Towards genetic dissection of neural crest specification and cartilage differentiation in zebrafish (Danio rerio) [Elektronische Ressource] / Michael Lang

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München





Towards Genetic Dissection of Neural Crest
Specification and Cartilage Differentiation
in Zebrafish (Danio rerio)





Michael Lang

aus

Münster-Hiltrup







2003

Erklärung

Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der
Promotionsordnung vom 29. Januar 1998 von Prof. Dr. R. Grosschedl
betreut.



Ehrenwörtliche Versicherung

Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.



München, am 12. September 2003



.................................................
(Michael Lang)




Dissertation eingereicht am 12. September 2003
1. Gutachter Prof. Dr. R. Grosschedl
2. Gutachter Prof. Dr. F. Eckardt-Schupp
Mündliche Prüfung am 19. Dezember 2003









How does newness come into the world? How is it born?
Of what fusions, translations, conjoinings is it made?
How does it survive, extreme and dangerous as it is?

Salman Rushdie (1988)








Table Of Contents

1 Commonly used abbreviations_________________________________________ 5
2 Introduction __________________________________________________________ 8
2.1 Zebrafish as a model organism ______________________________________________ 8
2.1.1 A genetic approach to the study of zebrafish embryonic development _____________________ 8
2.1.2 The vertebrate craniofacial skeleton 10
2.1.2.1 ebrate pharyngeal arches 10
2.1.2.2 The zebrafish craniofacial skeleton _____________________________________________ 11
2.2 The Neural Crest_________________________________________________________ 13
2.2.1 Neural crest induction 13
2.2.2 Neural crest specification: Progressive fate restriction ________________________________ 13
2.2.3 Neural crest migration and the organization of cranial neural crest streams ________________ 16
2.2.4 Neural crest differentiation and derivatives _________________________________________ 18
2.2.5 Hindbrain patterning and pharyngeal arch specification _______________________________ 19
2.2.6 Pharyngeal arch patterning _____________________________________________________ 21
2.3 Cartilage development ____________________________________________________ 23
2.3.1 Chondrogenesis ______________________________________________________________ 23
2.3.2 The extracellular matrix________________________________________________________ 24
2.3.3 Proteoglycans________________________________________________________________ 24
2.4 Strategies for the molecular dissection of neural crest derivative development in
zebrafish 27
2.4.1 Morphological and molecular analyses of ENU-induced zebrafish mutations ______________ 27
2.4.2 Genetic mapping of ENU-induced mutations _______________________________________ 27
2.4.3 Gene identification by candidate and positional cloning approaches _____________________ 31
3 Aims of the thesis ___________________________________________________ 32
4 Materials and Methods _______________________________________________ 33
4.1 Materials _______________________________________________________________ 33
4.1.1 Chemicals __________________________________________________________________ 33
4.1.2 Radionucleotides _____________________________________________________________ 34
4.1.3 Buffers and Solutions__________________________________________________________ 35
4.1.4 Oligonucleotides 36
4.1.5 Enzymes____________________________________________________________________ 38
4.1.6 Kits________________________________________________________________________ 38
4.1.7 Vectors_____________________________________________________________________ 38
4.1.8 Biological Materials___________________________________________________________ 39
4.1.8.1 Zebrafish strains____________________________________________________________ 39
4.1.8.2 Bacterial strains 39
4.1.9 Equipment __________________________________________________________________ 40
4.2 Methods ________________________________________________________________ 41
4.2.1 Genetic mapping _____________________________________________________________ 41
4.2.1.1 Genomic DNA Preparation ___________________________________________________ 41
4.2.1.2 Total Genome Scan by genotyping with microsatellite markers _______________________ 41
4.2.1.3 PCR 42
4.2.1.4 Agarose gel electrophoresis 42
4.2.1.5 Single-stranded conformation polymorphism (SSCP) analysis ________________________ 43
