikingly, cells were shown to exhibit membrane continuity
and an exchange of membrane bound fluorescent proteins could be observed between
cells. In addition, the authors witnessed vesicles moving through these extensions
from cell to cell.
Fig.3. Models explaining morphogen gradient formation in Drosophila wing imaginal discs. (A) Diffusion. The morphogen
(red) is released from its source (blue cell) and diffuses through the extracellular space. (B) Planar transcytosis. The
morphogen is internalized and resecreted by the receiving cells, while some of it is degraded. (C) Cytonemes. Long
filopodia extending to the morphogen source provide a means of direct transport of the morphogen to the target cell. (D)
Argosomes. Membrane particles travel from cell to cell by internalization and resecretion. Morphogens may travel in
association with these membrane particles. Modified from (Tabata and Takei, 2004).
Alternative ways of gradient formation
A recent report suggested an mRNA gradient, which is subsequently translated into a
protein gradient as a mechanism of establishing different morphogen concentrations
over a field of cells. The authors examined the distribution of FGF8 during chick
somitogenesis, which at high levels is important for maintaining an immature cellular
state in the posterior tailbud region, while lower levels more anteriorly trigger somite
maturation. Use of an intronic probe revealed that fgf8 was only transcribed in the tail
bud (Dubrulle and Pourquie, 2004). However, fgf8 mRNA was also found in more
anterior regions of the embryo in what appeared to be a graded distribution.
Therefore, cells moving out of the tailbud seemed to gradually loose FGF8 by
progressively decreasing concentrations of fgf8 mRNA.
INTRODUCTION
20
Such a mechanism most probably cannot be attributable for gradient formation in
the Drosophila wing imaginal disc, where the morphogen source is static. However,
in other developmental contexts, such as in limb formation, morphogen producing
cells have been shown to proliferate and move (Summerbell et al., 1973).
Interestingly, a recent report, in which the authors fate mapped the descendants of Shh
producing cells in the limb showed that these cells contributed extensively to the two
most posterior digits of the limb, previously shown to require high levels of Shh
signaling. Cells of the third digit were also derived from shh expressing cells, albeit to
a lesser extend. This argues for a temporal component in specifying digit identity.
Cells that receive the Shh signal for a longer time would be specified as posterior
digits, while the specification of anterior digits, which do not contain descendants of
shh expressing cells, depends on Shh concentration. Interestingly, in this model of a
“temporal” gradient, an mRNA gradient of shh might also contribute to the observed
concentration differences. However, the authors did not directly address this issue.
Taken together, much controversy exists on how morphogen molecules travel
from cell to cell and how they set up a gradient of concentration. With the advent of
new imaging techniques making in vivo observations possible and improved
immunohistochemical stainings, which allow the visualization of endogenous levels
of morphogen molecules, much will be learned in the future in this exciting field of
developmental biology.
The Hedgehog signaling pathway
In order to address the question how morphogen molecules travel through receiving
tissue, I decided to focus on the analysis of Shh, the vertebrate homologue of the hh
gene. hh was first identified in Drosophila as a member of the segment polarity class
of genes (Nusslein-Volhard and Wieschaus, 1980). In wild-type flies, the anterior half
of every segment carries spiky processes, called denticles. In the hh mutant, the entire
larval cuticle is covered with denticles and was therefore called ‘hedgehog’ (Mohler,
1988).
It is now well established that Hedgehog signaling is required for patterning
neighboring tissues (Ingham and McMahon, 2001; Mann and Beachy, 2004). These
processes range from cartilage differentiation, hair follicle development, myotome
INTRODUCTION
21
and sclerotome differentiation and limb morphogenesis to the specification of
different neuronal cell types. In vertebrates, members of the Hh family have dorsoventral patterning activities over the entire rostro-caudal extent of the neural tube
(Chiang et al., 1996; Jessell, 2000; Tanabe and Jessell, 1996). Studies on neural tube
development also provided evidence for Shh acting as a morphogen, either by
showing that different concentrations of Shh could activate different target genes
(Ericson et al., 1997; Marti et al., 1995) or by blocking the Shh pathway in different
regions in the neural tube (Briscoe et al., 2001).
In addition to dorso-ventral patterning, Shh signaling has also been implicated in
determining left-right asymmetry. In chick, shh is expressed on the left side of the
embryo (Levin, 1997; Levin, 1998). In zebrafish, even though endogenous shh
expression is bilateral, overexpression of shh on the right side of the embryo leads to
reversal of heart and gut looping (Schilling et al., 1999). This together with the
finding that embryos compromised in formation of midline structures which secrete
Shh, such as the notochord exhibit laterality defects (Halpern et al., 1993; Talbot et
al., 1995) suggests that Shh might also play a role in determining the left-right axis in
zebrafish.
In vertebrates, several members of the hh family have been identified. To date,
four Xenopus, three mouse, two human, and four zebrafish homologues are known
(Ingham and McMahon, 2001). The most extensively studied is shh, which is present
in all vertebrate species.
The Hh Signal Transduction Pathway
Hh proteins are transcribed as pre-proproteins with a signal sequence attached to the
N-terminus of the protein (Fig. 4). This signal sequence is cleaved in the secretory
pathway (Lee et al., 1992; Tabata et al., 1992), producing a precursor protein. This
undergoes a further autoproteolytic cleavage, which generates a 19 kDa N-terminal
peptide and a 26 kDa C-terminal peptide (Bumcrot et al., 1995; Lee et al., 1994;
Mann and Beachy, 2004). During this process, a cholesterol moiety is attached to the
N-terminal peptide (Porter et al., 1996b).
Subsequently, an acetyltransferase catalyzes the addition of a palmitate to the Nterminus of this N-terminal fragment, producing a double lipid modified protein
INTRODUCTION
22
(Chamoun et al., 2001; Pepinsky et al., 1998). All known signaling activity resides
within this N-terminal peptide (Bumcrot et al., 1995; Lee et al., 1994; Porter et al.,
1996a; Porter et al., 1996b) that is released from the cell, a process that requires
dispatched function (Burke et al., 1999).
Fig.4. Autoproteolytic
cleavage of Hh family
proteins.
(A) H h genes encode
precursor proteins of about
45 kDa, which undergo
autoproteolytic cleavage,
yielding a 19 kDa N-terminal
fragment that possesses all
known signaling functions.
The C-terminal end of the
protein, which also directs
the addition of cholesterol to
a C-terminal glycine on the
N-terminal
fragment,
catalyzes the cleavage
reaction. Subsequently, the
N-terminus
becomes
palmitoylated involving an
acyltransferase (ski). (B)
Mechanism
of
Hh
autoproteolysis. The reaction
proceeds via a thioesther
intermediate, leading to a
nucleophilic attack of the
cholesterol. After (Mann and
Beachy, 2004)
A transmembrane receptor complex that consists of at least two molecules,
Patched (Ptc) and Smoothened (Smo) is thought to transduce the Hh signal
intracellularly (Hooper and Scott, 1989; Nakano et al., 1989; van den Heuvel and
Ingham, 1996). Ptc belongs to a transporter family of proteins related to bacterial
transporters (Taipale et al., 2002) and harbors a sterol sensing domain (Martin et al.,
2001; Strutt et al., 2001). Mutational analysis indicated that Ptc acts as a negative
regulator of Hh signaling (Ingham, 1993; Ingham et al., 1991; Johnson et al., 1995).
Biochemical binding assays also provided evidence for Hh binding to Patched
(Marigo et al., 1996; Stone et al., 1996). In contrast, Hh did not bind to Smo. In
addition, Ptc was shown to inhibit diffusion of Hh molecules (Chen and Struhl, 1996).
Therefore, Ptc serves a double function in sequestering Hh molecules and in
transducing the signal (Stone et al., 1996).
Smo itself is a seven-pass transmembrane protein similar to the large family of Gprotein coupled receptors (Alcedo et al., 1996; van den Heuvel and Ingham, 1996)
23
INTRODUCTION
that is implicated in mediating a wide variety of signaling cascades. smo mutants have
a phenotype very similar to hh mutants (van den Heuvel and Ingham, 1996). These
results suggest that Smo is constitutively active, but normally repressed by Ptc.
Binding of Hh to Ptc antagonizes Ptc function, thereby allowing Smo to signal. A
more recent analysis indicated that Ptc acts to remove Smo from the cell surface in
unstimulated cells and causes dephosphorylation of Smo (Denef et al., 2000). In
contrast, binding of Hh to Ptc results in removal of Ptc from the cell surface, which
leads to an accumulation and phosphorylation of Smo (Denef et al., 2000). This
suggests an indirect mechanism of Ptc activity, a finding that was further supported by
the result that Ptc could act substoichiometrically, possibly by transporting a small
molecule, to inhibit Smo (Taipale et al., 2002). However, the view on Ptc acting
solely on inhibiting Smo function while Hh bound Ptc is rendered inactive was
recently challenged by a report showing that the ratio of bound to unbound Ptc protein
determines the cellular response to Hh (Casali and Struhl, 2004).
Interestingly, a recent report implicated vesicular trafficking as an important way
of regulating Shh signaling. Mice that carry a mutation in the Rab23 gene show
upregulation of Shh pathway genes even in the absence of Shh ligand (Eggenschwiler
et al., 2001). Furthermore, Rab23 was shown to colocalize with Ptc, but not with Smo
in cell culture (Evans et al., 2003), but altered Rab23 localization did not have an
effect on the trafficking of either Ptc or Smo. Therefore, Rab23 is thought to function
downstream of Hh signaling mediated by Smo.
The newest player in the Hh signaling pathway are interflagellar transport
proteins, which have been shown to be required for Hh signal transduction (Huangfu
et al., 2003). This requirement was shown to be downstream of Rab23 function, but
was suppressed by a mutation in Gli3.
Fig. 5. Hh signal transduction
pathway.
Hh proteins are lipid modified in the
producing cells and found on the plasma
membrane, possibly in rafts. Disp is
needed for the release of Hh proteins,
which have been proposed to form
multimers. HSPGs bind to Hh proteins
and so does Ptc, a transmembrane
protein shown to inhibit Smo activity.
Upon Hh binding this inhibition is
released and Smo signals via cos2 and
Ci to activate target gene transcription.
Rab23 is thought to influence Hh signal
transduction, possibly by regulating
vesicular trafficking.
INTRODUCTION
24
Cytoplasmic Components Of The Hh Signaling Pathway
Smo transduces the Hh signal via a complex of several cytoplasmic proteins that are
associated with microtubules in unstimulated cells. The components of this complex
include Fused (Fu) (Therond et al., 1993), a putative serine-threonine protein kinase,
Suppressor of Fused [Su (Fu) ] (Preat et al., 1993), and Costal-2 (Sisson et al., 1997).
Costal-2 mediates the interaction with microtubules (Robbins et al., 1997). This
complex in turn interacts with the only known transcription factor in the cascade, ci
(Orenic et al., 1987).
Ci exists in two forms, one is a 75 kDa protein, lacking the C-terminus of the fulllength protein. It is present in cells not exposed to Hh and acts as a transcriptional
repressor (Aza-Blanc et al., 1997), therefore named CiR. Formation of this repressor
form requires phosphorylation of specific serine-threonine residues by PKA (Chen et
al., 1998). This leads to subsequent phosphorylation by other kinases, namely GSK3
and CK1 family kinases (Jia et al., 2002; Lum et al., 2003; Price and Kalderon, 2002).
This phosphorylated form of Ci is thought to be cleaved by the proteasome, a process
that requires Slimp (Jiang and Struhl, 1998), ultimately yielding CiR. CiR retains its
DNA binding motif, shuttles to the nucleus and transcriptionally represses Hh target
genes. Upon exposure to Hh, Smo protein is stabilized and interacts with cos2 via
sequences in its C-terminal part (Jia et al., 2003; Lum et al., 2003; Ogden et al.,
2003). This interaction leads to a loss of Ci processing and accumulation of the fulllength Ci protein (Aza-Blanc et al., 1997). In turn, Ci leaves the cytoplasm and enters
the nucleus, where it activates Hh target genes. This activation was shown to require
CREB-binding protein, which acts as a transcriptional coactivator, essential for ci
function (Akimaru et al., 1997).
In contrast to the presence of one ci gene in Drosophila, three orthologs of ci
exist in vertebrates. The first was isolated as an amplified gene in a human glioma
line (Kinzler et al., 1987), hence it was named gli. The three members of the gli
family are expressed in a distinct combinatorial manner in specific tissues (Ruiz i
Altaba, 1998) and influence Hh signal transduction during development (Ruiz i
Altaba, 1999).
INTRODUCTION
25
Gli-1 and Gli-2 are normally expressed near the source of Hh signaling, Gli-3 and
sometimes Gli-2 are expressed in more distal regions (Ruiz i Altaba, 1999).
Interestingly, Gli-3 was shown to act as a repressor of Hh signaling and is expressed
in the dorsal neural tube (Ruiz i Altaba, 1998) and in the limb buds (Buscher et al.,
1997). Therefore, the dual function of Ci as a transcriptional activator and repressor
has been subdivided in vertebrates, where three Gli genes with activating and/or
repressing functions exist.
Distribution of Hh proteins
Biochemical analysis showed that Hh proteins are secreted molecules (Lee et al.,
1992), but in contrast to this the amino terminal signaling domain remained tightly
cell-associated when expressed in cultured cells (Lee et al., 1994; Porter et al., 1995).
In Drosophila embryos, however, protein could be detected outside producing cells in
large punctate structures (Lee et al., 1994; Taylor et al., 1993), which were found to
be mostly shared between two or three cells (Tabata and Kornberg, 1994). A more
recent report showed that these large punctate structures localize to apical regions of
receiving cells, a process that requires cholesterol modification of Hh proteins (Gallet
et al., 2003).
Furthermore, endocytosis seems to play a role in shaping the Hh protein gradient.
Preventing endocytosis in the larval cuticle by using a temperature sensitive shibire
allele revealed that Hh proteins accumulate in expressing cells when they cannot be
internalized anymore (Tabata and Kornberg, 1994). This gains further support from
studies in cell culture demonstrating that Shh proteins are found in endocytic vesicles
(Incardona et al., 2000). In addition, Hh proteins have been shown to be present in
endocytic compartments in fly wing imaginal discs (Torroja et al., 2004).
Endocytosed Hh was subsequently targeted to lysosomal degradation. Blocking of
endocytosis lead to accumulation of Hh proteins in the extracellular space. Thus,
endocytosis seems to be required for Hh gradient formation, possibly by regulating
Hh degradation (Torroja et al., 2004). Interestingly, the same process has been
described to function in controlling the asymmetric gradient of Wg in embryonic
segments (Dubois et al., 2001) and in restricting the spreading of Fgf8 (Scholpp and
Brand, 2004).
INTRODUCTION
26
In vertebrates, Shh proteins are thought to form multimeric complexes which,
despite the lipid modifications of individual proteins, allow them to be released from
producing cells. The presence of multimeric Shh could be confirmed by biochemical
analysis (Chen et al., 2004; Zeng et al., 2001) and isolation of a multimeric form of
Shh from vertebrate limb buds (Zeng et al., 2001). Improved immunohistochemistry
on sectioned mouse neural tube tissue revealed the presence of Shh outside producing
cells in a graded manner (Gritli-Linde et al., 2001), but a detailed analysis of the
subcellular Shh distribution in vertebrate embryos is still missing.
Defects in Hh Signaling and Human Disease
From the identification of gli-1 in a human glioma line, it became apparent that Hh
signaling could play a crucial role in development and as an oncogen. Several types of
human cancers are caused by mutations in components of the Hh signaling pathway,
such as Sporadic Basal Cell Carcinoma (Dahmane et al., 1997) and other sporadic
cancers (Ruiz i Altaba et al., 2002).
Furthermore, as inferred from the developmental function of Hh signaling,
several human congenital malformations are associated with mutations of the Hh
pathway. The most common is holoprosencephaly. It results from incomplete
cleavage of the forebrain (prosencephalon) into right and left hemispheres (Muenke
and Beachy, 2000; Roessler and Muenke, 2003). This leads to a variety of birth
defects, including median lip cleft, single nostril and cyclopia. Holoprosencephaly is
the most frequent brain anomaly in humans. It has a prevalence of 1 per 10,000 to
20,000 live births and of 1 per 250 during early embryogenesis. Many genes that are
associated with this disease have been identified in humans using data obtained from
genetic analysis in Drosophila, zebrafish and mouse (Muenke and Beachy, 2000).
This highlights the importance of studying zebrafish development both for
understanding developmental processes per se, and as a tool to understand the basis of
human genetic diseases (Dooley and Zon, 2000), such as hematological disorders
(Shafizadeh and Paw, 2004), aging (Kishi, 2004), muscular dystrophy (Bassett and
Currie, 2004) and bacterial infections (Miller and Neely, 2004).
INTRODUCTION
27
Zebrafish Mutations in Hh Pathway Components
Several mutations in members of the Hh pathway, which cause the same somite
phenotype have been identified in the zebrafish (van Eeden et al., 1996). Instead of a
distinct ‘chevron’ shape, the myotomes in Hh mutants are ‘u-shaped’, leading to the
name ‘u’-type mutants. These mutants include you (you), you-too (yot), sonic-you
(syu), chameleon (con), u-boot (ubo), and iguana (igu) (Brand et al., 1996; van Eeden
et al., 1996), several of which have been cloned.
yot encodes gli2 (Karlstrom et al., 1999) and syu encodes the zebrafish shh
ortholog (Schauerte et al., 1998). Interestingly, the phenotype of syu mutant fish is
much less severe when compared to the mouse knockout of the shh gene (Chiang et
al., 1996). This suggests redundant functions of other hh homologs in zebrafish such
as tiggy-winkle hedgehog (twhh), that is expressed in the shield, the floorplate and the
ventral midline of the midbrain (Ekker et al., 1995), as well as echidna hedgehog
(ehh), that is exclusively expressed in the notochord and its precursors (Currie and
Ingham, 1996). Furthermore, con was shown to encode zebrafish dispatched1
(Nakano et al., 2004), igu to encode a novel zinc finger containing protein Dzip1
(Sekimizu et al., 2004; Wolff et al., 2004) and detour (dtr) (Brand et al., 1996) to
encode zebrafish gli1 (Karlstrom et al., 2003).
In addition, mutations in slow muscle omitted (smu) have been isolated and
characterized (Barresi et al., 2000). The affected gene encodes a smo ortholog in
zebrafish (Chen et al., 2001; Varga et al., 2001). The mutants display strong
phenotypes in all regions that are thought to depend on Hh signaling. This could
indicate that most, if not all Hh signals converge on this part of the receptor complex.
Thus, the smu mutant might be a helpful tool to study processes that are thought to
depend on Hh signaling, but could so far not be analyzed because of redundant Hh
function. I used smu mutant embryos to address the question whether Hh signaling is
required for cell specification in gastrulating embryos. Furthermore, I addressed the
functionality of a SHH-venus fusion protein by rescue of syu mutant zebrafish.
INTRODUCTION
28
Relevance of the extracellular matrix in morphogen
signaling
Many morphogen molecules have been shown to interact with components of the
extracellular matrix, namely Heparan Sulfate Proteoglycans (HSPGs). The proteins
known to interact with HSPGs include members of the Wingless/Int (Wnt), fibroblast
growth factors (FGF), transforming growth factor (TGF) and Hedgehog (Hh)
families (Blackhall et al., 2001; Kramer and Yost, 2003; Nybakken and Perrimon,
2002). HSPGs are thought to regulate morphogen signaling in at least two ways: by
binding to the respective ligand and thereby influencing its distribution and by
regulating accessibility of receptor molecules. Therefore, I decided to study the
influence of HSPGs on morphogen and specifically Shh spreading and signaling in
zebrafish by identifying enzymes implicated in HSPG synthesis.
Synthesis of HSPGs
The synthesis of HSPGs depends on the addition of a glycosaminoglycan chain to a
core protein (Kjellen and Lindahl, 1991). Three core proteins are known, namely
syndecan, glypican and perlecan. The following addition of glycosaminoglycans is
catalyzed by glycosyltransferases belonging to the EXT (Exostosin) family (Lind et
al., 1998). Subsequently, the attached sugar residues are modified by a N-sulfation
reaction, followed by an epimerisation and an O-sulfation (Baeg and Perrimon, 2000)
(Fig. 4), ultimately yielding a great diversity of molecules with different sugar chain
lengths and sulfation patterns.
INTRODUCTION
29
Fig. 4. Synthesis of HSPGs. The linkage region of the core protein is covalently bound to alternating GlcNAc and GlcA
residues. These are further modified by N -deacetylation/ N -sulfation, epimerization and O -sulfation, yielding a great
structural diversity. From (Baeg and Perrimon, 2000).
EXT family of glycosyltransferases
The most extensively studied HSPG modifying enzymes are those belonging to the
EXT family. To date, several members of the EXT family have been cloned and
identified in humans. These include EXT1, EXT2, EXT3 and three EXT-like genes
(Cook et al., 1993; Hecht et al., 1995; Le Merrer et al., 1994; Van Hul et al., 1998;
Wise et al., 1997; Wuyts et al., 1997). In addition, orthologous genes for EXT1 and
EXT2 were identified in mouse, Xenopus, C. elegans and Drosophila, where the EXT1
homologue was named tout-velu (ttv) (Bellaiche et al., 1998; Clines et al., 1997; Han,
2001; Katada et al., 2002; Lin and Wells, 1997; Stickens and Evans, 1997; The et al.,
1999). However, to date no ext homologues are known in zebrafish and only one
member of the Ext1 family was found in every species examined so far. In contrast to
this, I could identify three ext1 family members in zebrafish, which are expressed in
mutually exclusive and overlapping domains.
Developmental functions of HSPGs
In humans, mutations in Ext1 genes cause HME (hereditary multiple exostoses),
benign tumors at the growths caps of the long bones (Zak et al., 2002). Mice that
carry a targeted deletion of the Ext1 gene die at gastrulation stages (Lin et al., 2000),
INTRODUCTION
30
which hampered a more detailed analysis. In a conditional loss of function situation,
which eliminated the Ext1 gene in the brain, a composite patterning phenotype similar
to the loss of function of multiple morphogen molecules was observed. In addition,
axon guidance defects were reported (Inatani et al., 2003). These phenotypes were
indicative of a broad specificity of HSPGs for various signaling pathways.
The organism in which HSPG function was most extensively studied is the
fruitfly. A surprising finding in Drosophila was that, in contrast to results obtained in
mammals, pathway specific HSPGs seem to exist. For instance, flies mutant for ttv
show a specific defect in the Hh pathway (Bellaiche et al., 1998; The et al., 1999).
Furthermore, HSPGs play a role in Wg and Dpp signaling during wing imaginal disc
development. In contrast to this, in embryonic dorso-ventral patterning, loss of
HSPGs only impairs Wg signaling, while Dpp functions normally (Baeg and
Perrimon, 2000). Therefore, HSPG function in flies does not only appear to be
pathway specific, but also to depend on the developmental context. The finding that
three zebrafish ext1 homologues are expressed in distinct tissues during development
might facilitate the analysis of vertebrate Ext1 genes.
31
Abbreviations
DAB
diaminobenzidine
Da
dalton
°C
degrees Celsius
C-terminus
carboxy-terminus (of a peptide)
DIG
digoxigenin
DNA
desoxyribonucleic acid
Dpp
decapentaplegic
ext1
exostosin1
Fgf
fibroblast growth factor
GFP
green fluorescent protein
GlcA
D-glucoronic
GlcNAc
N-acetyl-D-glucosamine
GPI
glycosylphosphatidylinositol
Hh
Hedgehog
HME
hereditary multiple exostoses
hpf
hours post fertilization
HRP
horseradish peroxidase
HSPG
heparan sulfate proteoglycan
Hyb
hybridization buffer
k
kilo
mAb
monoclonal antibody
min
minutes
MHB
midbrain-hindbrain-boundary
MyHC
slow muscle myosin heavy chain
N-terminus
amino-terminus (of a peptide)
PAGE
polyacrylamid gel electrophoresis
PCR
polymerase chain reaction
PBS
phosphate buffered saline
PBST
phosphate buffered saline with Tween
PFA
paraformaldehyde
acid
ABBREVIATIONS
ptc1
patched1
RACE
rapid amplification of cDNA ends
RNA
ribonucleic acid
SDS
sodium dodecyl sulfate
Shh
Sonic Hedgehog
TILLING
Targeting Induced Local Lesions IN Genomes
TNT
tunneling nano tubes
UTR
untranslated region
Wg
wingless
wt
wildtype
YFP
yellow fluorescent protein
32
33
Materials and Methods
Chemicals and solutions
All chemicals, if not noted otherwise, were purchased from the companies Merck,
SIGMA, Roth, and Applichem.
ABC-Kit
Vectastain (Vecta Laboratories)
anti-DIG antibody
Roche 1093274
anti-fluorescein antibody
Roche 1426338
BM Purple
Roche 1442074
DAB
Diaminobenzidine (SIGMA)
2% blocking reagent (Roche 1096176)
dissolved in MABT
5 mM NaCl, 0.17 mM KCl, 0.33 mM
CaCl2 x 2 H2O, 0.33 mM MgSO4 x 7
H2O, 10-5% Methylenblue
DIG block
E3 embryo medium
Fast Red substrate
Roche 1496549
-
Hyb
50% deionized formamid; 5x SSC pH
6.0; 0.1% Tween-20
Hyb+
Hyb- + 0.5 mg/ml torula (yeast) RNA; 50
µg/ml heparin
LB-Medium
0.5% yeast extract (Gibco BRL), 1%
Tryptone (Difco), 200 mM NaCL, H2O
ad 1 liter
MABT
100 mM maleic acid; 150 mM NaCl
adjusted to pH 7.5 with NaOH, 0.1%
Tween-20
PBS + 0.1% Tween 20
PBST
PFA
4 % paraformaldehyde in 100 mM
phosphate buffer, pH 7.4
proteinase K
Sigma P 6556
rhodamine dextran (“mini-ruby”)
Molecular Probes/Invitrogen, D-3312
SDS-PAGE
sodium dodecyl sulfate polyacrylamid gel
electrophoresis
0.1% SDS, 0.375 M Tris-Cl, pH 8.8, 12%
Acrylamide
SOC-Medium
LB (SIGMA L3397) 1.55g; 250mM
KCL; 2M MgCl2; 1M Glucose; H2O ad
34
MATERIALS AND METHODS
100 ml
20x SSC
for 1 liter: 175.3 g NaCl; 88.2 g Nacitrate (x2 H2O), pH adjusted to 6.0 with
1 M citric acid
SSCT
SSC + 0.1% Tween-20
Torula RNA
Sigma R 6625
tungsten wire
Clark Electromedical Instruments TW103 and WPI TGW1510
Fish husbandry and care
Zebrafish were kept at a constant light-dark cycle (14 h light/10 h dark). For
breeding, 2 fish were put in a mating container. To avoid parental cannibalism the
cage contained a sieve that separated parents from eggs. The eggs were collected and
transferred to methylene blue egg water to prevent the growth of fungi and bacteria.
