Functional divergence of Midkine growth factors [Elektronische Ressource] : non-redundant roles during neural crest induction, brain patterning and somitogenesis / vorgelegt von Daniel Liedtke

Daniel Liedtke

Sujets

• Biologie

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2008
• Nombre de lectures: 54
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Functional divergence of Midkine growth factors:
Non-redundant roles during neural crest induction,
brain patterning and somitogenesis





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




vorgelegt von
Daniel Liedtke
aus
Fulda



Würzburg, 2007

























Eingereicht am:

Mitglieder der Promotionskommission:
Vorsitzender:
Gutachter: Prof. Dr. Christoph Winkler
Gutachter: PD Dr. med. Ute Felbor

Tag des Promotionskolloquiums:

Doktorurkunde ausgehändigt am:















Darin besteht das Wesen der Wissenschaft.
Zuerst denkt man an etwas, das wahr sein könnte
– und dann sieht man nach, ob es der Fall ist, und im Allgemeinen ist es nicht der Fall.

Bertrand Russell
Index 1
Index
Index__________________________________________________________ 1

Summary ______________________________________________________ 5

Zusammenfassung ______________________________________________ 6

1. Introduction ________________________________________________ 7
1.1. Differentiation of the vertebrate brain and spinal cord _______________________ 7
1.1.1. Structure of the vertebrate brain ________________ 7
1.1.2. Induction of neural tissues _________________ 8
1.1.3. Patterning of the brain ________________________ 9
1.1.4. Pattf the vertebrate spinal cord __________________ 10
1.2. Development and functions of neural crest cells________ 12
1.2.1. Induction of neural crest cells _____________________________________ 12
1.2.2. Molecular networks involved in ncc differentiation _ 14
1.2.3. Development of the neural crest in zebrafish _____________ 15
1.2.4. Development of sensory neurons in zebrafish____________ 17
1.2.5. Open questions in ncc and sensory neuron development ________________ 18
1.3. Gradients and clockworks in somitogenesis ___________ 19
1.4. The zebrafish as a model system _______________________________________ 21
1.5. Gene duplication in zebrafish __________ 22
1.6. Midkine and Pleiotrophin ____________________________________________ 23
1.6.1. Midkine and pleiotrophin genes in vertebrates _____ 23
1.6.2. Midkine and pleiotrophin genes in zebrafish______________ 25
1.7. Aim of the PhD thesis ________________________________________ 26

2. Material___________________________________________________ 29
2.1. Fish maintenance and breeding ________________________________________ 29
2.2. Bacterial strains_____________________29
2.3. Morpholinos___________________________________________29
2.4. Antibodies_____________31
2.5. Oligonucleotides_______________________________________31
2.6. Kits_______________________________31
2.7. Enzymes__________________________________32
2.8. Chemicals_________________________32
2.9. Technical devices______________________________________32
Index 2

3. Methods___________________________________________________ 34
3.1. Microbiological methods_____________________________________________34
3.1.1. Sterilization________34
3.1.2. Growth media_____________
3.1.3. Bacteria cultivation and long time storage ____________________ 34
3.1.4. Chemically competent bacteria________________________35
3.1.5. Bacterial transformation__________
3.2. Molecular biological methods___________________35
3.2.1. Plasmid DNA amplification and isolation_________ 35
3.2.2. DNA purification___________________________________36
3.2.3. DNA and RNA precipitation ___________________
3.2.4. Determination of nucleic acid concentrations _____________ 36
3.2.5. Agarose gel electrophoresis____________________36
3.2.6. Gel extraction of DNA fragments ______________________ 37
3.2.7. DNA sequencing________________37
3.2.8. RNA extraction________________________38
3.2.9. Reverse transcription_____________
3.2.10. Polymerase chain reaction (PCR) __________ 39
3.2.11. Enzymatic DNA digestion________________________________________40
3.2.12. Capped RNA synthesis __________________________________________ 40
3.2.13. Riboprobe synthesis______________40
3.2.14. Whole-mount RNA in situ hybridization ________________ 41
3.2.15. Immunostaining_____________________________44
3.2.16. Cartilage staining_______________________44
3.3. Chemical treatments of zebrafish embryos 45
3.3.1. DEAB treatment_______________________________________________45
3.3.2. SU5402 treatment___
3.3.3. LiCl treatment_____________46
3.4. Microinjection into zebrafish embryos ___________________________ 46
3.4.1. Collection of embryos ___________________ 46
3.4.2. Microinjection______46
3.4.3. Cultivation of injected embryos____________________________________ 47

