Identification of novel regulators of neuronal programmed cell death

The Institute of Anatomy and Cell Biology
Department of Molecular Embryology
Albert-Ludwigs University, Freiburg
Identification of novel regulators of neuronal programmed cell death
Dissertation submitted to
the Faculty of Biology
Albert-Ludwigs University, Freiburg im Breisgau
to obtain the degree of Doctor rerum naturalium
Presented by
Judith Sophie Koszinowski
Supervisor: Prof. Dr. Kerstin Krieglstein
Freiburg, 2014
http://d-nb.info/1064672795
Table of contents
1
Introduction
1.1
Programmed Cell Death during Development
9
1.1.1
The discovery of genes involved in Programmed Cell Death
1.1.2
Vertebrate Programmed Cell Death
10
a)
The extrinsic apoptotic pathway
12
b)
The intrinsic apoptotic pathway
12
Autophagy
13
1.1.3
2
9
9
1.2
Retinoic acid in the regulation of cell death and differentiation
14
1.3
The chicken embryo as a model organism in developmental studies
18
1.3.1
Development of the chicken embryo
18
1.3.2
The avian ciliary ganglion
19
1.3.3
Development of the avian ciliary ganglion
21
1.3.4
Programmed cell death during ciliary ganglion development
21
a)
Synapse formation and cell death in the ciliary ganglion
21
b)
Trophic factors and their effects on neuronal survival and differentiation
22
c)
The role of TGFp in neuronal survival and death
25
1.4
RCAS vectors in the study of chicken development
26
1.5
Aim of the study
29
Materials and Methods
2.1
30
Materials
30
2.1.1
Chemicals and reagents
30
2.1.2
Solutions and buffers
32
2.1.3
Kits
2.1.4
Vectors
33
2.1.5
Cells
33
2.1.6
Polymerases
33
2.1.7
Restriction enzymes
33
2.1.8
Antibodies
34
2.1.9
Equipment
34
2.1.10
Consumables
35
2.1.11
Software
35
2.2
Methods
2.2.1
Embryos..
!
33
36
36
2.2.2
Cell counts during programmed cell death in the chicken ciliary ganglion....... 36
2.2.3
Hematoxylin and eosin staining
36
2.2.4
Dissection of chicken ciliary ganglia for the microarray
36
2.2.5
RNA extraction for the microarray
37
2.2.6
Gene expression analysis
38
2.2.7
Quantitative real-time PCR (qPCR)
38
a)
RNA extraction for qP.CR
38
b)
Reverse Transcription of RNA
39
c)
Primers for qPCR
40
d)
Primer quality test
41
e)
Primer efficiency tests
f)
Validation of gene expression by qPCR
42
In situ hybridization
43
a)
In situ hybridization probe design
43
b)
In situ probe PCR
43
c)
DNA Gel Extraction
45
d)
A-addition
45
e)
Ligation
46
f)
Transformation of competent cells
46
g)
Bacterial culture and Mini-Prep
46
h)
Sequencing
47
2.2.8
'.
42
".
i) Glycerol stocks
47
j) Linearization of plasmids
47
k)
48
1)
. m)
Purification of linearized plasmids
In vitro transcription of linearized plasmids
48
In situ hybridization
49
2.2.9
Culture of DF-1 cells
50
2.2.10
Verification of BID and RARB expression in DF-1 cells
51
2.2.11
siRNA experiments
51
a)
siRNA design
51
b)
Splitting of DF-1 cells for siRNA experiments
52
c)
Transfection of DF-1 cells with siRNA
d)
Fixation of cells
53
e)
RNA extraction from DF-1 cells
53
:
52
f)
qPCR of BID and RARB expression in DF-1 cells
53
In vivo shRNA knockdown of candidate genes
54
a)
shRNA oligo design and PCR
54
b)
Ligation of shRNA oligos into pRFPRNAiC
56
c)
Cloning of the MOEC into RCASB
57
d)
Transfection of DF-1 cells with RCAS virus
57
e)
Concentration of RCAS virus
58
f)
Infection of chicken embryos with RCAS virus
58
2.2.13
Bromodeoxyuridine (BrdU) injection
58
2.2.14
Immunohistochemistry
59
2.2.15
Cell counts of infected ganglia
59
2.2.16
Statistical procedures
60
2.2.12
Results
61
3.1
Neuron numbers during programmed cell death in the chicken ciliary ganglion
61
3.2
Gene expression data analysis
62
3.3
Validation of candidate gene expression in the ciliary ganglion by qPCR
68
3.4
Validation of transcript localization to the ciliary ganglion
70
3.5
siRNA-mediated knockdown of BID and RARB in DF-1 cell culture
72
3.6
Transfection of DF-1 cells with RCAS virus
74
3.7
In vivo shRNA-knockdown of candidate genes
76
3.7.1
Construction and injection of shRNA-expressing virus
76
3.7.2
Validation of successful virus-infection of the ciliary ganglion
77
3.7.3
Ciliary ganglion neuron counts of RCASBshRNA injected embryos
77
3.7.4
Investigation of developmental alterations after BID knockdown
78
a)
Decrease in neuronal apoptosis after BID knockdown
79
b)
Proliferation in the CG after BID knockdown
80
Investigation of developmental alterations after ACVR2B knockdown
81
a)
Decrease in neuronal apoptosis after ACVR2B knockdown
81
b)
Proliferation in the CG after ACVR2B knockdown
82
c)
Differentiation-related changes after ACVR2B knockdown
84
Investigation of developmental alterations after RARB knockdown
85
a)
Morphological abnormalities after RARB knockdown
85
b)
Delay in neuronal apoptosis after RARB knockdown
87
c)
Proliferation in the CG after RARB knockdown
88
3.7.5
3.7.6
4
d)
Increased Cash1-expression in E14 ciliary ganglia after RARB knockdown.... 90
e)
ChAT-expression after RARB knockdown
91
f)
Decrease in somatostatin-like immunoreactivity after RARB knockdown
92
Discussion
4.1
94
Differential gene expression in the chicken CG during programmed cell death
4.1.1
94
Analysis of gene expression
94
a)
Microarray setup
94
b)
Gene expression analysis
96
c)
Genes selected for further analysis
98
4.1.2
Validation of gene expression regulation
100
4.1.3
Validation of transcript expression levels and neuronal localization
101
4.2
In vitro siRNA-mediated knockdown of BID and RARB
102
4.3
Design and evaluation of the RCAS RNAi approach
102
4.4
Effects of candidate gene knockdown in vivo
104
4.4.1
Knockdown of ATG2B has no effect on CG neuron number....
104
4.4.2
Knockdown of BCL-XL has no effect on CG neuron number
104
4.4.3
BID knockdown inhibits neuronal PCD
105
4.4.4
ACVR2B knockdown affects CG neuron differentiation and cell death
106
4.4.5
RARB knockdown leads to a delay in cell death
109
4.4.6
RARB knockdown affects neuronal differentiation in the CG
110
5
Conclusions
114
6
Summaiy
115
7
References
117
8
Appendix
128
9
Acknowledgements
i
129