The eggs were maintained at 28.5°C until the desired stages. Fish were dechorionated
after fixation until the 18-somite stage. Later stages were dechorionated prior to
fixation, using watchmaker forceps. Embryos for transplantation were raised at 25°C
once they had reached the 50% of epiboly stage to slow down development. They
were dechorionated and kept in agar-coated Petri dishes to prevent them from sticking
to the plastic.
In vitro RNA transcription
I used the mMessage mMachine kit (Ambion) to produce mRNA for injection into
zebrafish embryos according to the manufacturer’s instructions. RNA was capped
with a cap analog and synthesized in vitro.
RNA injections
To overexpress genes in zebrafish embryos, I injected mRNA into 1 cell stage
embryos using borosilicate capillaries with filament (TW-100 4F, WPI) and air
pressure. I coinjected Mini-Ruby Rhodamine (Molecular Probes) as a lineage tracer
with the capped RNA to follow injected cells in a host embryo. In addition, mini-ruby
molecules are covalently linked to biotin. This allows detection of injected cells after
35
MATERIALS AND METHODS
fixation. For blastomere injections, I coinjected Rhodamine dextrane with a molecular
weight of 2x106 (Molecular Probes). The high molecular weight prevents diffusion of
the dye between individual blastomeres, which at this stage are still connected via
cytoplasmic bridges (Kimmel and Law, 1985).
Microneedles were prepared using a Flaming-Brown puller. For injections, the
capillaries were backfilled with RNA and lineage tracer. After pulling, small
openings at the microneedle tip were created by either cutting with a razor blade or by
using a pair of forceps to break off the tip. For the injection procedure, a special agar
ramp containing vertical furrows was prepared as described by Weinberg in the
zebrafish book (Westerfield, 1995; Figure 1). After filling the ramp with eggwater,
the embryos were aligned in the furrows animal pole up. After penetration of the
blastomere with the needle, a droplet of mRNA with a volume of about 2 nl was
injected into the yolk just underneath the blastodisc of each egg.
Morpholino injections
The following anti-sense morpholino oligonucleotides (GeneTools) were used:
Gene
Morpholino Sequence
name
Amount
used
rab23 rab23-ATG
5’-CCTCCTCCAGCATGACTGCGCTGTG-3’
12 pg
rab23 rab23-UTR
5’-TAGTGTATCCAGGTTAAGTTGTTTC-3’
12 pg
ext1a
ext1a-ATG
5’-TTTGGCCTGCATGTGTCCTCAGATC-3’
4 pg
ext1a
ext1a-UTR
5’-GCTGATGAATCCAAGGGTCTCAACT-3’
4 pg
ext1b
ext1b-UTR
5’-TTTGGAAGTTTGTCCCTCAACACCA-3’
4 pg
ext1c
ext1c-ATG
5’-TCTTGCGTGCCTGCATGAGGCTGTC-3’
4 pg
The ATG translational start codon is highlighted in bold. The morpholinos were
diluted in water with 0.1% phenol red to visualize the solution and injected in the yolk
at the 1-2 cell stage.
MATERIALS AND METHODS
36
Cell transplantation
To assess the inductive properties of cell populations expressing signaling molecules
at ectopic positions, cells were transplanted into host embryos. I used capillaries
without a filament (TW-100 4, WPI). These were beveled on a Narishige needle
beveller, creating a 45 degree angle to facilitate penetration of the embryo. The inner
diameter of the tip was slightly larger than the transplanted cells. The needle was
placed in a needle holder attached to a syringe with an airtight tube. The needle holder
was then mounted on a micromanipulator (Narishige). By varying the air pressure in
the syringe, I could transfer cell from one embryo to the next. Shield stage embryos
were immobilized before transplantation by putting them in rows of triangular shaped
depressions which were generated in agarose ramps. The agarose coated dish was
filled with 1x Ringer’s and the transplantation was carried out using a binocular.
About 10 - 50 cells were drawn into the needle, avoiding the uptake of yolk. The
cells were transferred to the host embryo and released at the animal pole, lateral to the
axis.
Synthesis of DIG or Fluorescein labeled probes
1 µ g of linearized template DNA was mixed with 2 µ l NTP mix, 2 µl 10x
transcription buffer (Roche) and 1µl RNAsin (Ambion) RNAse inhibitor. The volume
was adjusted to 18µl with H2O. Finally, 2 µl of the appropriate RNA polymerase were
added. After 2 h incubation at 37°C, 2 µl 0.2 M EDTA pH 8.0, 2.5 µl 4 M LiCl and
75 µl ice-cold 100 % ethanol were added, vortexed quickly and incubated for at least
30 min at –20°C to precipitate the RNA. RNA was then pelleted by 30 min
centrifugation at 13000 rpm at 4°C in a standard bench top centrifuge. After washing
with 70% ethanol and brief air drying, the RNA was resuspended in 100 µl H2O and
diluted to 500 µl with Hyb+. Routinely, a 1:100 dilution of this stock was used in in
situ hybridization protocols. I used probes for the following genes: ext1a (this study),
ext1b (this study), ext1c (this study), ptc1 (Concordet et al., 1996), pax2.1 (Krauss et
al., 1991), pax6 (Puschel et al., 1992), foxa2 (axial/HNF3) (Korzh, 2001; Strahle et
al., 1993) and shh (Krauss et al., 1993), lefty2 (Bisgrove et al., 1999) and gata4
(Stainier, D., unpublished) (Laverriere et al., 1994).
MATERIALS AND METHODS
37
Whole mount in situ hybridization
Whole mount in situ hybridization provides a tool to tissue specifically detect gene
expression in an embryo. Detailed protocols for in situ hybridization have been
previously described (Reifers et al., 1998). Briefly, embryos were fixed overnight at
4°C in 4 % PFA. They were then washed in PBST and transferred into 100%
methanol at –20°C for permeabilization. Before hybridization, the embryos were
rehydrated and rinsed in PBST. They were transferred into hybridization buffer that
contained formamide and SSC and incubated at 68°C. Addition of torula RNA
blocked unspecific binding sites and Tween 20 inhibited the embryos from sticking to
each other. After prehybridization, the DIG-labeled probe was added and the embryos
were incubated over night at 68°C on a shaker. They were then washed in decreasing
concentrations of SSC and finally transferred to PBST. Before -DIG antibody
(Roche) incubation, the embryos were rinsed for an hour in blocking solution to
prevent unspecific binding. To increase signal intensity, an anti-DIG antibody
coupled to alkaline phosphatase was used. The embryos were then stained with BM
purple (Roche) in MABT. Images of whole mount in situ hybridized embryos were
taken under an Olympus BX2 microscope using Metamorph software (Universal
Imaging). Final processing of the images was done with Adobe Photoshop software.
Double whole mount in situ hybridization
To detect two mRNAs simultaneously (Hauptmann and Gerster, 1994; Hauptmann
and Gerster, 2000), I labeled one mRNA with DIG, the other with fluorescein. This
allowed detection of the DIG probe by an anti-DIG antibody and detection of
fluorescein by an anti-fluorescein antibody (Roche). After simultaneous incubation of
the embryos with both probes, detection of the first probe was carried out following
the standard in situ hybridization protocol and BM purple coloration reaction. The
antibody complex (e.g. against DIG) was then stripped off, using an acidic glycine
solution. The second probe (e.g. fluorescein labeled) was then detected using an APcoupled anti fluorescein antibody and Fast Red (Roche) coloration reaction.
MATERIALS AND METHODS
38
Immunohistochemistry
I used immunohistochemistry to detect the distribution of specific proteins in
embryos. A primary antibody binds to a specific epitope and can then be detected by
a secondary antibody.
Subsequently, I amplified the signal intensity using a
biotin/avidin system (ABC-Kit). It exploits the high binding affinity of avidin to
biotin. A secondary antibody coupled to biotin is used and later incubated with a
solution containing avidin and biotinylated horseradish peroxidase, creating a stable,
high molecular weight complex. The horseradish peroxidase activity transforms DAB
into a brown color precipitate that can be detected using bright field microscopy. In
order to detect acetylated tubulin after in situ hybridization, I used a mouse
monoclonal -acetylated antibody (SIGMA). The antibody was added to the -DIG
antibody in a 1:1000 dilution and detected as described previously (Lauderdale et al.,
1997). In brief, after color development of the in situ, embryos were post fixed in 4%
PFA for 1 hour and rinsed 4 times 5 minutes in PBST. They were then blocked for
one hour in blocking buffer (2 mg/ml BSA, 0.1% Triton X-100, 1% goat serum in
PBS, pH 7.4). Subsequently, the primary antibody was detected using the anti-mouse
ABC kit (Vector laboratories) and DAB.
In addition, I used antibodies against Prox1 (Glasgow and Tomarev, 1998),
engrailed (mAb 4D9) (Patel et al., 1989) and slow MyHC (Devoto et al., 1996). The
stainings were carried out as previously described (Roy et al., 2001).
Biotin detection of transplanted cells
In order to detect transplanted mini-ruby-biotin labeled donor cells in a host embryo, I
used the ABC kit as follows: After in situ hybridization, embryos were 3 times
washed in PBST for 5 min. Then they were incubated in the diluted AB complex (8 µl
A + 8 µl B in 1 ml PBST, prepared 30 min in advance) for 45 min. They were washed
3 times for 5, 10 and 15 min and subsequently transferred to 0.1M Tris/HCl, pH 7.4.
The color reaction was carried out with DAB according to the manufacturer’s
instructions. The reaction was stopped by several washes in PBST. The embryos were
then cleared in 70% glycerol.
MATERIALS AND METHODS
39
Confocal microscopy
I used a Zeiss LSM 500 confocal microscope for the detection of Shh-venus in live
zebrafish embryos. I collected Z-stacks over time which I later assembled using
Volocity (Improvision) or Imaris (Bitplane) software. I oriented the embryo animal to
the top and took z-sections of about 20 µm, which at this stage of development
roughly equals one cell diameter. These z-sections were assembled into movies
(available on CD) and selected time points were assembled into composites.
Heat shock transformation of bacteria
To transform chemically competent E. coli XL-1 blue cells I used a heat shock at
42°C. This facilitated the uptake of plasmid DNA by the bacteria. The plasmids
contained ampicillin resistance genes as selection marker. Plasmid DNA was diluted
to 50 ng/µl in H2O and 1 µl was added to 200 µl of competent E. coli XL-1 blue cells.
The cells were incubated on ice for 20 minutes, then transferred to a water bath for
heat shock at 42°C (30 s), and chilled on ice for another 5 minutes. Finally, I added
0.5 ml of SOC medium and incubated the cells on a shaker for 1 h at 37°C. To
distinguish single colonies after overnight incubation 10 µl were plated on selective
agar plates and incubated at 37°C. The next morning, colonies were selected and
grown in a suitable amount of LB medium for Mini, Midi or Maxi Plasmid
preparations.
Plasmid preparations
I used Quiagen kits for Mini, Midi and Maxi Plasmid preparations according to the
manufacturer’s instruction. In brief, bacteria were harvested by centrifugation and
resuspended in an alkaline lysis buffer. After neutralization a precipitate of genomic
DNA and protein formed, which was removed by filtration. The plasmid DNA stayed
in solution. It was absorbed on a filter and washed before elution. Finally, the
recovered DNA was resuspended in double distilled water (dH20) and diluted to
approximately 1 µg/µl.
MATERIALS AND METHODS
40
Agarose Gel Electrophoresis
To detect DNA fragments of lengths of about 1-6 KB, I used 1% agarose gels
prepared with TAE buffer. To visualize the DNA fragments, I added 0.5 µg/ml
ethidiumbromide to the liquid agarose. Ethidiumbromide intercalates into the stacked
bases of the DNA molecule and emits orange light when illuminated with ultraviolet
light. To estimate the running time of the gel, I added gel-loading buffer to the DNA.
It contained glycerol and two dyes: Bromphenol blue (dark blue) co-migrated with
DNA of approximately 1 KB, whereas xylene cyanol FF (turquoise) indicated 500 bp
DNA fragments. To estimate the approximate size of the bands, I used a 1KB marker
from Invitrogen.
Extraction of DNA from agarose gels
Single bands of DNA were extracted from agarose gels by cutting out a piece of
agarose that contained a specific band. Subsequently, I used the QuiaQuick Gel
Elution (Quiagen) kit according to the manufacturer’s instructions. The DNA was
resuspended in 30 µl of dH2O and used for either TOPO cloning or subsequent
ligation reactions.
Restriction digest
The restriction reaction contained DNA, a specific enzyme and was buffered
according to the manufacturer’s instructions. To generate linear DNA templates for
mRNA or in situ probe synthesis, about 10 µg of DNA were digested in a volume of
100 µl. Restriction analysis of Miniprep DNA was carried out in a volume of 50 µl
using about 1 µg of DNA. In general, the digestion reaction was incubated at 37°C
for 2-3 hours. After complete digestion, the reaction was stopped by incubation at
68°C for 20 minutes.
Dephosphorylation and ligation of DNA fragments
DNA fragments, e.g. inserts and vectors bearing complementary ends to one another
can be ligated using a DNA ligation kit (Takara). If the vector is linearized using only
MATERIALS AND METHODS
41
one restriction enzyme, the ends of the linear vector have to be dephosphorylated in
order to prevent religation of the vector. To do so, I used shrimp alkaline phosphatase
(Amersham). 5 µg of DNA were digested using the appropriate restriction enzyme.
The DNA was then purified with a PCR Purification kit (Quiagen) and eluted in 51 µl
of dH2O. 6 µl of buffer were added and 3 µl of phosphatase. After incubation for 30
min at 37 °C, the reaction was stopped for 20 min at 68 °C. Subsequently, the DNA
was gel purified using a Gel Extraction kit (Quiagen) and resuspended in 30 µl of
dH2O. For the ligation reaction, 50 ng of Vector were used at a 3-5 times molar excess
of insert.
TOPO cloning
I used TOPO (Invitrogen) vectors according to the manufacturer’s instructions to
subclone PCR amplified DNA fragments. In brief, DNA fragments were amplified
using taq polymerase possess 5´A overhangs. These are complementary to T
overhangs of activated TOPO vectors. Bound topoisomerases facilitate the ligation
reaction. For PCR-fragments that were amplified using a proofreading polymerase
that does not create 5’A overhangs, TOPO ZeroBlunt (Invitrogen) vectors were used.
E. coli XL-1 blue cells were transformed with the vector using a heat shock protocol.
Subsequently, I screened for positive clones by colony PCR or Mini preparations.
Preparation of cDNA
I prepared complementary DNA (cDNA) from zebrafish message RNA (mRNA) in
order to amplify and clone genes. The preparation of the mRNA was carried out using
DynaBeads (Dynal) according to the manufacturers instructions. In brief, total RNA
was extracted from zebrafish larvae using Trizol (Gibco). To this end, larvae of
different developmental stages were ground up in a mortar in the presence of liquid
nitrogen prior to Trizol treatment. After purification of the total RNA, mRNAs were
specifically bound to magnetic beads harboring polyT sequences by virtue of their
polyA end structures. This mRNA was then used for cDNA synthesis using a reverse
transcriptase (Invitrogen).
42
MATERIALS AND METHODS
Database searches
To identify zebrafish homologues for EXT1 genes, I searched the publicly available
zebrafish genome databases with the mouse EXT1 gene, making use of the BLAST
algorithm. I retrieved sequences from NA6265 (ext1a), ctg9593 (ext1b) and NA205
(ext1c)
from
the
ZON
laboratory
Blast
server
(http://134.174.23.160/zfBlast/PublicBlast.htm). These encode parts of each of the
zebrafish ext1 homologues, which were subsequently amplified using PCR. The
complete open reading frames were amplified by RACE PCR.
PCR
I used PCR to specifically amplify DNA pieces or whole genes of interest. The
standard protocol included 10ng of template DNA, 10 mM dNTPs, 50 mM MgCl2 (in
10x PCR buffer from Invitrogen), 20 pmol of each forward and reverse primers and 5
units Taq polymerase in a total volume of 25 µl. The amplification reaction was
carried out in a Perkin Elmer 9600 thermal cycler using the following cycling
parameters:
Step
Program step
Temperature
Time
1
Denaturing
94 °C
2 min
2
Denaturing
94 °C
30 sec
4
Annealing
Primer dependent
30 sec
5
Elongation
72 °C
1 min/kb
The program was cycled between step 2 and 5 for 35 times. Subsequently, the
DNA was analyzed using agarose gel electrophoresis. For more demanding PCR
amplifications, such as amplification of genes from cDNA, a touchdown protocol in
combination with a polymerase mixture of proofreading and taq polymerase
(Advantage taq, Clontech) was used. For this protocol the cycling parameters were
adjusted as follows:
43
MATERIALS AND METHODS
Step
Program step
Temperature
Time
1
Denaturing
94 °C
2 min
2
Denaturing
94 °C
30 sec
4
Annealing
72 °C
2 min
5
Denaturing
94 °C
30 sec
6
Go to 4, 5 times
-
-
7
Denaturing
94 °C
30 sec
8
Annealing
66 °C
30 sec
9
Elongation
68 °C
2 min/kb
10
Go to 7, 22 times
-
-
Again, annealing temperatures had to be adjusted according to the primers used.
However, only primers with an annealing temperature above 68 °C were used for
touchdown PCR applications. In general, this protocol greatly increases the specificity
of the PCR reaction. For the zebrafish ext1 genes, I used cDNA from 15-somite stage
embryos and the following primers in the touchdown protocol:
ext1a fwd 5’-GCAGGAGTTGAGACCCTTGGATTCATCAGC-3’,
ext1a rev 5’-TGCCGTGTTTGCATGTGGTAAGGAGAACC-3’,
ext1b fwd 5’-TGGTGGTGTTGAGGGACAAACTTCCAAACG-3’,
ext1b rev 5’-CGTGTCCGAGCCGATGCCTGTCAA-3’,
ext1c fwd 5’-CCTGCTGAAGCTCCCGGCGTGGC-3’,
ext1c rev 5’-GGAAACGATGTCCTTGCCATTGTGTATGTGATG-3’.
To amplify zebrafish rab23, I used a degenerate primer PCR. Upon analysis of
sequence homologies within the known Rab23 proteins, I designed primers which
allowed for less stringent binding due to base pair wobbling. The primers had the
following sequences:
rab23degen fwd 5’-GARGAYGTCMGCYTNATGYTN-3’ ,
rab23degen rev 5’-CARRTCDATGTTCTTYTGNAC-3’.
Abbreviations are: R: AG, Y: CT, M: AC, N: ATGC. With these primers I could
amplify a DNA fragment of 210 bp length and homology to other RAB23 proteins.
From this sequence I designed primers for RACE PCR.
MATERIALS AND METHODS
44
RACE PCR
In order to amplify the full length sequences of only partially cloned genes, I used a
RACE PCR kit (GENERACER Invitrogen). This protocol allows the amplification of
both 5’ and 3’ ends of cDNAs. Briefly, oligonucleotides with known sequences were
added to both the 5’ and the 3’ end of the respective cDNA. Primers complementary
to these oligonucleotides and provided in the kit allowed the amplification of the
respective end of the cDNA when used together with a gene specific primer. I used 3’
RACE PCR to amplify zebrafish ext1a, ext1b and ext1c genes, respectively. In order
to clone zebrafish rab23, I used both 3’ and 5’ RACE reactions. The primers used
were as follows:
ext1a 5’-GGCAAGCGCTATTTGACTGGGATTGGGTCTGAC-3’,
ext1b 5’-TTGACAGGCATCGGCTCGGACACG-3’,
ext1c 5’-AGATGCTCGCTGTGACAACCAGGA-3’.
To amplify the 5’ and 3’ ends of rab23, I used the following primers:
Rab23 5primeRACE 5’-GATGGCATCAAACTCCTCCTGCC-3’
Rab23 3prime RACE 5’-CGTCTGTTTCATGTTATGGGACACGGC-3’.
Upon cloning and sequencing of the fragments, I could design primers to amplify the
full length rab23 ORF. The primers used were as follows:
rab23 fwd 5’-TATGCCAGAGTCTCCCGCTTCCCGCA-3’
rab23 rev5’-ACGTGAGCGAGGGCAGCGTCTGACAGA-3’.
Site Directed Mutagenesis
I used a site directed mutagenesis kit (Stratagene) to introduce single amino acid
changes or to introduce restriction enzyme sites in particular genes. This was achieved
by designing two complementary primers which harbor the desired mutations. With
these, I performed a PCR with a proofreading enzyme. Subsequently, the DNA was
subjected to a restriction digest with DpnI. This methylation sensitive enzyme only
digests the methylated template DNA. Therefore, in the following transformation,
only mutated, newly synthesized Plasmid DNA will be amplified. These plasmids
were then analyzed for the desired mutation by restriction digest and sequencing.
MATERIALS AND METHODS
45
Generation of Shh-GFP fusion proteins
Bearing the autoproteolytic activity of the Shh proteins in mind, I generated Shh
fusion proteins with fluorescent proteins either attached to the N-terminus of the Nterminal fragment or to the C-terminus of the N-terminal fragment. For the N-terminal
fusion, I used a site directed mutagenesis approach to introduce a NotI site at position
33 of the protein. The primers used were as follows:
ShhNotIfwd 5’-GGTCCTGGCAGAGGCTACGGCGGCCGCAGACATCCGAAG
AAGCTG-3’,
ShhNotIrev 5’-CAGCTTCTTCGGATGTCTGCGGCCGCCGTAGCCTCTGCCA
GGACC-3’.
In addition, GFP (Clontech) was modified with NotI restriction sites at the N and
C terminus by using the following primers:
GFPNotIfwd 5’-TGCACTCAGATTGTCAAGGCGGCCGCATGGTGAGCAAGG
GCGAGGAG-3’
GFPNotIrev 5’-ACTCTATCTGACAGAAAGCGGCCGCGATACTTGTACAGCTC
GTCCAT-3’.
Subsequently, the modified GFP was cloned into TOPO vectors. After restriction
digest and gel purification, GFP was cloned into linearized ShhT7TSNotI. The correct
orientation of the insert was determined by restriction digest and sequencing, yielding
Shh-NGFP. For the C-terminal fusion protein, I introduced XhoI and ClaI sites at
position 194 of Shh. In addition, XhoI and ClaI restriction sites were added to “venus”
(Nagai et al., 2002). Primers used were as follows:
ShhXhoIClaIfwd 5’-CGGTTGCTGCGAAACTCGAGTATCGATTCTGGGGGCTG
TTTC-3’,
ShhXhoIClaIrev 5’-GAAACAGCCCCCAGAATCGATACTCGAGTTTCGCAGCA
ACCG-3’.
venusXhoIfwd 5’- AAACTCGAGATGGTGAGCAAGGGCGAG-3’,
venusClaIrev 5’-TTTATCGATATACTTGTACAGCTCGTCCAT-3’.
After restriction digest, “venus” was cloned into XhoI/ClaI linearized ShhT7TS,
yielding Shh-venus.
The Shh-promoter constructs were cloned by digestion of the Shh-promoter
vector (R. Ertzer) with XhoI and SnaBI. Subsequently, Shh-venus, Shh-NGFP or Shh
MATERIALS AND METHODS
46
was modified with XhoI and SnaBI sites at the 5’ and 3’ end and subcloned into
TOPO vectors. The primers used were:
ShhXhoIfwd 5’-ATTCCACTCGAGGCCACCATGCGGCTTTTGACG-3’ and
ShhSnaBIrev 5’-ATAATAATTACGTATCAGCTTGAGTTTACTGA-3’.
The inserts were recovered by digestion of the respective vector with XhoI and
SnaBI and cloned into linearized Shh-promoter vector, yielding Shh-promoter-Shhvenus and Shh-promoter-Shh. DNA was injected to obtain embryos transiently
expressing Shh or Shh-venus in the endogenous Shh domain. In addition, embryos
injected with Shh-promoter-Shh-NGFP were raised for the generation of stable
transgenic lines.
Cloning of DN-Ezrin
Vector pUAST-ezrin(1-280)GFP (D. Alexandre) was digested with EcoRI and XhoI.
The retrieved fragment was cloned into EcoRI/XhoI linearized pCS2+, yielding
pCS2+DN-ezrin. For mRNA generation, the vector was digested using KpnI and Sp6
polymerase.
Western Blotting
For western blotting, about 10 embryos were loaded per lane of a standard 12% SDS
PAGE gel. The gel was run for 1 hour at 130 volts. Subsequently, the proteins were
transferred to a nitrocellulose membrane using a semi-dry blotting system (BioRad)
according to the manufacturer’s instructions. The amount of protein was assessed by
ponceau red staining. The membrane was then blocked in 5% fat-free milk in PBST
for 1 hour. I washed the membrane for 3 x 10 min in PBST prior to incubation for 1
hour with the primary -GFP antibody (SantaCruz, GFP-FL, sc 8334) in 5% fat-free
milk in a 1:200 dilution. The antibody was washed off 3 x 10 minutes in PBST
followed by incubation with the secondary -rabbit-HRP coupled antibody (SIGMA)
for 30 minutes. The secondary antibody was then washed off with PBST for 3 x 10
minutes. Detection was carried out using an ECL kit (Amersham) according to the
manufacturer’s instructions. The blot was then used to expose a film for 5 minutes.
47
MATERIALS AND METHODS
List of plasmids used in this study
Plasmid
Shh-NGFP
Vector backbone
ShhT7TS
Insert
Cloning Linearize
sites
with
GFP
NotI
BamHI
venus
XhoI/
BamHI
NotI inserted
at pos. 33
Shh-venus
ShhT7TS
XhoI/ClaI inserted
ClaI
at pos. 194
Shh-promoter-Shh-venus
I-SceI-LL-Shh-GFP- shh-venus
XhoI/
AraA-AraB-AraC
SnaBI
Shh-promoter-Shh-NGFP I-SceI-LL-Shh-GFP- shh-NGFP
Shh-promoter-Shh
XhoI/
AraA-AraB-AraC
SnaBI
I-SceI-LL-Shh-GFP- shh
XhoI/
ArA-ArB-ArC
SnaBI
YFP-GPI
in pCS2+
YFP-GPI
XhoI
NotI
GFP-GPI
in pCS2+
GFP-GPI
XhoI
NotI
DN-Ezrin
in pCS2+
DN-ezrinGFP EcoRI/
KpnI
XhoI
Ext1a
pCRTOPOII
ext1a partial
for in situ probe use
(1 kb)
EcoRI
NotI
EcoRI
SpeI
EcoRI
SpeI
Sp6 pol.