4. Results ____________________________________________________ 49
4.1. Analysis of mdkb expression during stages of ncc induction _________________ 49
4.2. Regulation of mdkb expression by neural crest inducing signals _______ 51
4.2.1. Loss of retinoic acid signaling represses mdkb expression _______________ 52
4.2.2. mdkb expression requires FGF signaling______________________ 53
4.2.3. FGF but not RA inhibition alters neural plate size _________ 54
4.2.4. Altered Wnt signaling has diverse effects on mdkb expression ____ 55
4.2.5. Delta-Notch signaling does not interfere with mdkb expression___________ 56
4.3. mdkb regulates ncc specification ________________________________ 57
4.3.1. Design and activity of the mdkb splice Morpholinos_______ 57
Index 3
4.3.2. Mdkb regulates earliest steps of ncc induction during gastrulation ________ 60
4.3.3. Misexpression of mdkb alters expression of prominent ncc specifiers ______ 61
4.3.4. Rescue of ncc defects in mdkb morphants by RNA co-injection __________ 63
4.3.5. Loss of ncc in mdkb morphants is not a consequence of increased apoptosis_ 65
4.3.6. Analysis of ncc recovery in late embryonic stages of mdkb morphants _____ 66
4.4. Mdkb regulates sensory neuron specification _____________________________ 69
4.4.1. Mdkb influences primary sensory neuron induction _ 69
4.4.2. Absence of sensory neuron recovery in mdkb morphants ________________ 70
4.5. Non-overlapping activities of Midkine growth factors during mid- and
hindbrain formation _________________________________________________ 73
4.5.1. Mostly non-overlapping expression patterns of mdka, mdkb and ptn
during zebrafish brain development ________ 73
4.5.2. Overexpression of midkine genes affects different aspects of brain patterning 77
4.5.3. Double and triple knockdown of Midkine factors: Design and activity of
mdka and ptn splice Morpholinos __________________________________ 81
4.5.4. Single knockdown of midkine genes reveals non-overlapping functions
during brain patterning __________________ 82
4.5.5. Combined knockdown reveals an exclusive role of mdka during MHB
establishment __________________________________________________ 84
4.5.6. Knockdown of ptn function results in hindbrain patterning defects _ 85
4.6. Somitogenesis is regulated by ptn ______________ 87
4.6.1. Combined knockdown of mdka, mdkb and ptn results in somite fusion _____ 87
4.6.2. Expression of midkine genes during somitogenesis in the paraxial and
presomitic mesoderm (PSM) ______________________________________ 88
4.6.3. Knockdown of ptn inhibits somite formation ______ 89

5. Discussion _________________________________________________ 93
5.1. Functions of Mdkb in neural crest and sensory neuron induction______________ 93
5.1.1. Spatiotemporal expression of mdkb is consistent with a role during ncc
and sensory neuron induction at the neural plate border _________________ 93
5.1.2. mdkb expression is regulated by known ncc-inducing signals, but not by
Delta-Notch signaling ___________________________________________ 94
5.1.3. Mdkb regulates neural crest and sensory neuron induction at the neural
plate border _______________ 98
5.1.4. Model of Mdkb action on cell induction at the neural plate border _______ 100
5.2. Restricted expression patterns of midkine genes during early brain development 102
5.2.1. Comparison of mdka, mdkb and ptn expression during early brain
development__________________________________________________ 102
5.2.2. Different aspects of zebrafish brain patterning are regulated by Midkine
growth factors ____________ 104
5.3. Ptn is essential for somite boundary formation ___________________________ 108
5.3.1. Combined knockdown of midkine and pleiotrophin function interferes
with somitogenesis_________________________________ 109
5.3.2. Triple knockdown of mdka, mdkb and ptn does not interfere with initiation
of the somitogenesis clock but affects somitomere maturation___________ 109
Index 4
5.3.3. Knockdown of Ptn function is responsible for somite ablation in triple
morphants ___________________________________