Dorso-ventral differentiation and specification of mesencephalon in early chick embryos [Elektronische Ressource] / vorgelegt von Naixin Li

Dorso-ventral Differentiation and Specification
of Mesencephalon in Early Chick Embryos











Dissertation zur Erlangung des
naturwissenschaftlichen Doktorgrades
der Bayerischen Julius-Maximilians-Universität Würzburg







vorgelegt von


Naixin Li

aus Tianjin, V. R. China




Würzburg, 2007





























Eingereicht am: ..................................................................................................14. June 2007


Mitglieder der Promotionskommission:

Vorsitzender: ..........................................................................................Prof. Dr. Martin Müller

Gutachter: Prof. Dr. Martin Heisenberg

Gutachter: Dr. Andrea Wizenmann

Tag des Promotionskolloquiums: .................................................................................................

Doktorurkunde ausgehändigt am: ................................................................................................









To My Parents
















































Table of Contents
Introduction 1

Early development of chick central nervous system..………………...….. 1
The formation of the neural tube……...…………....……………... 1
The differentiation of the neural tube.……….……..……………... 2
The development of the three embryonic axes....…..……………... 3
Regionalization of the developing CNS.………..…...……….…… 5
Neuronal migration during neurogenesis…….….……….……..… 7
Dorso-ventral patterning of the central nervous system.....……...….….… 8
Determining dorso-ventral cell types in the spinal cord…….…..… 8
The paired-box transcription factor Pax7..…………….….….…… 11
Shh in the development of central nervous system.…………….… 13
A small GTP-binding protein Rab23…………….…..……...…….. 15
Methods of gene repression in chick embryos...……………….…....…… 16
RNA interference……………………...……….…………….…… 16
Morpholino oligonucleotides……………………………………… 19
Why still working with chick embryos?..………………………………... 20
Aim of the project……………………………………...………………… 21
Materials 23

Avian embryos……………………………………………………….…… 23
Bacterial Strains……...…………………………………………………… 23
Clones…………………………………………………………………….. 24
Plasmids………………..…………………………………………………. 25
Morpholinos………………………………………………………….…… 26
Oligonucleotides………………………………………………………….. 27
Size Standard.…..………………………………………………………… 28
Enzymes…………………………………………………………………... 29
Fluorescent Probes…….………………...………………………………... 30
Chemicals…………………………………………………………….…… 30
Reagents……………………………………………………..…….. 30
Solutions, Media and Buffers………………………………..……. 32
Kits…………………………………………………………..…….. 35
Antibodies………………………………………………………………… 35
Antibodies for Immunohistochemistry……..………..……………. 35 or RNA in situ hybridisation…..……...…….……….. 36
Genes for RNA probes…………………………………………………… 36
Apparatus and Instruments……………………………………………….. 37
Softwares and Websites…………………………...……………………… 38

i Table of Contents
Methods 39

1. Embryo and tissue preparations.……………..………………………... 39
1.1. in vivo manipulations…...…….……………...………………… 39
Transplantation….………….………………..……………… 39
In ovo electroporation……………..………….…..…………. 41
1.2. in vitro manipulations………….……………………………….. 43
Co-culture………………….….…………..………………… 43
in vitro aggregation assay……………….……….....……….. 43

2. Molecular Techniques……………………...………………………….. 44
2.1. Isolation and determination of plasmid DNA…………………... 44
Mini preparation of plasmid DNA………..………..………... 44
Midi/Maxi preparation of plasmid DNA………..………..….. 44
Endotoxin-free plasmid extraction…………………………... 45
DNA concentration………………………………………….. 45
Agarose gel electrophoresis…………….…………………… 45
Gel extraction of DNA……….……..……………………….. 45
DNA quantitation………………….....……………………… 45
2.2. Isolation and cloning of embryogenetic genes & fragments…..... 46
Isolation of total RNA..……………………………………… 46
RT-PCR…………………………….……………….……….. 46
Polymerase chain reaction..…………………………………. 47
Restrictive digestion.…….………………….….……………. 48
Dephosphorylation of linearized plasmid DNA…………….. 48
Ligation……………….…………………………….……….. 49
Preparation of competent cells……...……………………….. 49
Transformation……….……………………………………… 49
Selection of colonies……………….……………….……….. 50
Bacterial culture...………………….………………………... 50
Glycerol stocks………………………………………………. 50
DNA sequencing………...………….……………………….. 50
2.3. In vitro transcription…………….……………………………… 51
Linearisation of the plasmid………………………………… 51
Phenol/chloroform extraction…………...………..…………. 51
DNA precipitation…………………………………………… 51
Synthesis of RNA-labelled probes...………………………… 51
RNA precipitation………………...…..…………….……….. 52