Ext1b
pCRTOPOII
ext1b partial
for in situ probe use
(900 bp)
T7 pol.
Ext1c
pCRTOPOII
ext1c partial
for in situ probe use
(800 bp)
T7 pol.
48
Results
Rationale:
Considerable efforts have been made in elucidating the mechanisms that lead to the
proper distribution of signaling molecules during embryonic development. Among
these signaling molecules, proteins of the Hh family have received much attention,
because of their unusual double lipid modification and involvement in human disease
(Ingham and McMahon, 2001; Mann and Beachy, 2004; McMahon et al., 2003).
However, even though several models for the spreading of Hh proteins exist, no
conclusive evidence is available to date that would explain how this membrane
tethered protein moves between cells and establishes a gradient of concentration. I
therefore decided to study the distribution of the vertebrate Hh homolog, Shh during
development in living zebrafish embryos by making use of a fluorescently labeled
Shh protein. By determining the subcellular localization and by following the
spreading of the protein in the embryo, I reasoned that I would gain insight into the
cellular mechanisms that govern Shh spreading. I chose gastrula stage embryos for
my analysis because of the easy accessibility for experimental manipulations.
Furthermore, the transparency of the embryo allowed me to carry out an analysis of
these experimental manipulations by making use of confocal microscopy.
In a second part of my thesis, I analyzed genes that are potentially implicated in
the distribution and the response of cells to Hh proteins, based on work in other
developmental model organisms. I focused on genes involved in the synthesis of
HSPGs and cloned three genes belonging to the EXT1 family of glycolsyltransferases.
These have been shown to be important for Hh trafficking in both invertebrate and
vertebrate systems (Bellaiche et al., 1998; Koziel et al., 2004; Rubin et al., 2002; The
et al., 1999). I addressed the question whether they are also involved in Shh signaling
during zebrafish development by analyzing their embryonic expression and their
response to elevated and reduced levels of Shh signaling.
Secondly, I cloned zebrafish Rab23, a small GTPase belonging to the Rab family
of proteins, which are important regulators of membrane trafficking (Zerial and
McBride, 2001). A mutation in mouse Rab23 causes an open brain phenotype and
upregulation of Shh target genes in the brain even in the absence of Shh ligand
(Eggenschwiler et al., 2001). By interfering with rab23 function in zebrafish embryos
49
RESULTS
using morpholino oligonucleotides, I addressed the question if rab23 also plays a role
in zebrafish Hh signaling.
Expression of shh and ptc1 at gastrula stages
To gain an understanding of Shh signaling at gastrula stages, it was important to
analyze the expression pattern of shh and its downstream targets at this developmental
stage. Therefore, I carried out in situ hybridizations with anti-sense probes for shh and
ptc1, a known downstream component of the Shh pathway, which has been shown to
directly respond to Shh signaling (Agren et al., 2004; Chen and Struhl, 1996;
Goodrich et al., 1996; Hahn et al., 1996).
Fig. 1. Expression of shh at 80%
of epiboly and tailbud stages.
Schematic drawings of 80% epiboly
(A) and tailbud (D) stages (Kimmel
et al., 1995) for comparison.
Embryos (A-E) are oriented dorsal to
the right. Flat mounted embryos (F,
J), anterior to the top. Cross sections
(G-I), dorsal to the top. The s h h
expression domain extends along
the anterior-posterior axis during
gastrulation (compare B, E and A, D,
arrowheads). At the 80% of epiboly
stage, the posterior third of the
embryonic axis expresses shh (B).
At this stage, s h h is confined to
involuting mesendodermal cells (C,
brackets). At tailbud stages, the
whole axis is positive for s h h
transcript (E, J). Cross sections (G-I)
at various anterior-posterior levels of
the embryonic axis reveal that shh
transcripts are localized in
mesendodermal cells. Note the
extensive antero-posterior expansion
of the s h h expression domain
(compare F, J) and the concomitant
thinning of the cell population
expressing s h h . Scale bars
represent
100
µ m.
en,
mesendoderm, ec, ectoderm.
Shh transcripts can be first detected at the 60% of epiboly stage in involuting
mesendodermal cells (Krauss et al., 1993). They continue to be expressed in this cell
population at the 80% of epiboly stage, where expression can be detected in the
posterior third of the embryonic axis (Fig. 1A-C, F). At the end of gastrulation, this
cell population has extended to the anterior part of the embryo. (Fig. 1D, E, G-J,
compare arrowheads). During this time, shh expressing cells undergo extensive cell
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50
movements known as convergent extension (Keller et al., 2000). Concomitant with
the anterior expansion, the width of the Shh source thinness (Fig. 1, compare F and J).
Therefore, the source of Shh signaling proteins is very dynamic during these early
stages of development.
A read out of the activity of Shh on the surrounding tissue is the transcriptional
response of its receptor ptc1. By analyzing the expression of ptc1 in zebrafish
gastrulae, I reasoned to obtain a genetic read-out of the distance Shh proteins travel at
this developmental stage. At the 80% of epiboly stage, ptc1 transcripts are present in
cells abutting the shh expression domain (Fig. 2A). Transcripts can be found in the
ectodermal tissue overlying the axial mesendoderm, which itself expresses ptc1 at
lower levels (Fig. 2B, C). In more lateral positions, both mesendodermal and
ectodermal cells contain ptc1 transcripts (Fig. 2D). In paraxial cell populations, ptc1
is more strongly expressed in mesendodermal cells (Fig. 2E).
Comparison of these expression domains of ptc1, which are found in close
proximity to sites of shh expression suggests that also in gastrulating embryos, ptc1
responds to Shh signaling. This is especially evident in a cross section (Fig. 2B)
where shh expressing cells (compare to Fig. 1H) would fill the axial tissue surrounded
by ptc1 positive cells. However, in addition to these ptc1 expression domains, which
abut tissue positive for shh transcripts, I also note weak ptc1 expression in paraxial
mesoderm, which is present at a considerable distance further away from the shh
source (Fig. 1B, E).
Expression of ptc1 at gastrula stages depends on s h h
signaling
To achieve a higher spatial resolution of the relationship between the expression of
shh and ptc1, I performed double in situ hybridization. Shh transcripts were visualized
in red, while ptc1 was detected in blue (Fig. 3A, B, E). In order to determine whether
the upregulation of ptc1 observed in tissues abutting the shh domain in gastrulating
embryos is dependent on Shh signaling, I also analyzed smu mutant embryos, which
carry a mutation in the canonical Hh receptor smoothened. In these embryos,
upregulation of ptc1 expression was not detected (Fig. 3C, D, F), providing evidence
for the Hh dependency of strong ptc1 expression at this stage of development.
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51
However, expression of ptc1 was still visible in paraxial mesodermal cells. This
suggests that the expression of ptc1 in paraxial cells is independent of functional Hh
signaling. This is especially evident in cross sections of wt (Fig. 3E) and smu mutant
embryos (Fig. 3F).
A close up of the middle region of wt embryos revealed that the limit of Shh
signaling must be in the range of up to 4 cell diameters away from its source, as
monitored by upregulation of ptc1 target gene transcription (Fig. 3B). Therefore,
genetic analysis of the spatial relationship between shh and ptc1 expressing cells in
combination with expression data obtained in smu mutant embryos suggests that Shh
proteins can exert their effect on both ectodermal and mesendodermal tissue and up to
several cell diameters away from their source.
Fig. 2. Expression of ptc1 at the 80% of epiboly stage.
(A) Flat mounted embryo, anterior to the top. Levels of cross and longitudinal sections are indicated with black bars. (B)
Cross section spanning axial and adaxial tissues. Ptc1 transcripts can be detected in ectodermal cells overlying the
involuting mesendodermal cells. In addition, adaxial cells of mesendodermal origin are positive for ptc1. Weaker
expression is present in paraxial mesoderm. (C) Longitudinal section at the midline level. Predominantly ectodermal cells
are positive for ptc1 transcripts (black arrow and white dotted line indicates separation of the two germ layers, also in D,
E). (D) In adaxial regions, ptc1 is expressed in both, mesendodermal and ectodermal cell populations. (E) In paraxial
regions, predominantly mesendodermal cells are positive for ptc1 transcripts. Scale bar represents 100 µm.
Shh can induce target gene activation in ectopic positions in
the embryo
I next wanted to address the question if Shh would be able to elicit similar target gene
activation in an ectopic position in the embryo. To this end, I transplanted cells from
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52
donors which I had injected with 100 pg of shh mRNA into host embryos at shield
stage. I let the embryos develop for an additional 3 hours to reach about 90% of
epiboly, fixed them and carried out an in situ hybridization for ptc1.
Fig. 3. smu is needed for Shh signaling at the tailbud stage.
Double in situ hybridization with probes detecting shh in red and ptc1 in blue. Wt embryos (A, B, E) and smu mutant
embryos (C, D, F). Embryos were flat mounted and oriented anterior to the top (A-D) or sectioned at the level
indicated (E, F). (A) ptc1 is expressed in cells around the shh expression domain in the midline. (B) Close up of the
indicated region in (A) reveals ptc1 upregulation of up to 3-4 cell diameters away from the axial mesoderm. (C) In
smu mutant embryos, induction of ptc1 is weaker and only present in the posterior region of the embryo. (D) Close up
of indicated region in (C). Note weak expression of ptc1 in mesodermal cells, but missing upregulation in adaxial
regions. (E) Cross section of embryo in (A). Upregulation of ptc1 can be detected in mesodermal (arrows) and
ectodermal (asterisk) cells surrounding the axial mesoderm expressing shh. Expression of ptc1 can also be detected
in more lateral paraxial mesoderm (arrowhead). (F) Cross section of smu mutant embryo. No ptc1 upregulation can
be detected (compare region marked by arrows with same region in (E)). However, weak expression in the paraxial
mesoderm persists (arrowhead). Scale bars represent 100 µm.
Transplanted cells were detected by coinjection of biotin with the shh RNA and
subsequent DAB staining. Normal ptc1 expression could be detected in the midline of
transplanted embryos (Fig. 4A, D). However, in addition, I frequently (61 out of 68
cases) observed patches of cells, which ectopically expressed ptc1 in mesodermal and
ectodermal cells (Fig. 4A, D, arrows). Codetection of transplanted cells in brown (Fig.
4C, F) revealed that these ectopic ptc1 expression domains (Fig. 4B, E) correlated
with regions of the embryo in which shh expressing cells were transplanted. The
ectopic ptc1 domains varied in size and relative position in relation to the embryonic
midline (Fig. 4, compare position in A and D, also B, E).
Nevertheless, the location of cells, which had started to express ptc1, was always
in the range of 3-4 cells away from the ectopic shh source. This was the same range of
target gene activation as observed in the endogenous domain in the axial mesoderm
(Fig. 3B). Therefore, I can conclude that at this stage of development mesodermal and
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53
ectodermal cells on the dorsal side of the embryo are competent to respond to Shh
signaling molecules and that the signaling range of Shh proteins in gastrulating
embryos is about 3-4 cell diameters within 3 hours. This range of Shh distribution is
also maintained in ectopic positions in the embryo. However, the means by which Shh
proteins reach their target cell remain unclear.
Fig. 4. Transplantation of shh expressing cells in ectopic positions induced expression of ptc1.
(A-F) Flat mounts of 2 embryos at the 90% of epiboly stage. In situ hybridized with ptc1 probe, staining in blue, anterior to
the top. (A, D) Overview. (B, E) Close up view of region with ectopic ptc1 expression. (C, F) Same regions as in (B, F)
with additional DAB detection of transplanted cell expressing shh (brown). The range of target gene induction around the
transplanted clone is 3-4 cells.
Analysis of fluorescent Shh fusion constructs
Mechanisms must exist that ensure the stable distribution of Shh proteins during the
dynamic gastrulation process and ensure the proper signaling range as revealed by in
situ hybridization. In order to directly visualize this distribution, I generated
fluorescently labeled Shh fusion constructs. I reasoned that this approach would
provide me with information concerning the subcellular structures Shh proteins
localize to and how these structures might relate to the process of Shh allocation.
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54
Cloning of Shh-NGFP and Shh-venus
Every modification introduced into a protein will change its properties. Shh is known
to be a signaling molecule interacting in a network of promoting and inhibitory
factors. It also undergoes a series of posttranslational modifications (see introduction).
Therefore, it was of importance to determine the signaling activity and the proper
processing of Shh fusion proteins. I constructed two different variants of fluorescent
Shh fusion proteins. In one case I cloned an enhanced GFP (Clontech) to the Nterminus of the N-terminal fragment after the signal sequence and the palmitoylation
site (Fig. 7B). This protein will be referred to as “Shh-NGFP”. I reasoned that by
leaving the palmitoylation site and the signal sequence of Shh in front of the GFP
intact, the protein should be correctly targeted to the secretory pathway and
subsequently modified.
A second fluorescently labeled Shh variant had a YFP derivative called “venus”
(Nagai et al., 2002) cloned in frame at the C-terminus of the N-terminal fragment in
front of the site of autoproteolysis (Fig. 7C). I will refer to this protein as Shh-venus. I
chose to use the “venus” protein because it was reported to have improved folding
properties and resistance to acidosis (Nagai et al., 2002). It has been previously shown
that the C-terminal fragment of the Shh precursor protein can direct the addition of
cholesterol to any other protein (Vincent et al., 2003) and that a similar Hh-GFP
fusion protein in Drosophila is properly processed (Torroja et al., 2004). I therefore
reasoned that the addition of a “venus” should not interfere with proper cleavage and
cholesterol modification.
The available crystal structure reveals the location of either cholesterol or
palmitoyl in respect to the rest of the protein (Fig. 5A). The GFP was inserted Cterminal to the putative palmitoylation site (palmitoyl indicated in purple). It would
therefore be the palmitoylated part of the resulting fusion protein. In the case of the Cterminal “venus” fusion, the fluorescent protein was inserted N-terminal to the site of
cholesterol addition (cholesterol indicated in yellow).
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RESULTS
Fig. 5. Shh and Shh fusion
constructs used in this study.
An arrow indicates the site of
autoproteolysis. (A) Wt s h h .
Indicated are the N- and C-terminal
parts of the proteins between which
autoproteolysis occurs. The crystal
structure of the N-terminal fragment
reveals sites of lipid modification.
Cholesterol is shown in yellow and
palmitoyl in purple (van Den Heuvel,
2001). (B) shh-NGFP . The GFP is
inserted at the N-terminus of the Nterminal fragment. (C) shh-venus.
The “venus” is inserted at the Cterminus of the N-terminal peptide.
(D, E, F) Shh promoter constructs
harboring either wt shh, shh-NGFP
or shh-venus in addition to the shh
promoter (2.2 promoter) and three
enhancer elements (A, B, C
enhancer) which control the
expression of shh in different tissues
(Muller et al., 1999).
In addition, I constructed plasmids containing the shh promoter and either shh (Fig.
5D), shh-NGFP (Fig. 5E) or shh-venus (Fig. 5F). I used these plasmids for expression
of the respective protein in the endogenous shh domain during zebrafish development
and for the generation of transgenic zebrafish.
Signaling strength of Shh-venus was comparable to
wt Shh
I next addressed whether the Shh-NGFP and Shh-venus fusion proteins retained their
signaling capacities. I addressed this issue using three different strategies: First I
ubiquitously overexpressed mRNA in embryos, secondly I created an ectopic source
of Shh-NGFP or Shh-venus expressing cells by transplantation in a host embryo and
thirdly I made use of the Shh promoter and expressed the respective fusion protein in
its endogenous domain in syu mutant fish, which carry a mutation in the shh gene. I
reasoned that if the fusion proteins retained their signaling capacities, I would expect
similar results in all three scenarios when compared to wt Shh.
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RESULTS
Ubiquitous overexpression of shh-venus or shh caused
similar morphological changes
For overexpression studies, I injected the same molar amount of either shh-venus or
shh-NGFP mRNA compared to wt shh, because the addition of GPF or “venus”
increases the molecular size of the signaling peptide by roughly a factor of two. When
I overexpressed 100 pg of Shh-venus in wt embryos, I observed similar morphological
changes at the 24 hpf stage compared to fish that were injected with 50 pg of wt shh
(Fig. 6). As reported earlier, the eyes exhibited severe dysmorphogenesis (Fig. 6,
black arrows) with an almost complete loss of pigmented retina (Barth and Wilson,
1995; Ekker et al., 1995; Krauss et al., 1993; Macdonald et al., 1995).
In addition, the folding of the mid-hindbrain-boundary (mhb) region (Fig. 6,
arrowheads) observed in wt embryos did not occur in either shh or shh-venus injected
embryos. Instead, the neural tube appeared as a straight rod. A third patterning defect
observable at the morphological level was a blocky appearance of the somites (Fig. 6,
white arrows). Therefore, I conclude that the morphological changes induced by
ubiquitous overexpression of 100 pg of shh-venus are comparable to the
overexpression of stoichiometric amounts of shh.
Table 1. Overexpression of shh, shh-venus or shh-NGFP altered
eye morphology.
Construct
Number of
Eyes severely
Eyes mildly
Eyes not
injected
embryos
affected
affected
affected
shh (50 pg)
69
62
7
0
shh-venus
88
67
21
0
87
0
32
55
(100 pg)
shh-NGFP
(100 pg)
In contrast to this, overexpression of shh-NGFP lead to considerably weaker
morphological changes (Fig. 7 and table 1). The optic stalk territory was broadened
when compared to wt embryos (Fig. 7A, B), but the response was more variable.
Often, only a slight expansion was observed, which never reached the same extend
observed in embryos overexpressing either shh or shh-venus.
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57
Fig. 6. Overexpression of shh-venus
induces similar morphological changes
when compared to
overexpression of wt
shh.
All embryos are at the
24 hpf stage, oriented
anterior to the left. Side
views (A, C, E), top
views (B, D, F). The
white asterisk marks
the otic vesicle. Wt
embryos (A, B) show a
morphological visible
constriction at the mhb
(A, black arrowhead),
an
eye
with
a
developed lens (A, B,
black arrow) and
chevron
shaped
somites (A, white
arrow).
Embryos
injected with 50 pg shh
RNA (C, D) lack the
mhb constriction (C,
black arrowhead) and
the eye is poorly
developed (C, D, black
arrow). In addition, the
somites have a blocky
appearance (C, white
arrowhead). Similarly,
embryos injected with
100 pg shh-venus RNA
(E, F) show loss of mhb
(E, black arrowhead),
impaired
eye
development (E, F,
black arrow) and blocky
somites (F, white
arrow).
This suggests that the addition of GFP to the N-terminus of the N-terminal
fragment of shh interferes with the signaling properties of the protein, possibly by
interfering with palmitoylation. In addition, marker analysis, transplantation
experiments and promoter driven expression of shh-NGFP did not result in target
gene activation (data not shown). For these reasons, I discontinued working with the
N-terminal fusion construct.
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RESULTS
Fig. 7. Signaling strength of SHH-NGFP is weaker when compared to wt SHH or SHH-CGFP.
Comparison of optic stalk tissue of wt (A), shh or shh-venus (B), and shh-NGFP (C, D) injected embryos. Top views, all
embryos are oriented anterior to the left. (A) The optic stalk connects the optic cup to the rest of the diencephalon
(bracket). (B) Upon overexpression of either shh or shh-venus the optic stalk tissue expands (arrow). (C) More weakly
affected embryo injected with shh-NGFP. Note the slight increase in size of the optic stalk (arrow). (D) Strongly affected
embryo overexpressing shh-NGFP . The optic stalk tissue is greatly expanded (arrow). In addition, the optic cup is
malformed. However, the overexpression effect is less pronounced when compared to the embryo in (B).
Alterations in marker gene expression induced by either shh
or shh-venus were comparable
To further characterize the observed morphological changes, I performed in situ
hybridization with markers known to be affected by shh signaling. These included
pax2.1, pax6 and foxa2. Pax2.1 and pax6 are expressed in mutually exclusive
domains in the eye, while foxa2 is a floor plate marker that has been previously
reported to respond to elevated levels of shh signaling (Krauss et al., 1993). In wt
embryos, pax2.1 is expressed in the optic stalk, located in the proximal part of the eye
(Fig. 8B, arrows). In contrast, pax6 expression is present throughout the retina, but
not in the optic stalk territory (Fig. 8H, arrowheads).
Table 2. Overexpression of either shh or shh-venus induced
ectopic expression of pax2.1.
Construct
Number of
Ectopic expression
Normal
injected
embryos
in eye
expression
shh (50 pg)
46
46
0
shh-venus (100 pg)
43
42
1
Upon overexpression of shh, pax2.1 expression expanded (Fig. 8C, D, arrows and
table 2). To the contrary, pax6 expression was reduced or lost (Fig. 8I, J, arrowheads
and table 3).
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RESULTS
Table 3. Overexpression of either shh or shh-venus repressed
pax6.
Construct
Number of
Expression in
Expression
Expression
injected
embryos
eye lost
barely visible
clearly visible
shh (50 pg)
57
18
27
12
shh-venus
57
15
18
24
(100 pg)
When I compared these embryos with embryos overexpressing shh-venus, I could
observe similar changes in the expression patterns of pax2.1 and pax6 (Fig. 8E, F; K,
L and tables 2 and 3). These findings indicate that shh-venus can induce the same
changes in the expression of known shh target genes.
Fig. 8. Expression of pax2.1 and pax6 in response to overexpression of shh or shh-venus.
24 hpf stages, all embryos are oriented anterior to the left. (A, C, E, G, I, K) Side views. (B, D, F, H, J, L) Top views. (A-F)
Expression of pax2.1. (G-L) Expression of pax6 . (A) In wt embryos, expression of pax2.1 can be detected in the optic
stalk tissue (arrow). (B) Pax2.1 expression is confined to the proximal retina (arrowheads). (C) Upon overexpression of
s h h , pax2.1 expression expands (arrow). (D) Pax2.1 can now be detected throughout the entire optic vesicle
(arrowheads). (E) Overexpression of shh-venus induces a similar increase in pax2.1 staining. (F) Expansion of pax2.1 to
proximal regions of the retina (arrowheads) in embryos overexpressing shh-venus). (G) Wt expression of pax6 in the optic
cup (arrow). (H) The entire optic cup expresses pax6 (arrowheads). (I) Repression of pax6 upon overexpression of shh
(arrow). (J) On one side, the expression of pax6 is lost in most of the eye (asterisk). On the other side, expression is
strongly diminished (arrowheads). (K) Repression of pax6 in embryos injected with shh-venus (arrow). Note a somewhat
weaker reduction compared to the embryo in (I). (L) Asymmetric repression of pax6. On one side, expression is strongly
diminished (asterisk), while on the other side, a broader expression domain persists (arrowheads).
However, I noted a slightly weaker response, especially in the case of pax6 to the
overexpression of shh-venus. For instance, repression of pax6 shifted from “barely
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RESULTS
visible” observed in most embryos overexpressing shh to “clearly visible” in the case
of shh-venus overexpression (Table 3).
When analyzing the expression of foxa2, I could observe a similar expansion of
foxa2 in embryos overexpressing either shh or shh-venus (Fig. 9 and table 5). While in
the wt, expression could be detected in the floorplate and the diencephalon (Fig. 9A,
arrow marks diencephalic expression), injection of either shh or shh-venus induced
ectopic foxa2 expression in the midbrain (Fig. 9B, C, arrows and table 5) and in the
rhombomeres (Fig. 9B, C, arrowheads).
Table 4. Overexpression of either shh or shh-venus induces
ectopic expression of foxa2 in the diencephalon.
Construct
Number of
Second foxa2
No second foxa2
injected
embryos
domain in
expression domain in
diencephalon
diencephalon
shh (50 pg)
45
18
27
shh-venus (100
33
15
18
pg)
Fig. 9. Expression of
f o x a 2 is similarly
altered
upon
overexpression of shh
and shh-venus.
Embryos at the 24 hpf
stage, oriented anterior
to the left. (A) Foxa2 is
expressed in the floor
plate and in the
diencephalon (arrow) in
wt
embryos.
(B)
Overexpression of shh
induced ectopic foxa2
expression domains in
the midbrain (asterisk)
and in the dorsal
rhombomeres
(arrowhead).
(C)
Overexpression of shhvenus induced similar
changes in f o x a 2
expression.
Therefore, I conclude that ubiquitously overexpressed shh-venus had signaling
properties comparable to shh as analyzed by morphological criteria and altered
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61
marker gene expression. However, I noted a slightly lower overall response to shhvenus, in particular in the case of pax6 (Table 3 and Fig. 8).
Autoproteolytic cleavage occurred normally in Shh-venus
To address the question if autoproteolytic cleavage occurred normally in Shh-venus
fusion proteins, I performed western blots of shh-venus injected and wt embryos with
an -GFP antibody, which also detects the “venus” protein (Nagai et al., 2002). The
Shh N-terminal protein has a molecular weight of about 19 kDa (Bumcrot et al., 1995)
and venus has a molecular weight of 27 kDa (Prasher et al., 1992) (Fig. 6A).
Therefore, I expected to detect a band of approximately 46 kDa. Indeed, a band
running just below the 50 kDa marker was visible in proteins extracted from shhvenus injected embryos at the 60% of epiboly stage (Fig. 10B). This band was also
very weakly present at the 24 hpf stage. In addition, a very weak band was visible at
60 kDa at 60% of epiboly. This band could represent the uncleaved form of the
protein. In the control lane, no bands were present. Therefore, I conclude that Shhvenus is properly cleaved and therefore probably also cholesterol modified.
Fig. 10. Autoproteolytic cleavage of Shh-venus occurs normally.
(A) Schematic representation of autoproteolysis of Shh-venus. The expected molecular size of 46 kDa corresponds to the
observed band on the western blot. (B) Western blot of zebrafish embryos injected with shh-venus and fixed at 60% of
epiboly (lane 3) or 24 hpf (lane 4) and probed with an antibody against GFP. Lane M Marker lane, numers are in kDa.
Lane C was loaded with uninjected control embryos. Left image ponceau staining to visualize total of loaded protein, right
image exposed western blot. 10 embryos were loaded per lane. A band is visible with a size of about 45 kDa.