4.2.1.6 Overgo Probing of High-Density BAC and PAC Filters _____________________________ 44
4.2.2 Molecular biology and cloning __________________________________________________ 47
4.2.2.1 RNA Isolation 47
4.2.2.2 RT-PCR 48
4.2.2.3 Restriction digest of DNA ____________________________________________________ 48
4.2.2.4 Ligation 49
4.2.2.5 Transformation_____________________________________________________________ 49
4.2.2.6 Synthesis of digoxygenin-labeled riboprobes for in situ hybridization __________________ 50
-3-
4.2.2.7 Capped mRNA synthesis (in vitro transcription)___________________________________ 51
4.2.3 Morphology and histology______________________________________________________ 52
4.2.3.1 Alcian Blue staining_________________________________________________________ 52
4.2.3.2 Ultramicrotomy and histological staining ________________________________________ 52
4.2.4 Molecular analysis of gene expression ____________________________________________ 54
4.2.4.1 Whole-mount in situ hybridization _____________________________________________ 54
4.2.4.2 Whole-mount immunohistochemistry ___________________________________________ 56
4.2.5 Functional assays _____________________________________________________________ 59
4.2.5.1 Microinjection of morpholino antisense oligonucleotides ____________________________ 59
4.2.5.2 Microinjection of capped mRNA_______________________________________________ 59
4.2.6 World Wide Web infrastructure__________________________________________________ 60
5 Results______________________________________________________________ 61
m452 5.1 Analysis of the brak (brk ) mutation _______________________________________ 61
m452 5.1.1 The brak mutation causes craniofacial defects and diminished melanophore pigmentation _ 61
m4525.1.2 The brak mutation maps to linkage group 14 ____________________________________ 63
m1885.2 Analysis of the mother superior (mos ) mutation _____________________________ 65
m188 5.2.1 Craniofacial cartilage elements are lost in mos mutant embryos ______________________ 65
m1885.2.2 Iridophores but not melanophores are greatly reduced in mos mutant embryos __________ 67
m1885.2.3 The mos mutation leads to the development of supernumerary neuromast organs ________ 68
5.2.4 Dorsal root ganglia and the enteric nervous system are greatly reduced in the absence
m188 of mos gene function _______________________________________________________ 69
5.2.5 Migratory cranial neural crest cells and pharyngeal arch primordia display severely reduced
m188 expression of dlx genes in mos mutant embryos___________________________________ 70
m188 5.2.6 Normal patterning of hindbrain rhombomeres in mos mutant embryos_________________ 72
m1885.2.7 The mutation in the mos locus leads to downregulation of key genes in neural crest
progenitor cells______________________________________________________________74
5.2.8 foxD3, a key regulator of neural crest specification, is not expressed in neural crest progenitor
m188 cells of mos mutant embryos _________________________________________________ 76
5.2.9 foxD3 transcripts are maternally deposited _________________________________________ 78
m1885.2.10 Genetic mapping of the mos locus _____________________________________________ 80
m188 5.2.11 Knockdown of foxD3 function phenocopies many aspects of the mos mutation __________ 82
m211, m641, m713, m7155.3 Analysis of the cartilage differentiation mutations round (rnd ) and
m299crusher (cru )__________________________________________________________ 85
m211 m2995.3.1 Comparative phenotypic characterization of the round and crusher mutations________ 85
m211 m2995.3.2 Comparative histological characterization of the round and crusher mutations _______ 88
m211, m641, m715 m2995.3.3 Mapping of the round and crusher mutations __________________________ 95
m211, m641, m7155.3.3.1 The round mutation maps to linkage group 21 95
m2995.3.3.2 Mapping of the crusher mutation____________________________________________ 98
m2995.3.3.2.1 Genetic mapping of the crusher mutation to linkage group 17 __________________ 98
m2995.3.3.2.2 Physical mapping of the crusher mutation _________________________________ 99
m2995.3.3.2.3 The sec23a gene is likely to be disrupted by the crusher mutation______________ 100
6 Discussion _________________________________________________________ 103
6.1 Zebrafish mutations as models fo