3. Histological examinations……………………………………………… 52
Dissection, fixation and mounting…………...…..………….. 52
Whole-mount immunostaining…….………………………… 52ount RNA in situ hybridization………………...…. 54
Vibratome section…………….…………………….……….. 55
Cresyl violet staining…….………………………………….. 55
Hematoxylin and eosin staining……….…….………………. 56

ii Table of Contents
Results & Discussions 57

Part I: DV Specification of Chick Midbrain 59


1. Expression patterns of Pax3/7, nkx6.1 and neural differentiation in the
mesencephalon………….……………………………….…...…….. 59
2. DV Polarity-reversed transplantation………………...……………….... 62
Homochronic grafts………………………………………...………. 62
Heterochronic grafts……..……………………………..…….…….. 68
3. Influence of floor plate and roof plate on the expression of ventral and
dorsal specific genes………….……….…………….……………... 70
4. Adhesion between ventral and dorsal midbrain cells.….…………….... 72
5. Long-term transplantation..………………………...…..…………........ 74
6. Discussion……………………………………………………….……... 76
Identity and autonomy along the DV axis….………………...…..… 76
Cell adhesion as a mechanism or read-out of DV regionalization..... 78
Progenitor dispersal………………………………………………… 79
Regionalisation as a feature of DV patterning in large structures….. 79

Part II: Function of Pax7 on Midbrain Expansion 81

1. Isolation and subcloning of chick Pax7 cDNA……………….…..….. 81
2. Influence of Pax7 on the size of mesencephalon……………………... 84
Overexpression of Pax7 in dorsal midbrain……..………..…....…. 84
Misexpression of Pax7 in ventral midbrain……..………..…....…. 84
Repression of Pax7 in dorsal midbrain.…..……..…………......…. 88
3. Pax7 promotes the neural proliferation in the midbrain……………… 91
Dividing precursor cells in the neural tube……..…..……...……... 91
More dividing cells by Pax7 overexpression……………………… 91
No effect on cell death.………………………………………….... 94
4. Analysis of Pax7 on the neural differentiation and specification.….… 95
Neural differentiation normally………………………………….... 95
Induction of Brn3a positive cells but not Isl-1 cells………………. 96
5. Pax7 - induced Pax3 co-localizes with Nkx6.1……..…………..…...... 99
6. Discussion……………………………………….………………..…... 102
Pax7 is involved in the neuronal proliferation……………..……... 102
A role of Pax7 on the specification of some dorsal neuronal
lineages………………………………………………….... 104
Cross-regulation between Pax3 and Pax7……..………………...... 106
Pax3/7 action on rapid expansion of the mesencephalon………… 107

iii Table of Contents
Part III: Expression and Function of Rab23 in Chick Nervous System 111

1. Expression pattern of Rab23 in chick embryos……...…………..….... 111
Preparation of antisense RNA probes for chick Rab23.………..… 111
Rab23 expression early in chick embryos..…………………....…. 112
A transient Rab23 expression in the notochord at early stages…... 113
Expression pattern of Rab23 in the neural tube………..……........ 113
2. Identification and analysis of chick Rab23 gene………….…....…..… 118
Identification of chick Rab23 cDNA…………………….……...... 118
Sequential analysis of chick Rab23 protein..........…………...…… 119
Phylogenetic anaylsis of Rab23 proteins.....….............….…..…… 122
3. Subcloning and ectopic expression of chick Rab23 gene.…………… 123
Construction of Rab23 expression plasmid..…………………..… 123
Ectopic expression of Rab23 in vivo…………………………….... 123
4. Rab23 specifies dorsal regional identity in early neural tube….…….. 126
Induction of dorsal specific genes Pax3/7…………….…….......... 126
No effect on ventral specific gene Nkx6.1……….....………..….... 127
5. Influence of Rab23 on early neurogenesis in dorsal midbrain……...... 131
No obvious effect on the dorsal Islet 1/2 cell subpopulation…..… 131
Specification of Brn3a expre