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Transplanted cells overexpressing shh-venus induced ptc1
The result that shh-venus has similar signaling properties when compared to shh was
obtained in experiments in which all cells of the embryo expressed the respective
protein. In order to address if the “venus” fusion might alter the signaling range of
Shh-venus, I made use of the transplantation assay in which I created an ectopic
source of cells overexpressing shh. I reasoned that I should see a comparable range of
target gene activation when transplanting cells overexpressing shh-venus.
I find that transplanted cells ectopically expressing shh-venus can induce ptc1 in
host embryos (Fig.11). The efficiency (66 out of 71 embryos) and the range of ptc1
induction were comparable to embryos into which I had transplanted cells
overexpressing shh (compare to Fig. 4). However, I frequently observed that for
strong induction of ptc1 cells had to be surrounded by shh-venus expressing cells.
This might suggest that the signaling strength of the fusion protein is somewhat
weaker when compared to wt shh.
Fig. 11. Cells expressing shh-venus can ectopically induce ptc1.
Flat mounted embryo at the 90% of epiboly stage, anterior to the top. (A) Patch of cells ectopically expressing ptc1
(arrow). (B) Close up view of clone indicated in (A). (C) Transplanted cells overexpressing shh-venus detected in brown.
Note expression of ptc1 in cells adjacent to the ectopic shh-venus source.
These findings are consistent with my observations concerning the ubiquitous
overexpression of either s h h or shh-venus. Nevertheless, in both situations, the
“venus” fusion protein elicited all observable effects of wt shh. Therefore, I conclude
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63
that shh-venus signaled to neighboring tissue as revealed by induction of its target
gene ptc1.
Expression of shh-venus in its endogenous domain partially
rescued loss of ext1c expression in syu embryos
The previous experiments were carried out in ectopic positions in the embryo, in
which shh signaling might be altered due to the absence of regulatory factors. I
therefore asked if shh-venus expressed in its endogenous domain could rescue the
phenotype of a known shh deletion in zebrafish, syu (Schauerte et al., 1998). To this
end, I constructed plasmids containing either shh for comparison, or shh-venus driven
by its endogenous promoter and enhancer elements (Muller et al., 1999) (gift from R.
Ertzer, Fig. 5).
I injected these into syu mutant fish, and essayed for the induction of ext1c, a
downstream target gene of shh signaling (Siekmann and Brand, 2004) in somitic cells
at the 15-somite stage. In wt embryos, ext1c is expressed in adaxial regions of the
posterior somites (Fig. 12A, B, arrows). In syu mutants, ext1c expression is absent in
posterior adaxial cells (Fig. 12D, E).
Fig. 12. Injection of shh (C) or shh-venus (F) can partially rescue ext1c expression in posterior adaxial cells.
Embryos at the 15 somite stage, oriented anterior to the left. (B, C, E, F) flat mounted embryos. Expression of ext1c (blue)
and shh (A-E) or shh-venus (F) (red) in wt (A, B) and syu mutant (C-F) embryos. Note loss of ext1c staining in posterior
somites of syu mutant embryos (compare arrows in A, D; brackets in B, E) and reduction in width of expression in more
anterior regions (compare bar in B, E). Injection of DNA containing the shh promoter and driving expression of shh (C) or
shh-venus (F) could partially rescue ext1c expression. Red cells in (C) were hybridized with an anti-shh probe, in (F) with
an anti-venus probe, revealing mosaic expression of the transgene in notochord cells (insets show fluorescent signal of
red probe). Adaxial cells close to a shh or shh-venus source express ext1c.
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64
In more anterior regions, the expression domain is narrowed (Fig. 12B, D,
compare black bars). If shh or shh-venus could replace the lacking endogenous shh in
syu embryos injected with either promoter construct, I would expect to detect adaxial
cells now expressing ext1c.
In all injected clutches examined, I could clearly distinguish syu mutant embryos
from wt embryos by variable loss of ext1c expression in the posterior part of the
embryo (Fig. 12C, F). This was probably due to the mosaic expression of the transient
shh or shh-venus expression. However, in several cases, I found expression of ext1c to
be present in some posterior somites. When I detected shh expression with a red probe
in those embryos, I found that notochord cells close to the ext1c expression site were
positive for the shh transgene (Fig.12C).
I obtained similar results when I had injected shh-venus and probed for venus
(Fig.12F, arrowheads). Therefore, injection of a promoter construct driving the
expression of either shh or shh-venus in its endogenous notochordal domain could
partially rescue the syu phenotype of loss of ext1c expression. Therefore, the shhvenus fusion construct could replace the function of wt shh if ubiquitously
overexpressed, transplanted in ectopic regions of the gastrulating embryo or driven
under the control of the endogenous shh promoter. For that reason, I conclude that
shh-venus has similar biological properties as wt shh.
Subcellular localization of Shh-venus
After the analysis of the signaling properties of Shh-venus, I asked to which
subcellular compartments the protein localizes and how it might move between cells.
Previous reports using biochemical and genetic tools showed that Shh is a secreted
protein and that it can act over a distance in embryonic tissue (Lee et al., 1992;
McMahon et al., 2003). In immunohistochemical studies in cultured cells,
overexpressed Shh proteins were predominantly detected at the cell surface (Roelink
et al., 1995), which is in agreement with them being lipid modified (Mann and
Beachy, 2004). In addition to their membrane association in producing cells, Hh
proteins were also found to accumulate at cellular junctions and in large punctate
structures in both the embryonic ectoderm and the wing imaginal discs in flies, (Gallet
et al., 2003; Porter et al., 1996a; Tabata and Kornberg, 1994). In addition, a recent
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report showed that a Hh-GFP fusion protein localized to similar punctate structures
(Torroja et al., 2004).
However, these localization studies were all performed in fixed specimens. I
therefore asked, if Shh-venus would also localize to similar structures in cells of
living zebrafish embryos. To address this question, I produced large clones of cells
overexpressing shh-venus by injecting one of 8-16 blastomeres of an early embryo
(Fig. 13). Together with the shh-venus RNA, I injected rhodamine as a lineage tracer. I
let the embryo develop until gastrulation stages and observed the distribution of Shhvenus in the living embryo using confocal microscopy. To obtain optimal spatialtemporal resolution, I acquired confocal z-stacks of about 20 µm each over time.
These sections were then processed using Imaris (Bitplane) and Volocity
(Improvision) software.
Fig. 13. Injection
scheme to produce
large
clones
expressing s h h venus.
(A) Embryo at the 16
cell stage. RNA is
injected together with a
lineage tracer into one
of 16 cells. (B) After 4
hours, the cell has
divided, producing a
large clone of cell
expressing the injected
RNA.
Shh-venus localized to punctate structures and filopodia
Upon analysis of the confocal images, I could distinguish the injected cells from the
surrounding tissue by virtue of “venus” expression. The cells containing most of the
venus fluorescence were also positive for the lineage tracer (Fig. 14).
In the injected cells, Shh-venus was found in punctate structures and large
patches inside the cell, but also in cortical regions (Fig.14 B, C, arrowheads). Protein
patches were randomly distributed over the cell’s surface, but were always present as
discrete entities and not as a uniform membrane label. Therefore, unlike reported for a
cholesterol modified GFP, which uniformly labels membranes in cultured cells
(Vincent et al., 2003), Shh-venus localization must depend on other factors besides
the cholesterol modification.
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Fig. 14. Close up of cells in
a gastrulating embryo
overexpressing shh-venus.
(A) Red channel showing
rhodamine lineage tracer and
highlighting cells that were
injected with s h h - v e n u s
mRNA. Note the incomplete
cytokinesis of the two cells in
the middle (marked by
asterisks).
(B) Green channel showing
the distribution of Shh-venus
in the embryo. Note the
punctate accumulation on the
surface of expressing cells
and outside of expressing
cells (arrowheads, compare
(B) to (C). Note the
fluorescent patch of Shhvenus present at the site of
cytokinesis (cells marked by
asterisks). Protein can also be
seen in a protrusion that
emerges from an expressing
cell into the extracellular
space (arrow).
(C) Overlay of red and green
channel, highlighting the
distribution of Shh-venus in
punctate
structures
(arrowheads), at cellular
junctions and in a filopodial
extension (arrow).
In addition to the association with expressing cells, fluorescent patches of Shhvenus protein were also detected outside these cells (Fig. 14B, C, arrowheads).
Strikingly, fluorescence often accumulated in regions of contact of two cells (Fig.
15B, arrowheads), as previously reported in Drosophila embryos (Tabata and
Kornberg, 1994). This accumulation was most obvious in dividing cells (Fig.14A-C,
cells marked by asterisks). Cells about to undergo cytokinesis could be distinguished
by continuous staining with the lineage tracer (Fig.14A, asterisks).
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At the site of cytokinesis, I observed a strong accumulation of Shh-venus protein
(Fig. 14B, C). Either the membrane composition or other specific properties of this
region of the dividing cell may account for the redistribution of Shh-venus proteins.
Fig. 15. Subcellular localization of SHH-venus in a gastrulating zebrafish embryo at the 70% of epiboly stage.
(A) Cells that were injected at the 8 cell stage were labeled by coinjection of rhodamine dextran as a lineage tracer
and visualized in the red channel. (B) Distribution of SHH-venus in the green channel. Note labeling of punctate
structures (arrowheads), filopodial extensions between cells (arrows) and accumulation of green fluorescence at cell
junctions (arrowheads).
From these findings, I conclude that the localization of Shh-venus at the cell
membrane, the accumulation in punctate structures and the buildup at cellular
junctions are in agreement with observations made in fixed tissues.
However, in addition to these previously described localizations for Shh proteins,
I observed Shh-venus in what seemed to be filopodial extensions reaching from cell to
cell (Fig. 14, arrow). These fluorescently labeled extensions were most easily seen
between injected cells (Fig. 15B, arrows). Nevertheless, they could also be observed
between an injected and a non-injected cell (Fig. 15, arrow in lower part of figure). In
some cases, the whole filopodial extension was brightly labeled (Fig. 15, arrows),
while in other cases, only parts of what I interpreted to be a protrusion connecting two
cells contained fluorescent signal (Fig. 14 arrow).
In these structures, the form and distribution of the protein patches varied.
However, due to the lack of continuous staining in many of these filopodial
extensions, it was often difficult to assign a localized accumulation of fluorescent
protein as to be present in a filopodium.
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Time lapse movies revealed traveling of Shh-venus along
filopodia
This analysis was greatly improved by making use of time lapse movies, in which I
could follow the route of specific protein patches. I analyzed 4 movies in total, where
I counted the number of injected cells derived from the single injected blastomere and
the number of filopodia present. Furthermore, I determined the number of filopodia
between injected and uninjected cells (Table 6).
Table 6. Confocal movies reveal Shh-venus to be present in
filopodia
Movie number
1
2
3
4
Number of injected cells
43
36
28
48
Number of filopodia
11
9
8
7
Number of filopodia between injected cells
6
5
4
4
I detected between 7 and 11 filopodial extensions in each of these movies. About
half of them were between injected and the other half between an injected and an
uninjected cell (Table 6). In higher magnification views (e.g. Fig. 16), I followed the
traveling of individual patches of fluorescence from one cell to a neighboring cell in a
time frame of about 15 minutes.
In the set of images presented here (movie on supplemental CD), I could observe
protein traveling from at least two cells (Fig. 16A-F, arrow and arrowheads). In one
case, the protein left the region of the embryo imaged (Fig. 16, arrowheads).
However, I could observe protein, which moved in discrete punctae. In addition, a
bright accumulation of SHH-venus at the cell cortex (Fig. 16E, F) changed its position
and moved towards the site where a putative filopodium was present (compare Fig.
16E, F, arrowheads with Fig. 16A).
The direct exchange of fluorescent protein was more obvious in a second case
(Fig. 16, arrows). Here, I could observe the emergence of a fluorescently labeled
extension from one cell, which reached into the extracellular space (Fig. 16A, arrow).
Subsequently, I could observe Shh-venus protein moving to another cell along what
seemed to be a preformed path, possibly a filopodium (Fig. 16A-F, compare arrows).
The form of the patches changed while traveling along the filopodium.
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Fig.16. Time lapse of shh-venus expressing cells.
Note the movement of a fluorescent particle (arrow) along a seemingly preformed path, possibly a filopodium,
connecting the two cells. Other punctae move out of the image region (arrowheads). The fluorescent particle forms
tubular structures (A, D), or fragments into several punctae (E), which then fuse again into a single particle (F). The
fluorescent structures (labeled by arrow and arrowheads) seem to originate from an accumulation of fluorescence on
an injected cell (A).
They sometimes filled the entire filopodium (Fig. 15B, arrows), but also
fragmented into punctae and merged again to form tubular looking structures
(compare Fig. 16A, F). However, the movement seemed to be directed from one cell
to the next. Strikingly, the time lapse movie revealed that a seemingly punctate
concentration of Shh-venus in the extracellular space (Fig. 16A, white arrow and also
visible in close up in Fig. 14B, C) was possibly part of a filopodial extension through
which protein moved between cells.
The distance covered in this case was between 30 and 50 µm, which would
correspond to one to two cell diameters at this stage of development. This would be
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70
consistent with a velocity between 38 and 55 nm/s. A recent report described the
existence of intercellular bridges between cultured cells, which the authors termed
tunneling nanotubes (TNT) (Rustom et al., 2004). The speed of particles moving
through these TNTs was reported to be 25.9 nm/s. My observations would be within
the range of this value.
These observations suggest that the spreading of Shh in gastrulating embryos
may occur via the exchange of discrete protein patches between cells. The in vivo
analysis of Shh-venus indicates that Shh protein is at least partially distributed by a
direct filopodial based mechanism.
Characterization of filopodial extensions in
gastrulating embryos
Filopodia have been shown to be present in zebrafish cells, which undergo
gastrulation movements (P. Herbomel, unpublished) (Ulrich et al., 2003). However,
they have not been linked to cell-cell signaling events. To further characterize these
filopodia, I asked which molecules apart from Shh-venus localize to them and if they
contain cytoplasm and membrane material.
Filopodia contained GFP-GPI but not cytosolic GFP
To this end, I used the same assay as for Shh-venus and generated clones of cells
either overexpressing cytosolic GFP or a GPI linked form of GFP or YFP. The GPI
linker targets proteins to the outer leaflet of the membrane (Cross, 1990; Keller et al.,
2001) and was previously shown to label traveling membrane fragments called
argosomes in Drosophila embryos (Greco et al., 2001). I reasoned that from these
experiments I would be able to distinguish between cytosolic and membrane
contribution to the observed filopodia. Previous reports showed that labeling single
neurons with cytosolic GFP also marked axonal projections (Higashijima et al., 2000;
Udvadia et al., 2001).
Therefore, I expected to observe cytosolic GFP signal in filopodial extensions of
gastrulating embryos. However, no fluorescence could be detected either outside
expressing cells or in filopodial extensions when overexpressing cytosolic GFP (Fig.
17A). In contrast, cells appeared rather round without any obvious connections
present between them.
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71
Fig. 17. Comparison of
distribution of cytosolic
GFP and GPI-GFP.
(A) Cytosolic GFP fills the
cells, which appear round
and do not show any
detectable protrusions.
(B) GFP-GPI uniformly
labels the membranes of
injected cells (section of cell
marked with asterisk) and is
visible in punctate structures
outside the injected cells
(arrowheads). In addition,
filopodia connecting two
cells can be seen (arrow).
This finding was supported by the observation that also the cytosolic lineage tracer
rhodamine could solely be detected in round appearing injected cells. In contrast to
that, GPI linked GFP was found to uniformly label the surface of injected cells (Fig.
17B). This was most obvious for cells cross-sectioned by the focal plane of the
microscope. In those cells, the red lineage tracer could be seen in the cytoplasm,
surrounded by green fluorescence (Fig. 17B, cell marked by asterisk). In addition to
this membrane labeling, I could observe filopodial extensions between cells (Fig.
17B, arrows) and punctate structures outside of the expressing cells (Fig. 17B,
arrowheads) similar to those, which were observed when overexpressing Shh-venus.
To further characterize the GFP-GPI membrane labeling, I recorded several
confocal time lapse movies of embryos overexpressing YFP-GPI and performed
image processing using Imaris (Fig. 18A, B) and Volocity software (Fig. 18C) for
three dimensional reconstruction of the confocal stacks.
Fig. 18. Visualization of filopodia in gastrulating zebrafish embryos.
(A) Rhodamine dextran as lineage tracer marked cells containing YFP-GPI in red. (B) Stack of confocal images
processed using Imaris software. Note filopodial extensions emanating from cells expressing YFP-GPI (arrows).
Sometimes filopodia not connected to any cell in the image plane can be seen (upper arrow). Often, dots, which fluoresce
more brightly, could be seen in these filopodia (arrowheads). (C) Dot-like structures (arrowheads) on filopodia (arrows)
visualized using Volocity software, presenting a higher contrast view.
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The injected cells were again colabeled with rhodamine as a lineage tracer. As
noted previously, no cellular protrusions were visible when examining the lineage
tracer alone (Fig. 18A). However, a dense and almost network like structure linking
individual cells was visible in both rendered images in the YFP channel (Fig. 18B, C,
arrows marking filopodia). On some filopodia, I could detect individual, brighter
punctae, which were more readily visible when using Volocity (Fig. 18B, C,
arrowheads). From these observations, I conclude that cells in gastrulating embryos
contain many filopodial extensions, which contain a high amount of membrane and
very little or no cytoplasm, as addressed by rhodamine and cytosolic GFP injections.
In addition, the stronger labeling of punctae within those cellular protrusions
suggests the presence of specialized, membrane containing structures within
filopodia. These structures might correspond to the punctae of extracellular Shh-venus
accumulation. In agreement with these findings, TNTs were reported to be devoid of
cytoplasmic proteins, but to contain membrane material, either as lipid containers or
as plasma membrane components (Rustom et al., 2004).
The distribution of Shh-venus differed from that of GPI-YFP
A recent report showed that also argosomes in flies, membrane exovesicles that were
described to be possible carriers for the spread of morphogens, are devoid of
cytoplasm and contain mostly membrane (Greco et al., 2001). However, the authors
did not report the existence of filopodial extensions. Interestingly, the staining pattern
of Shh-venus partially differed from that of YFP-GPI. While in the case of Shh-venus,
the staining appeared concentrated in discrete patches on the cell’s surface (Fig. 14,
15), GPI-YFP uniformly labeled the whole membrane of expressing cells (Fig. 17).
Previous reports showed that GPI linked proteins are sorted into raft membrane
microdomains, which are rich in sphingolipids and cholesterol (Brown and Rose,
1992; Simons and Ikonen, 1997). Because of the cholesterol modification of Shh, I
expected to find a similar distribution when comparing Shh-venus with GPI-GFP.
However, the unlike distribution of the two proteins and the comparison with the
localization of cholesterol modified GFP (Vincent et al., 2003) suggests that the Shh-
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venus might localize to a subset of rafts and that additional mechanisms must account
for the observed Shh-venus localization.
Filopodia contained actin
I wanted to address the question if actin is involved in the formation of filopodial
extensions in zebrafish, as reported for TNTs (Rustom et al., 2004) and for filopodial
extensions that form during Drosophila development (De Joussineau et al., 2003;
Jacinto et al., 2002; Milan et al., 2001; Ramirez-Weber and Kornberg, 1999). To this
end, I overexpressed actin-GFP together with a GPI-linked red fluorescent protein. I
observed GFP fluorescence mainly in the cytosol (Fig. 19A).
Fig. 19. Actin-GFP localized to
cellular protrusions marked by
RFP-GPI (A, B). Extracellular
localization of Shh-venus
containing filopodia (C, D).
(A) Cells overexpressing actin-GFP.
Most of the fluorescence could be
seen in the cytoplasm. However,
cellular protrusions also contained
small
amounts
of
green
fluorescence (arrows).
(B) Overlay of the image in (A) with
the red channel, highlighting cell
membranes. Note the colocalization
of actin-GFP with the red
fluorescence at sites of filopodial
extensions (arrows).
(C) Green channel showing
localization of Shh-venus. Note the
filopodial extension (arrow).
(D) Overlay of red channel showing
cells expressing s h h - v e n u s
(cytoplasmic rhodamine dextrane
staining) and cell outlines
(palmitoylated-RFP) with Shh-venus
in green. Note the overlay of Shhvenus with cell outlines (arrow).
However, unlike in the case of expression of cytosolic GFP, weak fluorescence
could also be detected in protrusions emanating from the cell (Fig. 19A, arrows). In
an overlay with the red channel, in which I detected cell membranes using RFP-GPI,
filopodial extensions became visible. These colocalized with actin-GFP (Fig. 19B,
arrows). Therefore, I argue that filopodial extensions of zebrafish cells contain actin
filaments.
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74
Filopodia extended in the extracellular space
I next asked whether these filopodia connect cells by taking a route through the
extracellular space or if they might penetrate through other cells. In order to do so, I
expressed shh-venus in an embryo in which the membranes of all cells were labeled
by a palmitoylated form of RFP. When I followed the path of individual filopodia, I
observed that they aligned with the membrane marker along the outlines of
neighboring cells (Fig. 19C, D, arrows). I therefore conclude that the filopodial
extensions form along the edges of neighboring cells and take their path through the
extracellular space. The same feature has been recently reported for filopodial
extensions emanating from the baso-lateral membranes of Drosophila wing imaginal
disc cells (DeMontis, F., personal communication).
Overexpression of DN-Ezrin diminished SHH signaling range
From these findings I speculate that Shh proteins might be transported in the
gastrulating zebrafish embryo in association with filopodial extensions reaching from
cell to cell. This mechanism would be in agreement with previous findings in
Drosophila, where cellular protrusions, termed cytonemes, have been described to
connect cells to the signaling centers at the anterior-posterior compartment boundary
in wing imaginal discs, a known site of Hh production (Ramirez-Weber and
Kornberg, 1999). One prediction this model makes is that upon interference with the
formation of these cellular protrusions, Shh signaling should be compromised.
To test this, I disturbed the formation of filopodia using a dominant negative form
of the ERM (for ezrin, radixin, moesin) protein Ezrin. This protein regulates
membrane microfilament interactions and was shown to inhibit filopodia formation in
Drosophila wing imaginal discs (De Joussineau et al., 2003). To test the function of
DN-Ezrin in zebrafish embryos, I overexpressed shh either alone or together with
DN-Ezrin in donor embryos and transplanted cells to host embryos at the 60% of
epiboly stage. At around 90% of epiboly, I assessed for the induction of ptc1 around
the clones.
I found that, as previously described, ptc1 was induced at a distance of up to 4
cells around the shh overexpressing clone (Fig. 20A,B). Clones, which I had also
injected with DN-Ezrin, induced ptc1 at an overall weaker level. Furthermore,
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75
upregulation of ptc1 was only detected up to two cell diameters away from the
transplanted cells (Fig. 20C-H). Therefore, coexpression of DN-Ezrin diminishes the
signaling range of Shh proteins. This reduction in signaling range is most likely
caused by a reduction of filopodia formation.
In summary, I conclude that by visualization of a Shh-venus protein in living
zebrafish embryos, I was able to observe protein being transported along filopodial
extensions. I could show that these extensions are present in cells of gastrulating
embryos. Further characterization revealed that these cellular protrusions contain
membrane and actin, but very little cytosol. These results suggest that Shh protein
possibly travels along filopodial extensions to reach target cells in gastrulating
embryos, a process disturbed in clones overexpressing a DN-form of Ezrin.
Fig. 20. Overexpression of shh together with DN-Ezrin reduces signaling range of Shh.
Clones of cells ectopically expressing ptc1 in embryos of 90% of epiboly (A-H). Brown staining (B, D, F, H) reveals
location of cells expressing either shh (B), or shh together with DN-ezrin (D, F, H). Note the reduced range of ptc1
activation induced by cells that were coinjected with shh and DN-ezrin.
These findings can be summarized in the following model: Normally, filopodial
extensions ensure proper Shh distribution and target gene activation (Fig. 21A, B). By
interfering with the formation of these filopodia, target gene activation is
compromised, because Shh cannot be transported anymore (Fig. 21C, D). This
filopodial based mechanism of Shh spreading would be one way of explaining how in
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a gastrulating embryo in which cells continuously change their positions a stable and
robust distribution of signaling molecules is assured.
Fig. 21. Model of Shh distribution
during zebrafish gastrulation.
(A) Shh protein is produced in a
localized source and travels along
filopodial extensions, which connect
individual cells.
(B) Upon reaching target cells, Shh
induces expression of target genes,
such as ptc1.
(C) In a clone injected with DNEzrin, cells fail to establish filopodial
connections between them.
(D) Without filopodial connections,
Shh cannot travel away from the
producing cells and the range of
target gene activation is diminished.
Identification of zebrafish genes possibly involved in
shh signaling
In order to analyze genes, which might play a role in the spreading of Shh proteins, I
focused on HSPGs, molecules present on the cell surface and in the extracellular
matrix. They are implicated in binding signaling proteins, including molecules
belonging to the Hh family. In addition, I carried out an analysis of zebrafish rab23, a
gene belonging to the Rab family of small GTPases and shown to play a role during
Shh signaling in the mouse (Eggenschwiler et al., 2001).
Cloning and expression of zebrafish ext1 genes
In other species examined, only one member of the Ext1 family is known to date. In
contrast to this, I identified three zebrafish ext1 genes by performing a BLAST search
of the publicly available zebrafish databases with mouse EXT1. These sequences
were amplified by PCR and subsequent RACE PCR to generate the full length open
reading frames. The predicted amino acid sequences of these proteins showed a high
homology to known EXT1 proteins of other species (Fig. 22B). EXT1a is 81%
identical to mammalian EXT1 proteins, while EXT1b is 83% identical. In contrast to
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these high values of identity, EXT1c shows only 62% identity. Zebrafish EXT1a and
EXT1b show 88% identity among themselves and only 61% identity to EXT1c.
Interestingly, these values are lower than the respective identities within the
mammalian clade. Here, EXT1 proteins show 95% identity (Fig. 22B).
These values differ greatly within the protein. After the first 7 amino acids, which
are conserved in all species, and the putative transmembrane domain, the N-terminus
up to amino acid 115 is less well conserved (Fig.22A). This portion is thought to form
a putative stem region, which separates the signal sequence from the catalytic portion
of the protein (Wei et al., 2000). Several mutations have been characterized in EXT1
genes affecting the glycosyltransferase activity (Wei et al., 2000). The amino acids
altered in these mutants are conserved in the zebrafish EXT1 paralogs (Fig. 22A,
asterisks), underlining the evolutionary conservation of all three proteins. In addition,
the phylogenetic tree (Fig. 1C) groups all three zebrafish EXT1 proteins in the EXT1
clade with high bootstrap support.
The phylogenetic analysis furthermore suggests that ext1a and ext1b arose during
a more recent duplication event in the zebrafish lineage, while ext1c is more diverged.
Thus, these findings indicate that ext1a, ext1b and ext1c are the orthologous zebrafish
genes of other known EXT1 genes. To analyze their expression during embryonic
development, I performed in situ hybridization with riboprobes for the respective
genes. As a control, I performed in situ hybridizations with sense probes of each gene
which did not produce a staining (data not shown).
Expression of ext1a
Ext1a message is maternally provided (data not shown) and present at sphere stage
(Fig. 23A). At about 80% of epiboly, ext1a is expressed in two ectodermal wings
animal to the germ ring.
These exclude the most dorsal structures, such as the axial mesoderm (Fig. 23B,
C). At the three somite stage, ext1a message is present in prospective forebrain
regions, while in putative midbrain regions it marks the outline of the neural plate
(Fig. 23G). Furthermore, three stripes of ext1a expressing cells can be detected in the
forming hindbrain. More posteriorly, ext1a is expressed in the tailbud and in the axial
mesoderm (Fig. 23G).
Fig. 22. Sequence comparison of EXT1 proteins. (A) Alignment of EXT1 proteins of different species. Amino acids that were shown to be critical for glycosyltransferase activity
of EXT1 proteins are conserved in all three zebrafish EXT1 proteins (asterisks). (B) Comparison of sequence identities between EXT1 proteins of different species. (C)
Phylogenetic tree of the different EXT1 proteins. All three zebrafish EXT1 proteins fall into the EXT1 clade with high bootstrap support. Cg, Cricetulus griseus, Dm, Drosophila
melanogaster, Dr, Danio rerio, Fr, Fugu rubripes, Hs, Homo sapiens, Xl, Xenopus laevis.
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Fig. 23. Expression
of ext1a (A, B, C) and
ext1b (D, E, F).
(A-F) Anterior is to the
top. (G) Anterior to the
left. Embryos in (B, E)
were opened on the
ventral side and flat
mounted with the
dorsal side facing the
viewer, animal pole to
the top. (A) Sphere
stage. (B) 80% of
epiboly.
Ext1a
transcripts can be
detected in two wings
in the prospective
hindbrain region lateral
to the embryonic
midline (marked by
asterisk). (C) Cross
section of the embryo
shown in (B). (D)
Sphere stage. (E) 80%
epiboly stage. Ext1b
RNA can be detected
in the embryonic
midline (marked by
asterisk) and in the
germ ring. (F) Cross
section of the embryo
in (E). (G) 3-somite
stage. Fb, forebrain,
hb, hindbrain, tb,
tailbud.
These expression domains are in close proximity to regions of fgf8 expression, as
revealed with a double in situ hybridization with ext1a (blue) and fgf8 (red) (Fig. 24).
The forebrain expression of ext1a is located posterior to fgf8 expressing row 1 cells
(Fig. 24A, arrow). Furthermore, the hindbrain domain of ext1a is partially
overlapping with fgf8 at the midbrain-hindbrain-boundary (mhb) (Fig. 24.B) and in
lateral regions of the neural plate (Fig. 24B, bracket). These tissues have been shown
to depend on Fgf8 signaling for proper patterning (Reifers et al., 1998). Thus, it is
possible that ext1a is involved in Fgf8 dependent signaling processes.
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80
Fig. 24. Relationship of ext1a to fgf8 expression.
Flat mounted embryos at the 1 somite stage,
oriented anterior to the left and in situ hybridized with
ext1a (blue) and fgf8 (red). (A) Expression of ext1a
can be detected close to regions of fgf8 expression,
such as row1 cells (arrow) and the mhb (arrowhead).
The tailbud (asterisk) is also positive for ext1a
transcripts, which will be more pronounced at slightly
later stages (compare Fig. 23G). (B) Close up of the
mhb region. Note overlapping and mutually
exclusive domains of ext1a and fgf8 in lateral
domains (bracket) and the embryonic midline
(arrow). Scale bars represent 100 µm.
Fig. 25. Expression of
ext1a (A, B, C, C’), ext1b
(D, E) and ext1c (F, G, H)
at the 16 somite stage.
Anterior is to the left. (A)
Expression of ext1a can be
detected in the eye, the
dorsal
neural
tube,
Kupffer’s vesicle (black
arrow) and the somites. (B)
Cross section at the level of
the 12th somite (C) Dorsal
view on the brain of the
embryo in (A). Krox20
demarcates rhombomeres
3 and 5 (inset). (C’)
Expression of ext1a in the
eye. Os, optic stalk. (D)
RNA encoding e x t 1 b is
expressed
more
ubiquitously with domains
of higher expression in the
tailbud region, the eye and
dorsal somites. (E) Section
at the level of the 9th
somite,
revealing
expression of ext1b in the
dorsal aspect of the somite.
(F) Alternating expression
domains of ext1c can be
detected in the somites. (G)
Cross section of the
embryo in (F) at the level of
the 16th somite. (H) Cross
section at the level of the
6th somite.
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81
From the 5 somite stage onwards, ext1a RNA can be detected in the somitic
mesoderm. This expression continues during somitogenesis (Fig. 25A). Cross sections
at the 16-somite stage reveal staining in two ventro-medial domains of the somites
(Fig. 25B, black arrowheads) and in a dorso-medial compartment, adjacent to the
neural tube (Fig. 25B, black arrows).
In addition to the somitic expression, ext1a message can be detected in a
segmental fashion in the dorsal neural tube (Fig. 25C). Double in situ hybridization
with Krox20 reveals that rhombomeres 4 and 6 express ext1a (Fig. 25C, inset).
Furthermore, the anterior hindbrain (Fig. 25C, black arrow) and the dorsal
diencephalon (Fig. 25C, white asterisk) are positive for ext1a message. In the eye,
expression consists of two domains: the distal retina (Fig. 25C’, white arrowhead) and
the optic stalk region (Fig. 25C’), as marked by double in situ hybridization with
pax2.1 in red (Fig. 25C’, inset shows fluorescent pax2.1 signal). Most posteriorly,
Kupffer’s vesicle exhibits elevated ext1a expression (Fig. 25A, black arrow).
At 24 hpf, strong cerebellar expression is detected (Fig. 26A, black arrow, Fig.
26D, black arrow) as well as expression in the optic stalk (Fig. 26A, black arrowhead,
Fig. 26F, black arrowhead) and the dorsal diencephalon (Fig. 26A, black asterisk). At
this stage, the anterior part of the epiphysis shows a stronger signal compared to the
posterior part (Fig. 26E, outlined with white dotted line). In general, ext1a expression
is confined to the dorsal part of the neural tube, as revealed by a cross section at the
level of the anterior spinal cord (Fig. 26C). Furthermore, expression can be detected
in the tail somites. At about 48 hpf, expression continues in the dorsal diencephalon
and in the cerebellum. In addition, mRNA can be detected in the otic vesicle (Fig.
26B, black arrow).
Expression of ext1b
Ext1b is also provided maternally (data not shown) and transcripts can be detected at
sphere stage (Fig. 23D). During gastrulation expression consists of two domains: the
germ ring and the axial mesoderm (Fig. 23E, asterisk). Cross sectioning at the level of
the germ ring confirms mesendodermal expression (Fig. 23F). During somitogenesis
stages expression is strongest in the tailbud and the posterior somites (Fig. 25D).
Cross section reveals transcripts in the myotomal part of the somites, while no
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82
expression is detected in the sclerotomal part and in adaxial tissues (Fig. 25E). In
addition, transcripts can be detected in the forming eye (Fig. 25D).
At the 24 hpf stage, cells of the neural tube up to the posterior limit of the
hindbrain (Fig. 26G, black arrow) are positive for ext1b transcripts. In addition, the
posterior somites and the tailbud (Fig. 26G, black arrowhead) continue to express
ext1b. Another mesodermal expression domain comprises cells in the region of the
anterior pronephric duct (Fig. 26I, black arrow). In the forming fin buds (Fig. 26J)
ext1b can also be detected. At about 48 hpf, the finbuds (Fig. 26H, black arrow) as
well as the brain are positive for ext1b transcripts.
Fig. 26. Expression of ext1a (A-F), ext1b (G-J) and ext1c (K-O) at 24 hpf (A-G, I-K, M) and 48 hpf (B, H, L, N, O). (A)
Expression of ext1a can be detected in the dorsal neural tube. (B) Expression in the otic vesicle (black arrow). (C) Cross
section of the embryo in (A) at the level of the anterior spinal cord. (D) Dorsal view of the embryo in (A) at the level of the
cerebellum (black arrow) and hindbrain. Ov, otic vesicle (E) Dorsal diencephalon (outlined with white dots, A, anterior, P,
posterior). (F) Cross section through the eye. Black arrowhead marks optic stalk. (G) Tail bud region (black arrowhead)
and anterior neural tube (black arrow). (H) The fins (black arrow) express ext1b. (I) Close up of cells belonging to the
anterior pronephric duct (black arrow). (J) Close up of the fin bud mesenchyme. (K) Transcripts can be detected in the
telencephalon (black arrow) and the rhombomeres (white arrowhead). (L) Expression is confined to the brain. (M)
Telencephalon (white asterisk) and the olfactory bulbs (marked by dotted white line) of the embryo in (A). (N) Expression
of ext1c in the retina. (O) Cross section of the embryo in (L) at the level of the otic vesicles (white arrowhead). The white
matter is marked by black arrow.
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83
Expression of ext1c
Ext1c message can be first detected at the beginning of somitogenesis, where it is
expressed in the forming somites. Later, at about the 16 somite stage (Fig. 25F),
expression is confined to adaxial cells in the posterior part of the embryo (Fig. 25G).
More anteriorly, cells adjacent to the neural tube and in ventromedial regions of the
somite express ext1c (Fig. 25H, black arrowheads). At the end of somitogenesis,
neural expression domains of ext1c appear, while the posterior somites continue to
express ext1c. Neural domains become more elaborate at the 24 hpf stage (Fig. 26K):
Transcripts can be detected in the ventral rhombomeres (Fig 26K, white arrowhead)
and the telencephalon (Fig. 26K, black arrow). Furthermore, close up of the
telencephalon (Fig. 26M, white asterisk) reveals that also cells of the olfactory bulbs
contain ext1c transcripts (Fig. 26M, black arrow).
This expression continues at the 36 hpf stage. Interestingly, co-staining of axonal
tracts with an -acetylated tubulin antibody reveals expression of ext1c to be present
in telencephalic cells that form the ventral and dorso-rostral clusters (Fig. 27B). These
are the cells from which the anterior and post optic commissures grow out. In
addition, strong staining of ext1c in the ventral parts of the hindbrain rhombomeres
coincides with the dorsal extension of axonal tracts emanating from the medial
longitudinal fascicle. Thus, the spatial correlation of ext1c expression with an axonal
tract marker suggests that ext1c might have a function in directing proper axon
growth. This would be in agreement with recent findings in mouse, which showed
that disrupting Ext1 function in neural tissue resulted in impaired axonal pathfinding
(Inatani et al., 2003).
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RESULTS
Fig. 27. Comparison of expression of ext1c and
axonal tract formation at 36 hpf.
(A) Schematic drawing of axonal tracts forming in
the developing zebrafish brain between 24 and 36
hpf (Devine and Key, 2003).
(B) Embryo at the 36 hpf stage, oriented anterior to
the left. Embryo in situ hybridized for ext1c mRNA in
blue. Costaining with -acetylated tubulin reveals
axonal tracts in brown. Note the strong expression of
ext1c in the cells of the drc. In addition, e x t 1 c
expression is confined to the ventral part of the
hindbrain rhombomeres (black bar). The dorsal
extend of ext1c expression coincides with the dorsal
extend of axonal tracts emanating from the mlf
(arrows).
Abbreviations: Ac, anterior commissure; drc, dorsorostral cluster; DVDT, dorsoventral diencephalic
tract; epi, epiphysis, MLF, medial longitudinal
fascicle; PC, posterior commissure; TPC, tract of the
posterior commissure; vcc, ventral-caudal cluster;
vrc, ventral rostral cluster.
At 48 hpf, transcripts can be detected in the brain (Fig. 26L). Cross section at the
level of the otic vesicle shows strong expression in the white matter (Fig. 26O, black
arrow) as well as in the otic vesicles (white arrowhead) and in more dorsally located
nascent neurons. At this stage, also the fin buds (data not shown) and the retina
express ext1c (Fig. 26N).
Taken together, my results reveal the existence of at least three different members
of the EXT1 family in zebrafish, which most likely arose during a genome duplication
event in the teleost lineage. Their expression throughout embryogenesis comprises
partially overlapping and mutually exclusive domains. In addition, the expression
patterns show a spatial shift over time: at somitogenesis stages, transcripts are present
in the somites, while at later stages of development, the brain and the fin buds express
ext1 genes.
Dependence of ext1a and ext1c on hedgehog signaling
Previous studies in Drosophila have shown that EXT1 genes are necessary for the
spreading of molecules belonging to the hh family (Bellaiche et al., 1998).
Furthermore, a study in mice suggested that EXT1 proteins might negatively regulate
shh signaling by synthesizing HSPGs which sequester the ligand (Koziel et al., 2004).
This negative regulation might suggest the existence of a feedback loop. For instance,
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85
overexpression of shh induces expression of the SHH receptor patched (Concordet et
al., 1996), which also sequesters the ligand (Chen and Struhl, 1996), while loss of shh
signaling leads to a downregulation of patched expression (Chen et al., 2001; Varga et
al., 2001). I therefore asked if ext1 genes respond to shh in a similar way.
To this end, I examined the effects of shh overexpression on zebrafish ext1 genes
using synthetically transcribed shh mRNA. I also addressed the effects of loss of
hedgehog signaling by analyzing the expression of ext1 genes in known mutants of
the hedgehog pathway in fish. These included smu (Chen et al., 2001; Varga et al.,
2001), and syu (Schauerte et al., 1998). The effect of altered hedgehog signaling was
assessed at the 14 somite stage. In a dorsal view of this stage, ext1a is more strongly
expressed in tissue closer to the midline (Fig. 28B). Increased hedgehog signaling
lead to ectopic expression of ext1a throughout most of the somite (Fig. 28B,C,
compare white brackets). However, different expression intensities of ext1a could still
be detected.
Fig. 28. Dependence of ext1a and ext1c expression on Shh signaling. Dorsal views of 14-somite stage embryos with
anterior to the left. Expression of ext1a (A-C) and ext1c (D-F) respectively. In smu mutant embryos, expression of ext1a
(A) and ext1c (D) is greatly reduced compared to wt embryos (B, E). Overexpression of shh results in an expansion of the
somitic expression domains of ext1a (C) and ext1c (F) (compare white brackets in B, C and E, F). Black lines indicate
extend of somites in (A-C).
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86
More medially located areas of the somites showed a stronger staining, which
might suggest that additional factors are required for the induction of strong ext1a
expression. In the case of ext1c, the effect of s h h overexpression was more
pronounced. Upon overexpression of shh, a large proportion of the somitic cells
expressed ext1c at uniformly high levels (Fig. 28E, F, compare white brackets).
Interestingly, expression of ext1b was found to be essentially normal in a gain of shh
function situation (data not shown).
In contrast to this, loss of shh signaling (shown for smu mutant embryos) lead to a
nearly complete loss of ext1a expression in the somites (Fig. 28A). Similarly, ext1c
expression could only be detected in the more anterior somites with a progressive loss
of expression towards posterior regions (Fig. 28D). Again, expression of ext1b was
not altered in smu mutant embryos (data not shown). Similar results were obtained
when analyzing syu mutant fish. However, I noted a less severe reduction in gene
expression of both ext1a and ext1c, probably reflecting a possible redundancy with
other members of the Hh family (data not shown and Fig. 12A, D).
Therefore, I conclude that expression of ext1a and ext1c in somites depends in
part on functional Hh signaling and responds to shh overexpression by an increase in
transcription. Interestingly, I noted ectopic expression of ext1a in the notochord of
smu mutant zebrafish at the 14-somite stage (Fig. 28A). At this stage, shh is produced
in notochord cells (Krauss et al., 1993). In wt embryos, axial mesoderm also
expresses ext1a at the beginning of somitogenesis (Fig. 25G), but this expression is
lost during later development (Fig. 26B). The prolonged expression of ext1a in the
notochord of smu embryos might reflect a differential response of the ext1a promoter
in notochord and somitic cells.
Knock-down of ext1a leads to curly-down body shape
Having established a tentative role of ext1a in Shh signaling, I asked if Shh dependent
processes are affected in a loss of ext1a function situation. To address this, I made use
of morpholino anti-sense oligonucleotides. Injection of 4 pg of a morpholino targeted
to the sequence around the translational start site of ext1a produced a curled-down
body phenotype at 24 hpf (Fig. 29).
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RESULTS
Fig. 29. Injection of e x t 1 a
morpholino causes a curled down
body shape.
All embryos at the 24 hpf stage.
Oriented anterior to the left (B, D). (A)
wt embryos, showing normal curl of the
body axis (red dots). (B) In wt, the
somites are chevron shaped (red dots).
(C) Morpholino injected embryos
display curled down body shape (red
dots). (D) In morpholino injected
embryos, the somites loose their
chevron shape and appear blocky (red
dots).
This is suggestive of a defect in Hh signaling which is necessary to organize
somite morphology (van Eeden et al., 1996) and differentiation of slow muscle cells
and muscle pioneers (Barresi et al., 2000; Wolff et al., 2003). However, analysis with
engrailed as a marker for muscle pioneers (Fjose et al., 1992) did not show any
alteration in either the number or the position of muscle pioneer cells in morpholino
injected embryos (Fig. 30E, F). Furthermore, addressing slow muscle cell formation
with an antibody recognizing slow myosin heavy chain (MyHC) (Devoto et al., 1996)
did not reveal gross alterations in ext1a morpholino injected embryos (Fig. 30A, B).
Rarely, a reduction in muscle pioneer staining of MyHC was observed (Fig. 30B,
arrow), but with a very low frequency (2 of 30 cases). I extended my analysis with a
prox1 antibody staining, which marks the nuclei of slow muscle cells (Glasgow and
Tomarev, 1998). Neither the number (23 ± 2), nor the position of slow muscle cells
was altered in morpholino injected embryos (Fig. 30A, B).
Fig. 30. Injection of ext1a morpholino does not cause an alteration of markers specific for muscle pioneers
or slow muscle cells.
Embryos at the 24 hpf stage and oriented anterior to the left. Wt embryos (A, C, E) or embryos injected with ext1a
morpholino (B, D, F). (A) MyHC staining reveals slow muscle cells in brown. Muscle pioneers are located at the
horizontal myoseptum at the midline of each somite and show a darker staining. (B) Morpholino injected embryo.
Normal staining of MyHC. However, in some somites, staining of muscle pioneer cells is weaker. (C) Wt prox1
staining. Nuclei of slow muscle cells are brown. (D) Morpholino injected embryo. (E) Engrailed staining marking
muscle pioneer cells aligned along the horizontal myoseptum. (F) No change in muscle pioneer cells number or
position in morpholino injected embryos.
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RESULTS
This suggests that the observed change in body morphology may not be due to
reduced somitic hedgehog signaling. In addition, injection of a second morpholino
(ext1a-UTR) targeted to the 5’UTR of ext1a did not produce a comparable phenotype.
Therefore, it remains to be determined what the cause of the downward curled body
shape is and if this phenotype is due to a loss of ext1a function.
Knockdown of ext1b and ext1c
Further attempts to interfere with ext1 gene function did not produce conclusive
results. Injection of a morpholino against the 5’UTR of ext1b did not interfere with
development and embryos morphologically resembled wt littermates at 24 hpf (data
not shown). The ext1c morpholino targeted around the translational start site lead to a
shortening of the embryonic axis at 24 hpf and a brain phenotype which was
characterized by a malformed midbrain-hindbrain-region (Fig. 31C, asterisk) and a
defect in telencephalon development (Fig. 31C, arrow).
Fig. 31. Injection of ext1 morpholino leads to a
shortened body axis and brain defects.
Embryos at the 24 hpf stage, oriented anterior to the
left. (A) Wt embryo. (B) Morpholino injected embryo.
The trunk region appears morphologically normal. The
tail is slightly curved upwards. (C) Close up of the head
region of a wt embryo, showing the mhb (asterisk) and
the telencephalon (arrow). (D) Close up of the head
region of a morpholino injected embryo. The body axis
is shortened (compare position of head in relation to
the yolk). Furthermore, the mhb is not properly
established (asterisk) and the telencephalon (arrow)
shows a morphological defect.
RESULTS
89
Analysis of zebrafish rab23
In addition to genes responsible for modifying the extracellular matrix, I analyzed
zebrafish rab23. In mouse, Rab23 was shown to play an important role in Shh
signaling (Eggenschwiler et al., 2001). I therefore asked if loss of rab23 function in
zebrafish would have a similar phenotype. To this end, I cloned zebrafish rab23,
determined its expression domains during embryonic development and carried out
knock down experiments using morpholino oligonucleotides.
Cloning of zebrafish rab23
In order to clone zebrafish rab23, I carried out database searches with the known
mouse Rab23 sequence. However, no matching sequences could be found. Therefore,
I decided to use a PCR strategy based on degenerate primers. With two of these I was
able to amplify a 210 bp fragment with high homology to mouse Rab23. This
fragment was used to design primers for RACE PCR. Subsequently, I amplified an
open reading frame with high sequence similarity to Rab23 from other species (Fig.
33, table 7).
More recently, I identified a GenScan predicted transcript (GENSCAN
00000025483) in the Zv4 zebrafish genome assembly, which has 100% sequence
identity to the previously amplified rab23. This transcript is located on chromosome
13 at position 17249-25583 on the fragment BX004843.10.1-131026 close to the
marker G39057 (Fig. 32). The predicted gene spans a region of 8.34 kb and consists
of 6 exons. Thus, the PCR amplified rab23 matches the genomic data.
Fig. 32. Genomic localization of rab23 (GENSCAN 00000025483), located on chromosome 13.
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RESULTS
Table 7. Alignment of Rab23 proteins from different species
Mm_Rab23
84
Hs_Rab23
84
93
Dm_CG2108
73
59
59
Gg_Rab23
83
84
88
54
Dr_Rab23
Mm_Rab23
Hs_Rab23
Dm_CG2108
The values indicate percent of amino acid identity. Dr, Danio rerio; Mm, Mus musculus; Dm, Drosophila
melanogaster; Gg, Gallus gallus.
An alignment of RAB23 proteins from different species illustrates the high
conservation of these proteins. This is particularly evident for the proteins’ N-termini,
while the C-termini are more diverged. Interestingly, the zebrafish RAB23 protein
shares a higher sequence identity with the Drosophila protein than the other vertebrate
proteins do. In addition, the sequence identity of the zebrafish protein to the other
vertebrate proteins is somewhat lower than among the other members of the
vertebrate group (Table 7).
Fig. 33. Sequence alignment of Rab23 proteins from different species. Note the high homology, especially in the Nterminal region of the protein up to amino acid 125. Dm, Drosophila melanogaster; Dr, Danio rerio; Gg, Gallus gallus; Hs,
Homo sapiens; Mm, Mus musculus.
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RESULTS
Expression of rab23 during zebrafish development
In order to determine the spatial and temporal expression of rab23, I performed in situ
hybridization with an anti-sense probe against zebrafish rab23. Rab23 is maternally
provided (Fig. 34A) and present at sphere stage (Fig. 34B). At the onset of
gastrulation, expression of rab23 is downregulated (Fig. 34C). During somitogenesis
stages (Fig. 34D), transcripts are present ubiquitously. At the 24 hpf stage, expression
can be detected in the developing neural tube, in the eye (Fig. 34E, arrow) and in the
heart (Fig. 34F, arrow). Thus, from the expression pattern I could not infer an
immediate relationship of rab23 to Shh signaling.
Fig. 34. Expression of zebrafish rab23
during development. All embryos were
in situ hybridized with an anti-sense probe
against rab23 and oriented dorsal up (AC), or anterior to the left (D-F). (A) 4 cell
stage, rab23 is maternally provided. (B)
Sphere stage embryo. (C) 50% of epiboly
stage, expression of r a b 2 3 is
downregulated. (D) 14-somite stage,
transcripts of r a b 2 3 are present
throughout the embryo. (E) 24 hpf stage,
note the stronger expression of rab23 in
the eye (arrow). (F) Close up of the left
side of a 24 hpf stage embryo. Note
stronger expression in the heart ventricle.
Knock-down of rab23 leads to laterality defects
In order to assess the function of rab23 during zebrafish development, I made use of
anti-sense morpholino oligonucleotides. Injection of 12 pg of a morpholino directed to
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RESULTS
the region around the ATG start codon of rab23 resulted in no morphological obvious
phenotypes up to the 24 hpf stage. At this stage, the heart tube starts to be oriented to
the left side of the body axis in wt embryos and beating of the heart commences
(Glickman and Yelon, 2002; Stainier et al., 1993; Yelon, 2001).
Fig. 35. Injection of 12pg of
r a b 2 3 morpholino causes
laterality defects.
(A, B) Embryos at the 36 hpf
stage, viewed from the front. The
heart tube is outlined with white
dots(C, D). Embryos at the 24
hpf stage, oriented anterior to the
left. (E, F) Embryos at the 28 hpf
stage, oriented anterior to the
top.
(A) Wt embryo exhibiting a
normal leftwards looping of the
heart (white dots). (B) Morpholino
injected embryo displaying an
inversed (rightward) looping of
the heart. (C) Embryo in situ
hybridized with a probe for lefty2,
which marks the left lateral plate
mesoderm. (D) In morpholino
injected embryos, lefty2 staining
is frequently detected in the right
lateral plate mesoderm. Inset
shows failure of lateral plate
mesoderm to turn to either side
of the embryo. (E) Wt embryo in
situ hybridized with a probe for
g a t a 4 , which detects the gut
primordium. The gut loops to the
left side in wt embryos. (F) In
morpholino injected embryos,
gata4 staining is often detected
on the right side of the embryonic
midline, revealing an inversion of
gut situs.
In the morpholino injected embryos, I observed a randomization of this laterality
on the morphological level and confirmed this finding by analysis of marker gene
expression. The nodal antagonist lefty2 is expressed in the left lateral plate mesoderm,
a cell population giving rise to heart precursors (Bisgrove et al., 1999; Stainier et al.,
1993). In situ hybridization with a lefty2 anti-sense probe revealed randomized
staining of lefty2 in morpholino injected embryos in comparable ratios as in the
embryos, which were scored by morphology. I could observe embryos in which the
left lateral plate mesoderm correctly expressed lefty2, but in others expression was
found on the right side (Fig. 35D).
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RESULTS
In addition, I frequently observed a symmetric expression of lefty2 along the
embryonic midline (Fig. 35D, inset). Mutant analysis in zebrafish revealed that initial
positioning of the heart and subsequent looping could occur independently of each
other (Chin et al., 2000). Therefore I asked whether heart looping was also affected in
rab23 morpholino injected embryos. I addressed the looping of the heart tube to either
side of the embryonic midline at the 36 hpf stage (Fig. 35, table 8). In wt embryos, the
heart looped to the left side of the axis (Fig. 35A).
Table 8. Injection of rab23 morpholino causes randomization of
heart situs
Number of
Heart loops left (wt)
Heart loops right
Heart in the middle
40
44
46
embryos
130
Embryos were injected with 12 pg of rab23 morpholino. Subsequently, the orientation of heart situs was
morphologically addressed at the 36 hpf stage. As a control, wt siblings were scored at the same age. 100%
showed leftward heart looping (31/31).
However, in morpholino injected embryos, I could observe looping to the left as
well as to the right and hearts that did not loop at all (Table 8). Therefore, interfering
with rab23 function causes an alteration of heart situs, which is also detected on the
level of marker gene expression.
Because of the expression of rab23 in the heart at the 24 hpf stage, I next asked if
the randomization of organ situs is restricted to the heart. To address whether other
organs that exhibit laterality were also affected by injection of a rab23 morpholino, I
carried out in situ hybridization with gata4, a marker known to be expressed in the gut
primordium in zebrafish (D. Stainier, unpublished) (Laverriere et al., 1994).
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RESULTS
Table 9. Gut situs is randomized in embryos injected with rab23
morpholino.
Number of embryos
Gut left (wt)
Gut right
Gut middle
52
26
20
6
Embryos were injected with 12 pg of rab23 morpholino and subsequently in situ hybridized at the 24 hpf
stage with an anti-sense probe for gata4. As a control, wt siblings were scored at the same age. 100%
showed leftward gut looping (28/28).
In wt embryos, the gut looped to the left side of the body (Fig 28E) (HorneBadovinac et al., 2003; Reiter et al., 1999). In contrast to this, morpholino injected
embryos displayed a randomization of gut situs as revealed by analysis of gata4
expression (Table 9). Thus, the randomization of organ situs observed in rab23
injected zebrafish embryos does not only affect the heart, but also other organs known
to exhibit a distinct laterality, such as the gut. Therefore, rab23 function is most likely
necessary for one of the early processes establishing body laterality.
95
Discussion
Signaling range of Shh
I have addressed the question of how Shh proteins are propagated from cell to cell in
gastrulating zebrafish embryos. Shh proteins have been shown to function in early
embryonic patterning by specifying ventral neural cell types and slow muscle cell
precursors. Furthermore, the establishment of a graded Shh distribution is
indispensable for later patterning events. These requirements seem to be hard to
reconcile with early embryonic development, where cells are motile and change their
position in respect to one another during the dynamic gastrulation process. Therefore,
mechanisms must exist which ensure proper Shh distribution in this developmental
context, which might differ from those controlling protein spreading in epithelial
tissues.
In order to characterize the range of Shh protein distribution at gastrula stages, I
have genetically determined the extend of Shh signaling. To this end, I analyzed the
spatial relationship between shh expression and one of its downstream target genes,
ptc1. My findings illustrate that Shh proteins signal from axial tissue to the
neighboring ectodermal and mesodermal cells in gastrulating embryos. ptc1
expression responds to Shh signaling, because in smu mutant embryos, which cannot
transduce Hh signals, ptc1 expression is reduced. The same is true for syu mutant
embryos in which the shh gene is deleted (data not shown). By measuring the extend
of target gene induction, I find that cells in a distance of about 3-4 cells activate ptc1.
I could confirm and extend these findings by creating an ectopic source of Shh
expressing cells in the embryo. Around the clone, ptc1 was also induced in a distance
of 3-4 cells from the ectopic Shh source. Therefore, the signaling range of Shh as
determined by target gene induction is 3-4 cell diameters in gastrulating zebrafish
embryos.
In other developmental contexts the signaling range of Hh proteins greatly varies.
Hh signaling in Drosophila larval segments is thought to be short range, mainly
acting in a juxtacrine fashion on neighboring cells (Alexandre et al., 1999). In contrast
to that, in Drosophila wing imaginal discs, Hh signals 8-10 cell diameters to induce
Dpp and ptc expression (Basler and Struhl, 1994; Chen and Struhl, 1996).
Interestingly, clones of cells ectopically expressing Hh in wing imaginal discs induce
DISCUSSION
96
the target gene dpp only at a distance of about 3-4 cell diameters (Strigini and Cohen,
1997). In vertebrates, Shh spreading is thought to be long range. For instance, in the
limb bud, Shh signals up to 30 cell diameters, a property thought to depend on its
cholesterol modification (Lewis et al., 2001) and the ability to form multimeric
structures (Chen et al., 2004; Zeng et al., 2001). The same long range signaling
properties were shown to be important for Shh function in neural tube development,
where Shh induces ventral cell types (Jessell, 2000) and acts as a chemoattractant for
commissural axons originating from dorsal positions within the neural tube (Charron
et al., 2003). Thus, in distinct developmental systems, the signaling ranges of proteins
belonging to the Hh class can differ considerably.
In addition, the target gene read out might not reflect the actual protein
distribution. The transcriptional response to Hh signaling is thought to have a lag time
of several hours due to the need of CiR degradation (Lum and Beachy, 2004).
Because I addressed the extend of target gene activation 3 hours after transplanting
Shh expressing cells, it is possible that Shh protein had moved beyond the ptc1
positive cells, but had not activated ptc1 expression yet. Alternatively, sequestering by
ptc1 might be effective enough to restrict Shh protein to the ptc1 positive cells,
hindering further spreading.
Control of Shh signaling range
The observations that Hh proteins have long and short range signaling properties
suggest that mechanisms exist which regulate the range of signaling. One mechanism
is the sequestering of Shh by its receptor Ptc (Chen and Struhl, 1996). In addition,
another molecule, called Hedgehog interacting protein (Hip) implicated in restricting
the range of Shh signaling has been identified in vertebrates (Chuang and McMahon,
1999). Furthermore, I have characterized three HSPG modifying enzymes, belonging
to the ext1 family, two of which respond to increased levels of Hh signaling. HSPGS
synthesized by these enzymes are implicated in binding Hh ligands and therefore
might also restrict Shh protein spreading. Thus, several feedback controls exist which
influence the spatial distribution of Shh signaling molecules. Which of these operate
in gastrulating zebrafish embryos needs to be determined. However, expression of
DISCUSSION
97
ext1b and later ext1a in axial cells might indicate that HSPGs are important in early
embryogenesis for regulating Shh distribution.
Signaling strength of fluorescent Shh fusion proteins
Having addressed the signaling range of Shh by genetic means, I directly visualized
Shh protein distribution by fluorescently tagging zebrafish Shh. I constructed two
fluorescent variants, one having a GFP cloned at the N-terminus of the N-terminal
fragment of Shh and the other by inserting “venus” N-terminal to the autoproteolytic
cleavage site. Subsequently, I addressed the signaling properties of these two
constructs. In the case of the N-terminal fusion, I found signaling strength to be
greatly reduced as addressed by morphological criteria and target gene induction.
Nevertheless, some upregulation of Shh target genes such as pax2.1 could be
observed in embryos ubiquitously overexpressing Shh-NGFP. To the contrary, clones
ectopically expressing Shh-NGFP never induced ptc1 in surrounding cells. Thus, in
transplanted cells, the seemingly reduced signaling strength of Shh-NGFP was
insufficient for target gene induction.
This reduced signaling strength was probably due to interference with the
palmitoylation reaction, shown to be necessary for full activation of the Shh pathway
(Pepinsky et al., 1998). Even though I left the known consensus palmitoylation
sequence (CGPGRG) intact, the exact spatial requirements for the acyltransferase
mediating the palmitoylation reaction are not known. Furthermore, this sequence is
short in comparison with other known lipid modification motifs, which are between
11 and 20 residues long (Fivaz and Meyer, 2003). Therefore, the putative
palmitoylation sequence might be incomplete and therefore unable to mediate
addition of a palmitoyl to a heterologous protein. Surprisingly, beads soaked in
bacterially produced Shh are able to induce target genes (Lopez-Martinez et al., 1995;
Zhang et al., 1999), even though they are lacking both lipid modifications. This
discrepancy is most likely explained by the high amounts of protein released from the
bead, which cannot be achieved by shh mRNA injections. Furthermore, the protein
released from beads is not subject to any sorting mechanism that has been shown to
deliver Hh proteins to the apical surface of Drosophila wing imaginal discs (Gallet et
DISCUSSION
98
al., 2003). In absence of lipid modifications, the protein was missorted and found to
spread further away from the source.
In contrast to these results, I found that Shh-venus signals essentially normal.
This was in agreement with a previous study in Drosophila, where the authors fused a
GFP to the corresponding location of Hh and showed that the resulting fusion protein
had normal signaling properties (Torroja et al., 2004). However, for Shh-venus I
noted a slight reduction in target gene response when compared to wt Shh. This might
be due to several reasons. The fusion protein might fold somewhat slower than the wt
counterpart. Therefore, the amount of available ligand might be lower, even though I
injected the same molar amount of Shh-venus. In addition, binding of Shh-venus to
the Ptc receptor might not be as stable as binding of wt Shh, because the addition of
the “venus” might alter the protein’s conformation. This gains particular relevance in
the light of the double lipid modification of Shh.
Subcellular localization of Shh-venus
When I analyzed the distribution of Shh-venus, I could detect fluorescence in distinct
punctate regions of expressing cells. These were randomly distributed over the cell’s
surface, but often accumulated in larger patches at cellular junctions. This was in
contrast to the expected localization of a lipid modified Shh protein. Lipid modified
proteins, which were either cholesterol modified (Vincent et al., 2003) or
palmitoylated (Iioka et al., 2004) were previously shown to label the entire plasma
membrane of expressing cells. Furthermore, earlier studies in fixed cultured cells
revealed that Shh protein accumulates at the cell membrane (Roelink et al., 1995), but
the authors did not report the presence of discrete regions of protein accumulation.
Therefore, the observed localization in discrete regions of the plasma membrane
contrasts with previous results in cell culture and furthermore indicates that the
subcellular localization of Shh proteins is most probably not only a direct
consequence of its lipid modifications. However, the localization of a doubly lipid
modified fluorescent protein has not been addressed. The punctate accumulation of
Hh proteins has been reported in fixed tissues (Gallet et al., 2003; Tabata and
Kornberg, 1994). Nevertheless, it remains to be determined if these punctae represent
endocytic compartments, as recently suggested in Drosophila (Torroja et al., 2004), or
compartments of the cell membrane.
DISCUSSION
99
Localization of Shh to raft lipid microdomains
Previous reports have addressed the relevance of membrane microdomains, called
lipid rafts, in respect to protein sorting and signaling (Simons and Ikonen, 1997).
Rafts contain a different lipid composition and cholesterol is thought to mainly reside
in these raft domains. Therefore, it is plausible to speculate that cholesterol modified
Shh proteins reside in rafts. Nevertheless, the finding that a cholesterol modified GFP,
which should also label rafts, uniformly marks the plasma membrane in non polarized
cells (Vincent et al., 2003) argues against this suggestion. However, it has not been
addressed if a cholesterol modified fluorescent protein targeted to the outer leaflet of
the plasma membrane by a signal sequence, as it is the case for Shh, would show a
different membrane labeling pattern.
In addition, I find that another membrane anchor, GPI, which has been reported
to target proteins to raft microdomains (Danielsen and van Deurs, 1995; Sargiacomo
et al., 1993) is also uniformly distributed in the plasma membrane. Therefore, if Shh
localizes to rafts, it does not identify the entire raft fraction. However, the observed
punctae might represent a subset of this population.
The estimates of raft size greatly vary. They range from 30-50 nm (Pralle et al.,
2000) to several micrometers (Schutz et al., 2000). Interestingly, a recent study using
Laurdan to monitor membrane fluidity in living cells has reported the accumulation of
raft clusters in certain membrane morphologies, such as filopodial extensions, regions
of cell-cell contact and adhesion points (Gaus et al., 2003). These structures strikingly
correlate with the observed localization of Shh-venus. Thus, different experimental
techniques used to determine the sub-cellular structure and localization of raft lipid
microdomains produce varying results. Nevertheless, my findings are in agreement
with previous raft localization studies performed in living cells (Gaus et al., 2003).
Shh-venus accumulation at cell junctions
In addition to the punctate localization of Shh-venus, I also observed accumulation of
fluorescent protein in regions of cell-cell contact, which is in agreement with previous
findings in Drosophila (Tabata and Kornberg, 1994). Interestingly, I detected the
strongest labeling of junctions between cells that were both descendants of an injected
single blastomere and therefore labeled by the lineage tracer. In addition, I found
DISCUSSION
100
targeting of Shh-venus protein to the site where cytokinesis of a dividing cell was
about to take place.
This suggests that the observed accumulation of Shh-venus at cellular junctions
of descendants of the injected blastomere might be a result of an earlier targeting of
Shh-venus protein during cell division and that cells after completion of cytokinesis
remain neighbors. Therefore, the localization of Shh-venus at the cleavage plane
might be a mechanism to insure that both daughter cells receive equal amounts of Shh
protein. This might be a way of assuring Shh distribution in contexts such as
development of the limb, where a temporal Shh gradient established by proliferating
ZPA cells was shown to contribute to Shh dependent digit formation (Harfe et al.,
2004). Furthermore, in Drosophila epidermal patterning, cell migration and protein
inheritance was shown to mediate Wg signaling (Pfeiffer et al., 2000), and a similar
mechanism was also suggested to play a role in Hh mediated cuticle patterning
(Alexandre et al., 1999).
Characterization of Shh-venus localization in filopodia
A third structure in which I found Shh-venus to accumulate was filopodial extensions.
These were present between injected cells, but could also be detected between
injected and uninjected cells. However, filopodial labeling between injected cells was
more intense. This was probably due to the larger amount of protein present when
both cells contained Shh-venus mRNA. Nevertheless, I cannot exclude the possibility
that the observed cellular extensions between injected cells represent sites of
incomplete cytokinesis. As mentioned earlier, Shh-venus is targeted to the cleavage
plane. When the cells now start to move away from each other, they might leave a
“trail” of Shh-venus behind. This would be different from a cell sending out a
protrusion to contact a neighboring cell. However, the existence of filopodia between
non injected cells and the results obtained from time lapse movies suggests that
incomplete cytokinesis cannot account for all of the observed cellular protrusions.
In time lapse movies of gastrulating embryos, I could follow the distribution of
Shh-venus. These movies revealed that accumulations of Shh-venus could be
exchanged between neighboring cells. Most strikingly, the time lapse movies also
revealed that punctae detected outside expressing cells in still images were actually
DISCUSSION
101
often associated with filopodial extensions along which protein was exchanged
between cells. Therefore, the punctate accumulations of Shh-venus might represent
specialized structures within such filopodia. This is in agreement with recent findings
that report the existence of filopodial extensions between cells in culture (Rustom et
al., 2004) and in Drosophila wing imaginal discs (Ramirez-Weber and Kornberg,
1999). However, the functional significance of these structures in respect to Hh
signaling remains obscure. Therefore, my time lapse movies provide first evidence for
the specific localization of Shh protein to these filopodial structures.
Argosomes have been described in Drosophila as membranous particles
implicated in the spread of morphogen molecules (Greco et al., 2001). They were
described as fusing and forming tubular structures, similar to the changes in shape I
observed for protein patches, which traveled along filopodia. Nevertheless, even
though filopodia are known to be present between cells of the wing imaginal disc in
apical regions (Ramirez-Weber and Kornberg, 1999), and in baso-lateral regions
(deMontis, unpublished observations) from which argosomes arise, the authors did
not detect an association of argosomes with filopodial structures. Nevertheless, it is
intriguing to speculate that argosomes are associated with filopodial extensions
present in wing imaginal discs. This notion gains further support from studies
addressing air sac development in Drosophila (Sato and Kornberg, 2002). Here, the
authors detected punctate structures along tracheoblast filopodia as well as at the tips
of such filopodia. The observed differences in labeling might be due to the different
membrane markers used.
Mechanisms of targeting Shh to distinct subcellular
compartments
The observed localization of Shh-venus in punctate structures and filopodia pose the
question of how Shh-venus is targeted to these domains. I already discussed the
possible association of Shh proteins with raft lipid microdomains. In addition, other
factors besides the lipid modifications of Hh proteins might play a role in targeting. It
is not clear which influence the cytoskeleton has on the observed localization. In
dividing cells an actin ring is present at the site of cytokinesis (Glotzer, 2001) where
Shh-venus accumulates. Furthermore, filopodia have been shown to form upon the
DISCUSSION
102
assembly of actin filaments (Kaverina et al., 2002) and I detected actin in zebrafish
filopodia. Therefore, the localized accumulation of actin might be in part responsible
for Shh-venus localization. This hypothesis might be tested by showing a
colocalization of Shh-venus with actin and by subsequent interfering with the actin
cytoskeleton and determining the changes in Shh-venus localization.
An additional protein, Dispatched, has been shown to be necessary for release of
Shh from producing cells (Burke et al., 1999). Dispatched possesses a sterol sensing
domain and might therefore be associated with lipid rafts. How it exerts its effect on
Hh is not clear, but it is tempting to speculate that targeting of Hh protein to sites of
filopodial formation might depend on Dispatched function. A recently identified
zebrafish dispatched mutant, called con (Brand et al., 1996; Nakano et al., 2004) will
help to clarify the effect of Dispatched function on Shh localization.
Interfering with filopodial formation diminishes Shh signaling
range
The observation that Shh-venus can be found in filopodial extensions present between
cells lead to the prediction that upon interfering with the formation of these filopodia,
I should observe an effect on the signaling range of Shh proteins. To address this
question, I have made use of a DN form of ezrin, which has been shown to disrupt the
formation of filopodia in Drosophila embryos (De Joussineau et al., 2003). In cells
surrounding a clone overexpressing DN-ezrin together with shh, I found a reduced
induction of the Shh target gene ptc1.
This is most probably due to reduced transport of the ligand Shh to the
surrounding tissue. However, I cannot exclude the possibility that the overexpression
of DN-ezrin has an effect on the transcription of shh and thereby lowers the amount of
available Shh ligand. Previous results in zebrafish provided no evidence for reduced
expression of RNAs when coinjected (Schmid et al., 2000). Nevertheless, direct
evidence is needed to address the effect of DN-ezrin on Shh transcription.
Furthermore, direct visualization of altered localization of Shh-venus upon coinjection
with DN-ezrin will help to clarify the effects of disturbed filopodia formation on the
signaling range of Shh.
DISCUSSION
103
Comparison of ext1 expression domains with other species
In order to find genes, which might influence Shh signaling in zebrafish, I cloned and
characterized the expression of HSPG modifying glycosyltransferases belonging to
the Ext1 family. I identified three members of this family, which are expressed in
distinct domains in developing zebrafish embryos.
These expression domains partially contrast with those found in other species.
For instance, the Drosophila EXT1 homolog, ttv is present ubiquitously (The et al.,
1999) and Xenopus EXT1 is expressed without tissue specificity during early
embryogenesis and at adult stages (Katada et al., 2002). Interestingly, the mouse
EXT1 expression pattern is more complex. In early embryonic development (5.5 to
7.5 d.p.c.), EXT1 expression is reported to be ubiquitous (Lin et al., 1998). Later,
transcripts accumulate in limb buds, regions of ossification, tail and brain (Inatani and
Yamaguchi, 2003; Lin et al., 1998; Stickens et al., 2000). As I report for zebrafish
ext1a, expression of mouse Ext1 is strongest in the cerebellum, which continues to
express Ext1 at postnatal stages (Inatani and Yamaguchi, 2003; Rubin et al., 2002). In
addition, the mouse forebrain contains Ext1 transcripts (Inatani and Yamaguchi,
2003). In zebrafish, I find ext1c present in these structures (Fig. 25K arrow, M), while
ext1a expression is lost in forebrain regions (Fig. 25A). Thus, different zebrafish ext1
genes are expressed in structures which both express the single mouse Ext1 gene.
Another site of mouse Ext1 expression is the limb buds, homologous to fin buds
in zebrafish. Here, ext1b and ext1c transcripts can be found, but no ext1a expression,
which might have become dispensable during evolution due to the continued
expression of the other two ext1 genes. From these findings I speculate that the
diversification of ext1 gene expression might provide another example of the
duplication-degeneration-complementation model (Force et al., 1999).
Relationship of ext genes to sites of Shh expression
The expression domains reported for the different ext1 genes suggest a relationship to
Hh signaling in zebrafish. For instance, expression of ext1c in the adaxial cells of the
somitic mesoderm overlaps with the expression of ptc1 (Lewis et al., 1999) and these
cells have been shown to require Hh signaling for their differentiation into muscle
pioneer cells (Blagden et al., 1997). Together with results obtained in Drosophila that
DISCUSSION
104
implicated the ext1 homolog in Hh signaling, it is likely that ext1c expressed by
adaxial cells plays a role in reception and/or restriction of notochord derived Hh
signaling.
However, muscle pioneers are known to exhibit a dynamic migratory behavior
(Cortes et al., 2003), a process for which changes in adhesiveness and therefore
interactions with the extracellular matrix are essential. HSPGs have been shown to
mediate these properties and are therefore thought to play a role in metastasis
(Sanderson, 2001). Thus, differential expression of ext1c in slow muscle precursor
cells might also be responsible for changes in HSPG structure and slow muscle cell
delamination and migration.
In the nervous system, expression of ext1a in the rhombomeres 2 and 4 coincides
with expression of ptc1 (Lewis et al., 1999) and high levels of ext1a can be detected
in the cerebellum, a structure known to be patterned by Shh signals (Rubin et al.,
2002). Furthermore, Shh has been shown to act as a chemoattractant for growing
axons (Charron et al., 2003). Strikingly, ext1a and ext1c are both expressed in regions
of axonal growth, such as the optic stalk, the olfactory bulbs and the ventral
rhombomeres. Therefore, these structures may rely on HSPGs to provide necessary
cues for growing axons.
Zebrafish ext genes respond to Shh signaling
From the observations that ext1 genes in zebrafish are expressed in regions of the
embryo that are patterned by Shh signaling, I asked if ext1 family genes respond to
elevated and reduced levels of this signaling pathway. When overexpressing Shh, I
find that the somitic expression of both ext1a and ext1c was upregulated.
Furthermore, in smu mutant embryos, expression of both genes was strongly reduced.
In contrast to these findings, expression of ext1b was not altered.
I therefore speculate that a putative regulatory link between zebrafish ext1a and
ext1c genes and hh signaling exists during vertebrate somite patterning. Alternatively,
the observed effects may be due to an indirect effect of slow muscle respecification,
which has been shown to occur upon shh overexpression (Blagden et al., 1997). The
finding that ext1b does not respond to elevated or reduced levels of shh signaling
underscores the divergence of different members of the ext1 family in zebrafish.
DISCUSSION
105
Relationship of ext1 gene expression to Fgf signaling
Despite regions of overlapping or adjacent expression between shh and ext1 family
members, differences also exist, especially during early embryonic development.
During early somitogenesis, ext1a is expressed in the prospective hindbrain, the
forebrain and tailbud regions, structures known to be patterned by Fgfs (Reifers et al.,
1998). Therefore, ext1a expression in these embryonic regions might regulate the
production of HSPGs necessary for reception of Fgf8 signaling molecules. This could
be clarified by analyzing the expression of known Fgf8 target genes upon interfering
with ext1 gene function.
Furthermore, Fgf8 has been shown to be expressed in tissues adjacent to muscle
cell pioneers (Reifers et al., 1998) and these cells were shown to be reduced in ace
mutant embryos, which lack Fgf8 signaling. Therefore, muscle pioneer differentiation
depends on Fgf8. This dependency might also rely on proper HSPG synthesis
catalyzed by ext1c.
Morpholino knock-down of zebrafish ext1 genes
The phenotype of ext1a morpholino knock-down embryos morphologically resembled
the u-type mutants, which are characterized by a downward curled body and loss of
chevron-shaped somites (Brand et al., 1996; van Eeden et al., 1996). Many of these
mutants were later characterized to be affected in components of the Hh signaling
pathway (Holley and Nusslein-Volhard, 2000; Nakano et al., 2004; Wolff et al.,
2004). Therefore, I reasoned that in ext1a knock down embryos, Hh signaling would
be compromised. However, muscle specific marker analysis did not reveal changes of
muscle subtype specification. Slow muscle cells and muscle pioneers, both known to
depend on distinct levels of Hh signaling (Wolff et al., 2003) formed normally.
This finding suggests that the curved down body shape is not caused by
interfering with muscle development. Nevertheless, it cannot be excluded that Hh
signaling is compromised in these morpholino injected embryos, because zebrafish
dtr mutants defective for gli1 (Brand et al., 1996; Karlstrom et al., 2003) also show a
curved down body shape, but correct muscle subtype differentiation. Furthermore,
ext1a and ext1c might function in a redundant way during muscle development,
DISCUSSION
106
because of their overlapping somitic expression domains. Most importantly, the
morpholino phenotype needs to be validated, because morpholinos can cause
unspecific phenotypes (Ekker and Larson, 2001). Injection of a second morpholino
did not phenocopy the effects of the first morpholino.
Knock-down of the two other ext1 genes resulted in no visible phenotype in the
case of ext1b and in a brain defect when interfering with ext1c function. This might be
suggestive of redundancy of ext1b with the other ext1 genes. Combinatorial knockdown of various ext1 genes would address this question. For ext1c, it would be
interesting to determine if axonal pathfinding is affected in morpholino injected
embryos. The expression pattern of ext1c and previous results in mouse (Inatani et al.,
2003) suggest a function of ext1c in axon guidance.
Interfering with Rab23 function alters organ situs
The analysis of Rab23 function revealed a role of this GTPase in determining organ
situs of both the heart and the intestine. This suggests that Rab23 functions early in
the pathway specifying the left-right body axis, a finding further supported by the
result that marker gene expression of early left side determinants is randomized. This
is somewhat unexpected, because a mutation in Rab23 in the mouse (Eggenschwiler
et al., 2001) was not reported to display randomized organ situs. In addition, the
mutant phenotype of upregulation of several Shh target genes was not observed in
morpholino injected zebrafish embryos. This might suggest a different action of
Rab23 in zebrafish compared to mouse. There is also the possibility that additional
Rab23 genes exist in zebrafish that might compensate for loss of one Rab23.
Shh has been implicated in determining left-right asymmetry in a variety of
species (Levin, 1997), including zebrafish (Schilling et al., 1999). Therefore, Rab23
might influence early Shh signaling events, such as left-right axis formation, but is
dispensable for later Shh signaling.
107
References
Agathon, A., Thisse, C. and Thisse, B. (2003). The molecular nature of the zebrafish
tail organizer. Nature 424, 448-52.
Agren, M., Kogerman, P., Kleman, M. I., Wessling, M. and Toftgard, R. (2004).
Expression of the PTCH1 tumor suppressor gene is regulated by alternative promoters
and a single functional Gli-binding site. Gene 330, 101-14.
Akimaru, H., Chen, Y., Dai, P., Hou, D. X., Nonaka, M., Smolik, S. M.,
Armstrong, S., Goodman, R. H. and Ishii, S. (1997). Drosophila CBP is a coactivator of cubitus interruptus in hedgehog signalling. Nature 386, 735-8.
Alcedo, J., Ayzenzon, M., Von Ohlen, T., Noll, M. and Hooper, J. E. (1996). The
Drosophila smoothened gene encodes a seven-pass membrane protein, a putative
receptor for the hedgehog signal. Cell 86, 221-32.
Alexandre, C., Lecourtois, M. and Vincent, J. (1999). Wingless and Hedgehog
pattern Drosophila denticle belts by regulating the production of short-range signals.
Development 126, 5689-98.
Aza-Blanc, P., Ramirez-Weber, F. A., Laget, M. P., Schwartz, C. and Kornberg,
T. B. (1997). Proteolysis that is inhibited by hedgehog targets Cubitus interruptus
protein to the nucleus and converts it to a repressor. Cell 89, 1043-53.
Baeg, G. H. and Perrimon, N. (2000). Functional binding of secreted molecules to
heparan sulfate proteoglycans in Drosophila. Curr Opin Cell Biol 12, 575-80.
Barresi, M. J., Stickney, H. L. and Devoto, S. H. (2000). The zebrafish slowmuscle-omitted gene product is required for Hedgehog signal transduction and the
development of slow muscle identity. Development 127, 2189-99.
Barth, K. A. and Wilson, S. W. (1995). Expression of zebrafish nk2.2 is influenced
by sonic hedgehog/vertebrate hedgehog-1 and demarcates a zone of neuronal
differentiation in the embryonic forebrain. Development 121, 1755-68.
Basler, K. and Struhl, G. (1994). Compartment boundaries and the control of
Drosophila limb pattern by hedgehog protein. Nature 368, 208-14.
Bassett, D. and Currie, P. D. (2004). Identification of a zebrafish model of muscular
dystrophy. Clin Exp Pharmacol Physiol 31, 537-40.
Bejsovec, A. and Wieschaus, E. (1995). Signaling activities of the Drosophila
wingless gene are separately mutable and appear to be transduced at the cell surface.
Genetics 139, 309-20.
Bellaiche, Y., The, I. and Perrimon, N. (1998). Tout-velu is a Drosophila
homologue of the putative tumour suppressor EXT-1 and is needed for Hh diffusion.
Nature 394, 85-8.
Bisgrove, B. W., Essner, J. J. and Yost, H. J. (1999). Regulation of midline
development by antagonism of lefty and nodal signaling. Development 126, 3253-62.
Blackhall, F. H., Merry, C. L., Davies, E. J. and Jayson, G. C. (2001). Heparan
sulfate proteoglycans and cancer. Br J Cancer 85, 1094-8.
REFERENCES
108
Blagden, C. S., Currie, P. D., Ingham, P. W. and Hughes, S. M. (1997). Notochord
induction of zebrafish slow muscle mediated by Sonic hedgehog. Genes Dev 11,
2163-75.
Brand, M., Heisenberg, C. P., Warga, R. M., Pelegri, F., Karlstrom, R. O.,
Beuchle, D., Picker, A., Jiang, Y. J., Furutani-Seiki, M., van Eeden, F. J. et al.
(1996). Mutations affecting development of the midline and general body shape
during zebrafish embryogenesis. Development 123, 129-42.
Briscoe, J., Chen, Y., Jessell, T. M. and Struhl, G. (2001). A hedgehog-insensitive
form of patched provides evidence for direct long-range morphogen activity of sonic
hedgehog in the neural tube. Mol Cell 7, 1279-91.
Brown, D. A. and Rose, J. K. (1992). Sorting of GPI-anchored proteins to
glycolipid-enriched membrane subdomains during transport to the apical cell surface.
Cell 68, 533-44.
Bumcrot, D. A., Takada, R. and McMahon, A. P. (1995). Proteolytic processing
yields two secreted forms of sonic hedgehog. Mol Cell Biol 15, 2294-303.
Burke, R., Nellen, D., Bellotto, M., Hafen, E., Senti, K. A., Dickson, B. J. and
Basler, K. (1999). Dispatched, a novel sterol-sensing domain protein dedicated to the
release of cholesterol-modified hedgehog from signaling cells. Cell 99, 803-15.
Buscher, D., Bosse, B., Heymer, J. and Ruther, U. (1997). Evidence for genetic
control of Sonic hedgehog by Gli3 in mouse limb development. Mech Dev 62, 17582.
Capdevila, J. and Izpisua Belmonte, J. C. (2001). Patterning mechanisms
controlling vertebrate limb development. Annu Rev Cell Dev Biol 17, 87-132.
Casali, A. and Struhl, G. (2004). Reading the Hedgehog morphogen gradient by
measuring the ratio of bound to unbound Patched protein. Nature 431, 76-80.
Chamoun, Z., Mann, R. K., Nellen, D., von Kessler, D. P., Bellotto, M., Beachy,
P. A. and Basler, K. (2001). Skinny hedgehog, an acyltransferase required for
palmitoylation and activity of the hedgehog signal. Science 293, 2080-4.
Charron, F., Stein, E., Jeong, J., McMahon, A. P. and Tessier-Lavigne, M.
(2003). The morphogen sonic hedgehog is an axonal chemoattractant that collaborates
with netrin-1 in midline axon guidance. Cell 113, 11-23.
Chen, M. H., Li, Y. J., Kawakami, T., Xu, S. M. and Chuang, P. T. (2004).
Palmitoylation is required for the production of a soluble multimeric Hedgehog
protein complex and long-range signaling in vertebrates. Genes Dev 18, 641-59.
Chen, W., Burgess, S. and Hopkins, N. (2001). Analysis of the zebrafish
smoothened mutant reveals conserved and divergent functions of hedgehog activity.
Development 128, 2385-96.
Chen, Y., Gallaher, N., Goodman, R. H. and Smolik, S. M. (1998). Protein kinase
A directly regulates the activity and proteolysis of cubitus interruptus. Proc Natl Acad
Sci U S A 95, 2349-54.
Chen, Y. and Schier, A. F. (2001). The zebrafish Nodal signal Squint functions as a
morphogen. Nature 411, 607-10.
REFERENCES
109
Chen, Y. and Struhl, G. (1996). Dual roles for patched in sequestering and
transducing Hedgehog. Cell 87, 553-63.
Chiang, C., Litingtung, Y., Lee, E., Young, K. E., Corden, J. L., Westphal, H.
and Beachy, P. A. (1996). Cyclopia and defective axial patterning in mice lacking
Sonic hedgehog gene function. Nature 383, 407-13.
Chin, A. J., Tsang, M. and Weinberg, E. S. (2000). Heart and gut chiralities are
controlled independently from initial heart position in the developing zebrafish. Dev
Biol 227, 403-21.
Chuang, P. T. and McMahon, A. P. (1999). Vertebrate Hedgehog signalling
modulated by induction of a Hedgehog-binding protein. Nature 397, 617-21.
Clines, G. A., Ashley, J. A., Shah, S. and Lovett, M. (1997). The structure of the
human multiple exostoses 2 gene and characterization of homologs in mouse and
Caenorhabditis elegans. Genome Res 7, 359-67.
Concordet, J. P., Lewis, K. E., Moore, J. W., Goodrich, L. V., Johnson, R. L.,
Scott, M. P. and Ingham, P. W. (1996). Spatial regulation of a zebrafish patched
homologue reflects the roles of sonic hedgehog and protein kinase A in neural tube
and somite patterning. Development 122, 2835-46.
Cook, A., Raskind, W., Blanton, S. H., Pauli, R. M., Gregg, R. G., Francomano,
C. A., Puffenberger, E., Conrad, E. U., Schmale, G., Schellenberg, G. et al.
(1993). Genetic heterogeneity in families with hereditary multiple exostoses. Am J
Hum Genet 53, 71-9.
Cortes, F., Daggett, D., Bryson-Richardson, R. J., Neyt, C., Maule, J., Gautier,
P., Hollway, G. E., Keenan, D. and Currie, P. D. (2003). Cadherin-mediated
differential cell adhesion controls slow muscle cell migration in the developing
zebrafish myotome. Dev Cell 5, 865-76.
Cross, G. A. (1990). Glycolipid anchoring of plasma membrane proteins. Annu Rev
Cell Biol 6, 1-39.
Currie, P. D. and Ingham, P. W. (1996). Induction of a specific muscle cell type by
a hedgehog-like protein in zebrafish. Nature 382, 452-5.
Dahmane, N., Lee, J., Robins, P., Heller, P. and Ruiz i Altaba, A. (1997).
Activation of the transcription factor Gli1 and the Sonic hedgehog signalling pathway
in skin tumours. Nature 389, 876-81.
Danielsen, E. M. and van Deurs, B. (1995). A transferrin-like GPI-linked ironbinding protein in detergent-insoluble noncaveolar microdomains at the apical surface
of fetal intestinal epithelial cells. J Cell Biol 131, 939-50.
De Joussineau, C., Soule, J., Martin, M., Anguille, C., Montcourrier, P. and
Alexandre, D. (2003). Delta-promoted filopodia mediate long-range lateral inhibition
in Drosophila. Nature 426, 555-9.
Denef, N., Neubuser, D., Perez, L. and Cohen, S. M. (2000). Hedgehog induces
opposite changes in turnover and subcellular localization of patched and smoothened.
Cell 102, 521-31.
Devine, C. A. and Key, B. (2003). Identifying axon guidance defects in the
embryonic zebrafish brain. Methods Cell Sci 25, 33-7.
REFERENCES
110
Devoto, S. H., Melancon, E., Eisen, J. S. and Westerfield, M. (1996). Identification
of separate slow and fast muscle precursor cells in vivo, prior to somite formation.
Development 122, 3371-80.
Dooley, K. and Zon, L. I. (2000). Zebrafish: a model system for the study of human
disease. Curr Opin Genet Dev 10, 252-6.
Driever, W., Solnica-Krezel, L., Schier, A. F., Neuhauss, S. C., Malicki, J.,
Stemple, D. L., Stainier, D. Y., Zwartkruis, F., Abdelilah, S., Rangini, Z. et al.
(1996). A genetic screen for mutations affecting embryogenesis in zebrafish.
Development 123, 37-46.
Dubois, L., Lecourtois, M., Alexandre, C., Hirst, E. and Vincent, J. P. (2001).
Regulated endocytic routing modulates wingless signaling in Drosophila embryos.
Cell 105, 613-24.
Dubrulle, J. and Pourquie, O. (2004). fgf8 mRNA decay establishes a gradient that
couples axial elongation to patterning in the vertebrate embryo. Nature 427, 419-22.
Eggenschwiler, J. T., Espinoza, E. and Anderson, K. V. (2001). Rab23 is an
essential negative regulator of the mouse Sonic hedgehog signalling pathway. Nature
412, 194-8.
Ekker, S. C. and Larson, J. D. (2001). Morphant technology in model
developmental systems. Genesis 30, 89-93.
Ekker, S. C., Ungar, A. R., Greenstein, P., von Kessler, D. P., Porter, J. A.,
Moon, R. T. and Beachy, P. A. (1995). Patterning activities of vertebrate hedgehog
proteins in the developing eye and brain. Curr Biol 5, 944-55.
Entchev, E. V. and Gonzalez-Gaitan, M. A. (2002). Morphogen gradient formation
and vesicular trafficking. Traffic 3, 98-109.
Entchev, E. V., Schwabedissen, A. and Gonzalez-Gaitan, M. (2000). Gradient
formation of the TGF-beta homolog Dpp. Cell 103, 981-91.
Ericson, J., Briscoe, J., Rashbass, P., van Heyningen, V. and Jessell, T. M. (1997).
Graded sonic hedgehog signaling and the specification of cell fate in the ventral
neural tube. Cold Spring Harb Symp Quant Biol 62, 451-66.
Evans, T. M., Ferguson, C., Wainwright, B. J., Parton, R. G. and Wicking, C.
(2003). Rab23, a negative regulator of hedgehog signaling, localizes to the plasma
membrane and the endocytic pathway. Traffic 4, 869-84.
Fivaz, M. and Meyer, T. (2003). Specific localization and timing in neuronal signal
transduction mediated by protein-lipid interactions. Neuron 40, 319-30.
Fjose, A., Njolstad, P. R., Nornes, S., Molven, A. and Krauss, S. (1992). Structure
and early embryonic expression of the zebrafish engrailed-2 gene. Mech Dev 39, 5162.
Force, A., Lynch, M., Pickett, F. B., Amores, A., Yan, Y. L. and Postlethwait, J.
(1999). Preservation of duplicate genes by complementary, degenerative mutations.
Genetics 151, 1531-45.
Gallet, A., Rodriguez, R., Ruel, L. and Therond, P. P. (2003). Cholesterol
modification of hedgehog is required for trafficking and movement, revealing an
asymmetric cellular response to hedgehog. Dev Cell 4, 191-204.
REFERENCES
111
Gaus, K., Gratton, E., Kable, E. P., Jones, A. S., Gelissen, I., Kritharides, L. and
Jessup, W. (2003). Visualizing lipid structure and raft domains in living cells with
two-photon microscopy. Proc Natl Acad Sci U S A 100, 15554-9.
Gibson, M. C. and Schubiger, G. (2000). Peripodial cells regulate proliferation and
patterning of Drosophila imaginal discs. Cell 103, 343-50.
Gilbert, S. (2003). Developmental Biology: Sinauer Associates, Inc.
Glasgow, E. and Tomarev, S. I. (1998). Restricted expression of the homeobox gene
prox 1 in developing zebrafish. Mech Dev 76, 175-8.
Glickman, N. S. and Yelon, D. (2002). Cardiac development in zebrafish:
coordination of form and function. Semin Cell Dev Biol 13, 507-13.
Glotzer, M. (2001). Animal cell cytokinesis. Annu Rev Cell Dev Biol 17, 351-86.
Goodrich, L. V., Johnson, R. L., Milenkovic, L., McMahon, J. A. and Scott, M. P.
(1996). Conservation of the hedgehog/patched signaling pathway from flies to mice:
induction of a mouse patched gene by Hedgehog. Genes Dev 10, 301-12.
Greco, V., Hannus, M. and Eaton, S. (2001). Argosomes: a potential vehicle for the
spread of morphogens through epithelia. Cell 106, 633-45.
Gritli-Linde, A., Lewis, P., McMahon, A. P. and Linde, A. (2001). The
whereabouts of a morphogen: direct evidence for short- and graded long-range
activity of hedgehog signaling peptides. Dev Biol 236, 364-86.
Grobstein, C. and Dalton, A. J. (1957). Kidney tubule induction in mouse
metanephrogenic mesenchyme without cytoplasmic contact. J Exp Zool 135, 57-73.
Haffter, P., Granato, M., Brand, M., Mullins, M. C., Hammerschmidt, M., Kane,
D. A., Odenthal, J., van Eeden, F. J., Jiang, Y. J., Heisenberg, C. P. et al. (1996).
The identification of genes with unique and essential functions in the development of
the zebrafish, Danio rerio. Development 123, 1-36.
Hahn, H., Christiansen, J., Wicking, C., Zaphiropoulos, P. G., Chidambaram,
A., Gerrard, B., Vorechovsky, I., Bale, A. E., Toftgard, R., Dean, M. et al. (1996).
A mammalian patched homolog is expressed in target tissues of sonic hedgehog and
maps to a region associated with developmental abnormalities. J Biol Chem 271,
12125-8.
Halpern, M. E., Ho, R. K., Walker, C. and Kimmel, C. B. (1993). Induction of
muscle pioneers and floor plate is distinguished by the zebrafish no tail mutation. Cell
75, 99-111.
Han, C., Lin, X., Zhao, C., Opoka, R., Wang, K. and Lin, X. (2001). Identification
of botv, a new segment polarity gene that encodes a protein of the putative tumor
suppressor EXT family. 42nd Annual Drosophila Research Conference (Washington,
D.C., Program No. 411).
Harfe, B. D., Scherz, P. J., Nissim, S., Tian, H., McMahon, A. P. and Tabin, C. J.
(2004). Evidence for an expansion-based temporal Shh gradient in specifying
vertebrate digit identities. Cell 118, 517-28.
Hauptmann, G. and Gerster, T. (1994). Two-color whole-mount in situ
hybridization to vertebrate and Drosophila embryos. Trends Genet 10, 266.
REFERENCES
112
Hauptmann, G. and Gerster, T. (2000). Regulatory gene expression patterns reveal
transverse and longitudinal subdivisions of the embryonic zebrafish forebrain. Mech
Dev 91, 105-18.
Hecht, J. T., Hogue, D., Strong, L. C., Hansen, M. F., Blanton, S. H. and Wagner,
M . (1995). Hereditary multiple exostosis and chondrosarcoma: linkage to
chromosome II and loss of heterozygosity for EXT-linked markers on chromosomes
II and 8. Am J Hum Genet 56, 1125-31.
Higashijima, S., Hotta, Y. and Okamoto, H. (2000). Visualization of cranial motor
neurons in live transgenic zebrafish expressing green fluorescent protein under the
control of the islet-1 promoter/enhancer. J Neurosci 20, 206-18.
Holley, S. A. and Nusslein-Volhard, C. (2000). Somitogenesis in zebrafish. Curr
Top Dev Biol 47, 247-77.
Hooper, J. E. and Scott, M. P. (1989). The Drosophila patched gene encodes a
putative membrane protein required for segmental patterning. Cell 59, 751-65.
Horne-Badovinac, S., Rebagliati, M. and Stainier, D. Y. (2003). A cellular
framework for gut-looping morphogenesis in zebrafish. Science 302, 662-5.
Houart, C., Westerfield, M. and Wilson, S. W. (1998). A small population of
anterior cells patterns the forebrain during zebrafish gastrulation. Nature 391, 788-92.
Huangfu, D., Liu, A., Rakeman, A. S., Murcia, N. S., Niswander, L. and
Anderson, K. V. (2003). Hedgehog signalling in the mouse requires intraflagellar
transport proteins. Nature 426, 83-7.
Iioka, H., Ueno, N. and Kinoshita, N. (2004). Essential role of MARCKS in cortical
actin dynamics during gastrulation movements. J Cell Biol 164, 169-74.
Inatani, M., Irie, F., Plump, A. S., Tessier-Lavigne, M. and Yamaguchi, Y.
(2003). Mammalian brain morphogenesis and midline axon guidance require heparan
sulfate. Science 302, 1044-6.
Inatani, M. and Yamaguchi, Y. (2003). Gene expression of EXT1 and EXT2 during
mouse brain development. Brain Res Dev Brain Res 141, 129-36.
Incardona, J. P., Lee, J. H., Robertson, C. P., Enga, K., Kapur, R. P. and
Roelink, H. (2000). Receptor-mediated endocytosis of soluble and membranetethered Sonic hedgehog by Patched-1. Proc Natl Acad Sci U S A 97, 12044-9.
Ingham, P. W. (1993). Localized hedgehog activity controls spatial limits of
wingless transcription in the Drosophila embryo. Nature 366, 560-2.
Ingham, P. W. and McMahon, A. P. (2001). Hedgehog signaling in animal
development: paradigms and principles. Genes Dev 15, 3059-87.
Ingham, P. W., Taylor, A. M. and Nakano, Y. (1991). Role of the Drosophila
patched gene in positional signalling. Nature 353, 184-7.
Jacinto, A., Wood, W., Woolner, S., Hiley, C., Turner, L., Wilson, C., MartinezArias, A. and Martin, P. (2002). Dynamic analysis of actin cable function during
Drosophila dorsal closure. Curr Biol 12, 1245-50.
Jessell, T. M. (2000). Neuronal specification in the spinal cord: inductive signals and
transcriptional codes. Nat Rev Genet 1, 20-9.
REFERENCES
113
Jia, J., Amanai, K., Wang, G., Tang, J., Wang, B. and Jiang, J. (2002).
Shaggy/GSK3 antagonizes Hedgehog signalling by regulating Cubitus interruptus.
Nature 416, 548-52.
Jia, J., Tong, C. and Jiang, J. (2003). Smoothened transduces Hedgehog signal by
physically interacting with Costal2/Fused complex through its C-terminal tail. Genes
Dev 17, 2709-20.
Jiang, J. and Struhl, G. (1998). Regulation of the Hedgehog and Wingless signalling
pathways by the F-box/WD40-repeat protein Slimb. Nature 391, 493-6.
Johnson, R. L., Grenier, J. K. and Scott, M. P. (1995). patched overexpression
alters wing disc size and pattern: transcriptional and post-transcriptional effects on
hedgehog targets. Development 121, 4161-70.
Karlstrom, R. O., Talbot, W. S. and Schier, A. F. (1999). Comparative synteny
cloning of zebrafish you-too: mutations in the Hedgehog target gli2 affect ventral
forebrain patterning. Genes Dev 13, 388-93.
Karlstrom, R. O., Tyurina, O. V., Kawakami, A., Nishioka, N., Talbot, W. S.,
Sasaki, H. and Schier, A. F. (2003). Genetic analysis of zebrafish gli1 and gli2
reveals divergent requirements for gli genes in vertebrate development. Development
130, 1549-64.
Katada, T., Oogami, S., Shima, N. and Kinoshita, T. (2002). cDNA cloning and
distribution of XEXT1, the Xenopus homologue of EXT1. Dev Genes Evol 212, 24850.
Kaverina, I., Krylyshkina, O. and Small, J. V. (2002). Regulation of substrate
adhesion dynamics during cell motility. Int J Biochem Cell Biol 34, 746-61.
Keller, P., Toomre, D., Diaz, E., White, J. and Simons, K. (2001). Multicolour
imaging of post-Golgi sorting and trafficking in live cells. Nat Cell Biol 3, 140-9.
Keller, R., Davidson, L., Edlund, A., Elul, T., Ezin, M., Shook, D. and Skoglund,
P. (2000). Mechanisms of convergence and extension by cell intercalation. Philos
Trans R Soc Lond B Biol Sci 355, 897-922.
Kerszberg, M. and Wolpert, L. (1998). Mechanisms for positional signalling by
morphogen transport: a theoretical study. J Theor Biol 191, 103-14.
Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B. and Schilling, T. F.
(1995). Stages of embryonic development of the zebrafish. Dev Dyn 203, 253-310.
Kimmel, C. B. and Law, R. D. (1985). Cell lineage of zebrafish blastomeres. I.
Cleavage pattern and cytoplasmic bridges between cells. Dev Biol 108, 78-85.
Kinzler, K. W., Bigner, S. H., Bigner, D. D., Trent, J. M., Law, M. L., O'Brien, S.
J., Wong, A. J. and Vogelstein, B. (1987). Identification of an amplified, highly
expressed gene in a human glioma. Science 236, 70-3.
Kishi, S. (2004). Functional aging and gradual senescence in zebrafish. Ann N Y Acad
Sci 1019, 521-6.
Kjellen, L. and Lindahl, U. (1991). Proteoglycans: structures and interactions. Annu
Rev Biochem 60, 443-75.
Korzh, V. P. (2001). [T-Box genes and developmental decisions that cells make].
Ontogenez 32, 196-203.
REFERENCES
114
Koziel, L., Kunath, M., Kelly, O. G. and Vortkamp, A. (2004). Ext1-dependent
heparan sulfate regulates the range of Ihh signaling during endochondral ossification.
Dev Cell 6, 801-13.
Kramer, K. L. and Yost, H. J. (2003). Heparan sulfate core proteins in cell-cell
signaling. Annu Rev Genet 37, 461-84.
Krauss, S., Concordet, J. P. and Ingham, P. W. (1993). A functionally conserved
homolog of the Drosophila segment polarity gene hh is expressed in tissues with
polarizing activity in zebrafish embryos. Cell 75, 1431-44.
Krauss, S., Johansen, T., Korzh, V. and Fjose, A. (1991). Expression of the
zebrafish paired box gene pax[zf-b] during early neurogenesis. Development 113,
1193-206.
Kruse, K., Pantazis, P., Bollenbach, T., Julicher, F. and Gonzalez-Gaitan, M.
(2004). Dpp gradient formation by dynamin-dependent endocytosis: receptor
trafficking and the diffusion model. Development 131, 4843-56.
Lander, A. D., Nie, Q. and Wan, F. Y. (2002). Do morphogen gradients arise by
diffusion? Dev Cell 2, 785-96.
Lauderdale, J. D., Davis, N. M. and Kuwada, J. Y. (1997). Axon tracts correlate
with netrin-1a expression in the zebrafish embryo. Mol Cell Neurosci 9, 293-313.
Laverriere, A. C., MacNeill, C., Mueller, C., Poelmann, R. E., Burch, J. B. and
Evans, T. (1994). GATA-4/5/6, a subfamily of three transcription factors transcribed
in developing heart and gut. J Biol Chem 269, 23177-84.
Le Merrer, M., Legeai-Mallet, L., Jeannin, P. M., Horsthemke, B., Schinzel, A.,
Plauchu, H., Toutain, A., Achard, F., Munnich, A. and Maroteaux, P. (1994). A
gene for hereditary multiple exostoses maps to chromosome 19p. Hum Mol Genet 3,
717-22.
Lee, J. J., Ekker, S. C., von Kessler, D. P., Porter, J. A., Sun, B. I. and Beachy, P.
A. (1994). Autoproteolysis in hedgehog protein biogenesis. Science 266, 1528-37.
Lee, J. J., von Kessler, D. P., Parks, S. and Beachy, P. A. (1992). Secretion and
localized transcription suggest a role in positional signaling for products of the
segmentation gene hedgehog. Cell 71, 33-50.
Levin, M. (1997). Left-right asymmetry in vertebrate embryogenesis. Bioessays 19,
287-96.
Levin, M. (1998). Left-right asymmetry and the chick embryo. Semin Cell Dev Biol
9, 67-76.
Lewis, K. E., Concordet, J. P. and Ingham, P. W. (1999). Characterisation of a
second patched gene in the zebrafish Danio rerio and the differential response of
patched genes to Hedgehog signalling. Dev Biol 208, 14-29.
Lewis, P. M., Dunn, M. P., McMahon, J. A., Logan, M., Martin, J. F., StJacques, B. and McMahon, A. P. (2001). Cholesterol modification of sonic
hedgehog is required for long-range signaling activity and effective modulation of
signaling by Ptc1. Cell 105, 599-612.
REFERENCES
115
Lin, X., Gan, L., Klein, W. H. and Wells, D. (1998). Expression and functional
analysis of mouse EXT1, a homolog of the human multiple exostoses type 1 gene.
Biochem Biophys Res Commun 248, 738-43.
Lin, X., Wei, G., Shi, Z., Dryer, L., Esko, J. D., Wells, D. E. and Matzuk, M. M.
(2000). Disruption of gastrulation and heparan sulfate biosynthesis in EXT1-deficient
mice. Dev Biol 224, 299-311.
Lin, X. and Wells, D. (1997). Isolation of the mouse cDNA homologous to the
human EXT1 gene responsible for Hereditary Multiple Exostoses. DNA Seq 7, 199202.
Lind, T., Tufaro, F., McCormick, C., Lindahl, U. and Lidholt, K. (1998). The
putative tumor suppressors EXT1 and EXT2 are glycosyltransferases required for the
biosynthesis of heparan sulfate. J Biol Chem 273, 26265-8.
Lopez-Martinez, A., Chang, D. T., Chiang, C., Porter, J. A., Ros, M. A., Simandl,
B. K., Beachy, P. A. and Fallon, J. F. (1995). Limb-patterning activity and restricted
posterior localization of the amino-terminal product of Sonic hedgehog cleavage.
Curr Biol 5, 791-6.
Lum, L. and Beachy, P. A. (2004). The Hedgehog response network: sensors,
switches, and routers. Science 304, 1755-9.
Lum, L., Zhang, C., Oh, S., Mann, R. K., von Kessler, D. P., Taipale, J., WeisGarcia, F., Gong, R., Wang, B. and Beachy, P. A. (2003). Hedgehog signal
transduction via Smoothened association with a cytoplasmic complex scaffolded by
the atypical kinesin, Costal-2. Mol Cell 12, 1261-74.
Macdonald, R., Barth, K. A., Xu, Q., Holder, N., Mikkola, I. and Wilson, S. W.
(1995). Midline signalling is required for Pax gene regulation and patterning of the
eyes. Development 121, 3267-78.
Mann, R. K. and Beachy, P. A. (2004). Novel lipid modifications of secreted protein
signals. Annu Rev Biochem 73, 891-923.
Marigo, V., Davey, R. A., Zuo, Y., Cunningham, J. M. and Tabin, C. J. (1996).
Biochemical evidence that patched is the Hedgehog receptor. Nature 384, 176-9.
Marti, E., Bumcrot, D. A., Takada, R. and McMahon, A. P. (1995). Requirement
of 19K form of Sonic hedgehog for induction of distinct ventral cell types in CNS
explants. Nature 375, 322-5.
Martin, V., Carrillo, G., Torroja, C. and Guerrero, I. (2001). The sterol-sensing
domain of Patched protein seems to control Smoothened activity through Patched
vesicular trafficking. Curr Biol 11, 601-7.
McDowell, N., Gurdon, J. B. and Grainger, D. J. (2001). Formation of a functional
morphogen gradient by a passive process in tissue from the early Xenopus embryo.
Int J Dev Biol 45, 199-207.
McMahon, A. P., Ingham, P. W. and Tabin, C. J. (2003). Developmental roles and
clinical significance of hedgehog signaling. Curr Top Dev Biol 53, 1-114.
Milan, M., Weihe, U., Perez, L. and Cohen, S. M. (2001). The LRR proteins
capricious and Tartan mediate cell interactions during DV boundary formation in the
Drosophila wing. Cell 106, 785-94.
REFERENCES
116
Miller, J. D. and Neely, M. N. (2004). Zebrafish as a model host for streptococcal
pathogenesis. Acta Trop 91, 53-68.
Mohler, J. (1988). Requirements for hedgehod, a segmental polarity gene, in
patterning larval and adult cuticle of Drosophila. Genetics 120, 1061-72.
Muenke, M. and Beachy, P. A. (2000). Genetics of ventral forebrain development
and holoprosencephaly. Curr Opin Genet Dev 10, 262-9.
Muller, F., Chang, B., Albert, S., Fischer, N., Tora, L. and Strahle, U. (1999).
Intronic enhancers control expression of zebrafish sonic hedgehog in floor plate and
notochord. Development 126, 2103-16.
Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba, K. and Miyawaki, A.
(2002). A variant of yellow fluorescent protein with fast and efficient maturation for
cell-biological applications. Nat Biotechnol 20, 87-90.
Nakano, Y., Guerrero, I., Hidalgo, A., Taylor, A., Whittle, J. R. and Ingham, P.
W. (1989). A protein with several possible membrane-spanning domains encoded by
the Drosophila segment polarity gene patched. Nature 341, 508-13.
Nakano, Y., Kim, H. R., Kawakami, A., Roy, S., Schier, A. F. and Ingham, P. W.
(2004). Inactivation of dispatched 1 by the chameleon mutation disrupts Hedgehog
signalling in the zebrafish embryo. Dev Biol 269, 381-92.
Nasevicius, A. and Ekker, S. C. (2000). Effective targeted gene 'knockdown' in
zebrafish. Nat Genet 26, 216-20.
Niehrs, C. (2004). Regionally specific induction by the Spemann-Mangold organizer.
Nat Rev Genet 5, 425-34.
Nusslein-Volhard, C. and Wieschaus, E. (1980). Mutations affecting segment
number and polarity in Drosophila. Nature 287, 795-801.
Nybakken, K. and Perrimon, N. (2002). Heparan sulfate proteoglycan modulation
of developmental signaling in Drosophila. Biochim Biophys Acta 1573, 280-91.
Ogden, S. K., Ascano, M., Jr., Stegman, M. A., Suber, L. M., Hooper, J. E. and
Robbins, D. J. (2003). Identification of a functional interaction between the
transmembrane protein Smoothened and the kinesin-related protein Costal2. Curr Biol
13, 1998-2003.
Orenic, T., Chidsey, J. and Holmgren, R. (1987). Cell and cubitus interruptus
dominant: two segment polarity genes on the fourth chromosome in Drosophila. Dev
Biol 124, 50-6.
Patel, N. H., Martin-Blanco, E., Coleman, K. G., Poole, S. J., Ellis, M. C.,
Kornberg, T. B. and Goodman, C. S. (1989). Expression of engrailed proteins in
arthropods, annelids, and chordates. Cell 58, 955-68.
Pepinsky, R. B., Zeng, C., Wen, D., Rayhorn, P., Baker, D. P., Williams, K. P.,
Bixler, S. A., Ambrose, C. M., Garber, E. A., Miatkowski, K. et al. (1998).
Identification of a palmitic acid-modified form of human Sonic hedgehog. J Biol
Chem 273, 14037-45.
Pfeiffer, S., Alexandre, C., Calleja, M. and Vincent, J. P. (2000). The progeny of
wingless-expressing cells deliver the signal at a distance in Drosophila embryos. Curr
Biol 10, 321-4.
REFERENCES
117
Porter, J. A., Ekker, S. C., Park, W. J., von Kessler, D. P., Young, K. E., Chen,
C. H., Ma, Y., Woods, A. S., Cotter, R. J., Koonin, E. V. et al. (1996a). Hedgehog
patterning activity: role of a lipophilic modification mediated by the carboxy-terminal
autoprocessing domain. Cell 86, 21-34.
Porter, J. A., von Kessler, D. P., Ekker, S. C., Young, K. E., Lee, J. J., Moses, K.
and Beachy, P. A. (1995). The product of hedgehog autoproteolytic cleavage active
in local and long-range signalling. Nature 374, 363-6.
Porter, J. A., Young, K. E. and Beachy, P. A. (1996b). Cholesterol modification of
hedgehog signaling proteins in animal development. Science 274, 255-9.
Portin, P. (2002). General outlines of the molecular genetics of the Notch signalling
pathway in Drosophila melanogaster: a review. Hereditas 136, 89-96.
Pralle, A., Keller, P., Florin, E. L., Simons, K. and Horber, J. K. (2000).
Sphingolipid-cholesterol rafts diffuse as small entities in the plasma membrane of
mammalian cells. J Cell Biol 148, 997-1008.
Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G. and Cormier,
M. J. (1992). Primary structure of the Aequorea victoria green-fluorescent protein.
Gene 111, 229-33.
Preat, T., Therond, P., Limbourg-Bouchon, B., Pham, A., Tricoire, H., Busson,
D. and Lamour-Isnard, C. (1993). Segmental polarity in Drosophila melanogaster:
genetic dissection of fused in a Suppressor of fused background reveals interaction
with costal-2. Genetics 135, 1047-62.
Price, M. A. and Kalderon, D. (2002). Proteolysis of the Hedgehog signaling
effector Cubitus interruptus requires phosphorylation by Glycogen Synthase Kinase 3
and Casein Kinase 1. Cell 108, 823-35.
Puschel, A. W., Gruss, P. and Westerfield, M. (1992). Sequence and expression
pattern of pax-6 are highly conserved between zebrafish and mice. Development 114,
643-51.
Raible, F. and Brand, M. (2004). Divide et Impera-the midbrain-hindbrain boundary
and its organizer. Trends Neurosc in press.
Ramirez-Weber, F. A. and Kornberg, T. B. (1999). Cytonemes: cellular processes
that project to the principal signaling center in Drosophila imaginal discs. Cell 97,
599-607.
Ramirez-Weber, F. A. and Kornberg, T. B. (2000). Signaling reaches to new
dimensions in Drosophila imaginal discs. Cell 103, 189-92.
Reifers, F., Bohli, H., Walsh, E. C., Crossley, P. H., Stainier, D. Y. and Brand, M.
(1998). Fgf8 is mutated in zebrafish acerebellar (ace) mutants and is required for
maintenance of midbrain-hindbrain boundary development and somitogenesis.
Development 125, 2381-95.
Reiter, J. F., Alexander, J., Rodaway, A., Yelon, D., Patient, R., Holder, N. and
Stainier, D. Y. (1999). Gata5 is required for the development of the heart and
endoderm in zebrafish. Genes Dev 13, 2983-95.
Rhinn, M. and Brand, M. (2001). The midbrain--hindbrain boundary organizer.
Curr Opin Neurobiol 11, 34-42.
REFERENCES
118
Rhinn, M., Lun, K., Amores, A., Yan, Y. L., Postlethwait, J. H. and Brand, M.
(2003). Cloning, expression and relationship of zebrafish gbx1 and gbx2 genes to Fgf
signaling. Mech Dev 120, 919-36.
Robbins, D. J., Nybakken, K. E., Kobayashi, R., Sisson, J. C., Bishop, J. M. and
Therond, P. P. (1997). Hedgehog elicits signal transduction by means of a large
complex containing the kinesin-related protein costal2. Cell 90, 225-34.
Roelink, H., Porter, J. A., Chiang, C., Tanabe, Y., Chang, D. T., Beachy, P. A.
and Jessell, T. M. (1995). Floor plate and motor neuron induction by different
concentrations of the amino-terminal cleavage product of sonic hedgehog
autoproteolysis. Cell 81, 445-55.
Roessler, E. and Muenke, M. (2003). How a Hedgehog might see
holoprosencephaly. Hum Mol Genet 12 Spec No 1, R15-25.
Roy, S., Wolff, C. and Ingham, P. W. (2001). The u-boot mutation identifies a
Hedgehog-regulated myogenic switch for fiber-type diversification in the zebrafish
embryo. Genes Dev 15, 1563-76.
Rubin, J. B., Choi, Y. and Segal, R. A. (2002). Cerebellar proteoglycans regulate
sonic hedgehog responses during development. Development 129, 2223-32.
Ruiz i Altaba, A. (1998). Combinatorial Gli gene function in floor plate and neuronal
inductions by Sonic hedgehog. Development 125, 2203-12.
Ruiz i Altaba, A. (1999). Gli proteins encode context-dependent positive and
negative functions: implications for development and disease. Development 126,
3205-16.
Ruiz i Altaba, A., Sanchez, P. and Dahmane, N. (2002). Gli and hedgehog in
cancer: tumours, embryos and stem cells. Nat Rev Cancer 2, 361-72.
Rustom, A., Saffrich, R., Markovic, I., Walther, P. and Gerdes, H. H. (2004).
Nanotubular highways for intercellular organelle transport. Science 303, 1007-10.
Sanderson, R. D. (2001). Heparan sulfate proteoglycans in invasion and metastasis.
Semin Cell Dev Biol 12, 89-98.
Sargiacomo, M., Sudol, M., Tang, Z. and Lisanti, M. P. (1993). Signal transducing
molecules and glycosyl-phosphatidylinositol-linked proteins form a caveolin-rich
insoluble complex in MDCK cells. J Cell Biol 122, 789-807.
Sato, M. and Kornberg, T. B. (2002). FGF is an essential mitogen and
chemoattractant for the air sacs of the drosophila tracheal system. Dev Cell 3, 195207.
Schauerte, H. E., van Eeden, F. J., Fricke, C., Odenthal, J., Strahle, U. and
Haffter, P. (1998). Sonic hedgehog is not required for the induction of medial floor
plate cells in the zebrafish. Development 125, 2983-93.
Schilling, T. F., Concordet, J. P. and Ingham, P. W. (1999). Regulation of leftright asymmetries in the zebrafish by Shh and BMP4. Dev Biol 210, 277-87.
Schmid, B., Furthauer, M., Connors, S. A., Trout, J., Thisse, B., Thisse, C. and
Mullins, M. C. (2000). Equivalent genetic roles for bmp7/snailhouse and
bmp2b/swirl in dorsoventral pattern formation. Development 127, 957-67.
REFERENCES
119
Scholpp, S. and Brand, M. (2004). Endocytosis controls spreading and effective
signalling range of Fgf8 protein. Curr Biol in press.
Schutz, G. J., Kada, G., Pastushenko, V. P. and Schindler, H. (2000). Properties of
lipid microdomains in a muscle cell membrane visualized by single molecule
microscopy. Embo J 19, 892-901.
Sekimizu, K., Nishioka, N., Sasaki, H., Takeda, H., Karlstrom, R. O. and
Kawakami, A. (2004). The zebrafish iguana locus encodes Dzip1, a novel zinc-finger
protein required for proper regulation of Hedgehog signaling. Development 131,
2521-32.
Shafizadeh, E. and Paw, B. H. (2004). Zebrafish as a model of human hematologic
disorders. Curr Opin Hematol 11, 255-61.
Shih, J. and Fraser, S. E. (1996). Characterizing the zebrafish organizer:
microsurgical analysis at the early-shield stage. Development 122, 1313-22.
Shimamura, K. and Rubenstein, J. L. (1997). Inductive interactions direct early
regionalization of the mouse forebrain. Development 124, 2709-18.
Siekmann, A. F. and Brand, M. (2004). Distinct tissue-specificity of three zebrafish
ext1 genes encoding proteoglycan modifying enzymes and their relationship to
somitic Sonic Hedgehog signaling. Dev Dyn in press.
Simons, K. and Ikonen, E. (1997). Functional rafts in cell membranes. Nature 387,
569-72.
Sisson, J. C., Ho, K. S., Suyama, K. and Scott, M. P. (1997). Costal2, a novel
kinesin-related protein in the Hedgehog signaling pathway. Cell 90, 235-45.
Spemann, H. (1938). Embryonic development and induction. New Haven
Connecticut: Yale University Press.
Stainier, D. Y., Lee, R. K. and Fishman, M. C. (1993). Cardiovascular development
in the zebrafish. I. Myocardial fate map and heart tube formation. Development 119,
31-40.
Stickens, D., Brown, D. and Evans, G. A. (2000). EXT genes are differentially
expressed in bone and cartilage during mouse embryogenesis. Dev Dyn 218, 452-64.
Stickens, D. and Evans, G. A. (1997). Isolation and characterization of the murine
homolog of the human EXT2 multiple exostoses gene. Biochem Mol Med 61, 16-21.
Stone, D. M., Hynes, M., Armanini, M., Swanson, T. A., Gu, Q., Johnson, R. L.,
Scott, M. P., Pennica, D., Goddard, A., Phillips, H. et al. (1996). The tumoursuppressor gene patched encodes a candidate receptor for Sonic hedgehog. Nature
384, 129-34.
Strahle, U., Blader, P., Henrique, D. and Ingham, P. W. (1993). Axial, a zebrafish
gene expressed along the developing body axis, shows altered expression in cyclops
mutant embryos. Genes Dev 7, 1436-46.
Strigini, M. and Cohen, S. M. (1997). A Hedgehog activity gradient contributes to
AP axial patterning of the Drosophila wing. Development 124, 4697-705.
Strigini, M. and Cohen, S. M. (2000). Wingless gradient formation in the
Drosophila wing. Curr Biol 10, 293-300.
REFERENCES
120
Strutt, H., Thomas, C., Nakano, Y., Stark, D., Neave, B., Taylor, A. M. and
Ingham, P. W. (2001). Mutations in the sterol-sensing domain of Patched suggest a
role for vesicular trafficking in Smoothened regulation. Curr Biol 11, 608-13.
Summerbell, D., Lewis, J. H. and Wolpert, L. (1973). Positional information in
chick limb morphogenesis. Nature 244, 492-6.
Tabata, T., Eaton, S. and Kornberg, T. B. (1992). The Drosophila hedgehog gene
is expressed specifically in posterior compartment cells and is a target of engrailed
regulation. Genes Dev 6, 2635-45.
Tabata, T. and Kornberg, T. B. (1994). Hedgehog is a signaling protein with a key
role in patterning Drosophila imaginal discs. Cell 76, 89-102.
Tabata, T. and Takei, Y. (2004). Morphogens, their identification and regulation.
Development 131, 703-12.
Taipale, J., Cooper, M. K., Maiti, T. and Beachy, P. A. (2002). Patched acts
catalytically to suppress the activity of Smoothened. Nature 418, 892-7.
Talbot, W. S., Trevarrow, B., Halpern, M. E., Melby, A. E., Farr, G.,
Postlethwait, J. H., Jowett, T., Kimmel, C. B. and Kimelman, D. (1995). A
homeobox gene essential for zebrafish notochord development. Nature 378, 150-7.
Tanabe, Y. and Jessell, T. M. (1996). Diversity and pattern in the developing spinal
cord. Science 274, 1115-23.
Taylor, A. M., Nakano, Y., Mohler, J. and Ingham, P. W. (1993). Contrasting
distributions of patched and hedgehog proteins in the Drosophila embryo. Mech Dev
42, 89-96.
Teleman, A. A. and Cohen, S. M. (2000). Dpp gradient formation in the Drosophila
wing imaginal disc. Cell 103, 971-80.
The, I., Bellaiche, Y. and Perrimon, N. (1999). Hedgehog movement is regulated
through tout velu-dependent synthesis of a heparan sulfate proteoglycan. Mol Cell 4,
633-9.
Therond, P., Busson, D., Guillemet, E., Limbourg-Bouchon, B., Preat, T.,
Terracol, R., Tricoire, H. and Lamour-Isnard, C. (1993). Molecular organisation
and expression pattern of the segment polarity gene fused of Drosophila
melanogaster. Mech Dev 44, 65-80.
Torroja, C., Gorfinkiel, N. and Guerrero, I. (2004). Patched controls the Hedgehog
gradient by endocytosis in a dynamin-dependent manner, but this internalization does
not play a major role in signal transduction. Development 131, 2395-408.
Turing, A. M. (1952). The chemical basis of morphogenesis. Philos. Trans. R. Soc.
London 237, 37-72.
Udvadia, A. J., Koster, R. W. and Skene, J. H. (2001). GAP-43 promoter elements
in transgenic zebrafish reveal a difference in signals for axon growth during CNS
development and regeneration. Development 128, 1175-82.
Ulrich, F., Concha, M. L., Heid, P. J., Voss, E., Witzel, S., Roehl, H., Tada, M.,
Wilson, S. W., Adams, R. J., Soll, D. R. et al. (2003). Slb/Wnt11 controls hypoblast
cell migration and morphogenesis at the onset of zebrafish gastrulation. Development
130, 5375-84.
REFERENCES
121
Vacquier, V. D. (1968). The connection of blastomeres of sea urchin embryos by
filopodia. Exp Cell Res 52, 571-81.
van Den Heuvel, M. (2001). Fat hedgehogs, slower or richer? Sci STKE 2001, PE31.
van den Heuvel, M. and Ingham, P. W. (1996). smoothened encodes a receptor-like
serpentine protein required for hedgehog signalling. Nature 382, 547-51.
van Eeden, F. J., Granato, M., Schach, U., Brand, M., Furutani-Seiki, M.,
Haffter, P., Hammerschmidt, M., Heisenberg, C. P., Jiang, Y. J., Kane, D. A. et
al. (1996). Mutations affecting somite formation and patterning in the zebrafish,
Danio rerio. Development 123, 153-64.
Van Hul, W., Wuyts, W., Hendrickx, J., Speleman, F., Wauters, J., De Boulle, K.,
Van Roy, N., Bossuyt, P. and Willems, P. J. (1998). Identification of a third EXTlike gene (EXTL3) belonging to the EXT gene family. Genomics 47, 230-7.
Varga, Z. M., Amores, A., Lewis, K. E., Yan, Y. L., Postlethwait, J. H., Eisen, J.
S. and Westerfield, M. (2001). Zebrafish smoothened functions in ventral neural
tube specification and axon tract formation. Development 128, 3497-509.
Vincent, S., Thomas, A., Brasher, B. and Benson, J. D. (2003). Targeting of
proteins to membranes through hedgehog auto-processing. Nat Biotechnol 21, 936-40.
Wei, G., Bai, X., Gabb, M. M., Bame, K. J., Koshy, T. I., Spear, P. G. and Esko,
J. D. (2000). Location of the glucuronosyltransferase domain in the heparan sulfate
copolymerase EXT1 by analysis of Chinese hamster ovary cell mutants. J Biol Chem
275, 27733-40.
Wienholds, E., van Eeden, F., Kosters, M., Mudde, J., Plasterk, R. H. and
Cuppen, E. (2003). Efficient target-selected mutagenesis in zebrafish. Genome Res
13, 2700-7.
Wise, C. A., Clines, G. A., Massa, H., Trask, B. J. and Lovett, M. (1997).
Identification and localization of the gene for EXTL, a third member of the multiple
exostoses gene family. Genome Res 7, 10-6.
Wolff, C., Roy, S. and Ingham, P. W. (2003). Multiple muscle cell identities
induced by distinct levels and timing of hedgehog activity in the zebrafish embryo.
Curr Biol 13, 1169-81.
Wolff, C., Roy, S., Lewis, K. E., Schauerte, H., Joerg-Rauch, G., Kirn, A.,
Weiler, C., Geisler, R., Haffter, P. and Ingham, P. W. (2004). iguana encodes a
novel zinc-finger protein with coiled-coil domains essential for Hedgehog signal
transduction in the zebrafish embryo. Genes Dev 18, 1565-76.
Wolpert, L. (1969). Positional information and the spatial pattern of cellular
differentiation. J Theor Biol 25, 1-47.
Wurst, W. and Bally-Cuif, L. (2001). Neural plate patterning: upstream and
downstream of the isthmic organizer. Nat Rev Neurosci 2, 99-108.
Wuyts, W., Van Hul, W., Hendrickx, J., Speleman, F., Wauters, J., De Boulle, K.,
Van Roy, N., Van Agtmael, T., Bossuyt, P. and Willems, P. J. (1997).
Identification and characterization of a novel member of the EXT gene family,
EXTL2. Eur J Hum Genet 5, 382-9.
REFERENCES
122
Yelon, D. (2001). Cardiac patterning and morphogenesis in zebrafish. Dev Dyn 222,
552-63.
Zak, B. M., Crawford, B. E. and Esko, J. D. (2002). Hereditary multiple exostoses
and heparan sulfate polymerization. Biochim Biophys Acta 1573, 346-55.
Zeng, X., Goetz, J. A., Suber, L. M., Scott, W. J., Jr., Schreiner, C. M. and
Robbins, D. J. (2001). A freely diffusible form of Sonic hedgehog mediates longrange signalling. Nature 411, 716-20.
Zerial, M. and McBride, H. (2001). Rab proteins as membrane organizers. Nat Rev
Mol Cell Biol 2, 107-17.
Zhang, Y., Zhao, X., Hu, Y., St Amand, T., Zhang, M., Ramamurthy, R., Qiu, M.
and Chen, Y. (1999). Msx1 is required for the induction of Patched by Sonic
hedgehog in the mammalian tooth germ. Dev Dyn 215, 45-53.
123
List of publications
A part of the data presented in this thesis has been published:
Siekmann, A.F. and Brand, M. (2004). Distinct tissue-specificity of three zebrafish
ext1 genes encoding proteoglycan modifying enzymes and their relationship to
somitic Sonic Hedgehog signaling. Developmental Dynamics, in press
Two other publications describing the localization of Shh proteins in gastrulating
embryos and the involvement of Rab23 in determining the left-right body axis are in
preparation.
124
Hiermit versichere ich, dass ich die vorliegende Arbeit ohne unzulässige Hilfe Dritter
und ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe; die aus
fremden Quellen direkt oder indirekt übernommenen Gedanken sind als solche
kenntlich gemacht. Die Arbeit wurde weder im Inland noch im Ausland in gleicher
oder ähnlicher Form einer anderen Prüfungsbehörde vorgelegt.
Dresden, den 30.09.2004
(Arndt Siekmann)