Analysis of the arachidonyl-CoA synthetase ACSL4a as a potential regulator of BMP expression and of the role of BMPs in timing of cell commitment along the dorsoventral axis of the gastrulating zebrafish embryo [Elektronische Ressource] / vorgelegt von Björn Renisch

Analysis of the arachidonyl-CoA synthetase ACSL4a as a potential regulator
of BMP expression and
of the role of BMPs in timing of cell commitment along the dorsoventral axis
of the gastrulating zebrafish embryo



I n a u g u r a l - D i s s e r t a t i o n
zur

Erlangung des Doktorgrades

der Mathematisch-Naturwissenschaftlichen Fakultät

der Universität zu Köln

vorgelegt von

Björn Renisch

aus Bad Soden a. Ts.


Köln, 2009
- 1 -








































Berichterstatter: Prof. Dr. Matthias Hammerschmidt
Berichterstatter: Prof. Dr. Siegfried Roth

Tag der mündlichen Prüfung: 27. November 2009
- 2 - Table of contents
1. Summary ................................................................................................................................ 6
1.2 Zusammenfassung ................................................................................................................ 8
2. General Introduction ............................................................................................................ 10
2.1. Early zebrafish development ......................................................................................... 10
2.2 The establishment of the dorsal-ventral axis in zebrafish .............................................. 12
2.3. The role of Bone Morphogenetic Proteins during gastrulation ..................................... 14
2.4 The role of Bone Morphogenetic Proteins in stem cell biology .................................... 18
2.5. Aim of the work ............................................................................................................ 20
3. Material and Methods ........................................................................................................... 21
3.1. Zebrafish lines and husbandary ..................................................................................... 21
3.2. Embryological methods ................................................................................................. 21
3.3. Molecular methods ........................................................................................................ 25
3.4. Materials ........................................................................................................................ 28
4. The role of acyl-coA-synthetase longchain family member 4 in dorsoventral patterning
during zebrafish gastrulation .................................................................................................... 31
4.1. Introduction ................................................................................................................... 31
4.1.1 The arachidonic acid pathway ................................................................................. 31
4.1.2 The cyclooxygenase pathway of prostanoid production ......................................... 32
4.1.3 The leukotriene and other pathway ......................................................................... 35
4.1.4 The enzyme acyl-coA-synthetase longchain family member 4 (ACSL4) .............. 35
4.1.5 Aim of the project ................................................................................................... 36
4.2. Results ........................................................................................................................... 38
4.2.1. Knock-down with morpholino SP3005a causes dorsalisation of the zebrafish
embryo .............................................................................................................................. 38
4.2.2. ACSL4a seems to be required for the maintenance of BMP signaling .................. 41
4.2.3. During gastrulation, acsl4a is mainly expressed and required in the Yolk Syncytial
Layer ................................................................................................................................. 44
4.2.4. Further morpholinos targeting acsl4a do not lead to dorsalisation ........................ 47
4.2.5. Injection of acsl4a RNA does not lead to a rescue of the dorsalisation caused by
morpholino SP3005a ........................................................................................................ 48
4.2.6. Knockdown of acsl4a shows no specific defects during gastrulation comparable to
loss of PGE signaling ...................................................................................................... 52 2
4.2.7. Morpholinos targeting genes downstream of acsl4a show no phenotype during
early development ............................................................................................................ 53
- 3 - Table of contents
4.2.8. Treatments with agonist and antagonist of the arachidonic acid pathway ............. 55
4.3. Discussion ..................................................................................................................... 58
4.3.1. Knockdown of acsl4a create a new role for arachidonic acid metabolism during
early embryonic development .......................................................................................... 58
4.3.2 acsl4a is expressed in the yolk syncytial layer of the gastrulating embryo, from
where it might regulate dorsoventral patterning of the blastoderm ................................. 60
4.3.3 The phenotype of SP3005a morphants might be caused by off-target effects ........ 62
4.3.4 Further components of the arachidonic acid cascade can most likely be excluded as
regulators of dorsoventral patterning ............................................................................... 63
4.3.5 Outlook and perspectives ........................................................................................ 66
5. Studies for cell commitment during early gastrulation stages ............................................. 67
5.1. Introduction ................................................................................................................... 67
5.1.1. During gastrulation cells are specified towards their final fate in a region-and time-
dependent manner ............................................................................................................ 67
5.1.2. Aim of the project .................................................................................................. 71
5.2. Results ........................................................................................................................... 73
5.2.1. Dorsal and ventral cells do not show significant commitment differences after
heterotopic transplantations at shield stage ...................................................................... 73
5.2.2. Dorsal and ventral cells do not show significant commitment differences after
heterotopic transplantations at the 60-70% epiboly stage ................................................ 78
5.2.3. Heterochronic transplantations of dorsal ectodermal cells from 60% epiboly stage
donors into ventralised blastula stage embryos ................................................................ 81
5.2.4. Heterochronic transplantations of ventral ectodermal cells from 60% epiboly stage
donors into dorsalised blastula stage embryos ................................................................. 83
5.2.4 Heterochronic transplantations of dorsal and ventral ectodermal cells of 80%
epiboly stage embryos ...................................................................................................... 86
5.3. Discussion ......................................................................................................................... 89
5.3.1. After heterotopic transplantations, both dorsal and ventral cells do not maintain
their initial fate and can integrate into the new tissues .................................................... 89
5.3.2. After heterochronic transplantations, both dorsal and ventral cells show an
increased tendency to lack the neural as well as the epidermal marker ........................... 93
5.3.3. Outlook and further perspective ............................................................................. 96
6. Large-scale screen using morpholino antisense nucleotides to identify new genes involved
in early development, pituitary or skin development ............................................................... 98
- 4 - Table of contents
6.1. Introduction ................................................................................................................... 98
6.2. Results ........................................................................................................................... 98
6.3. Discussion ................................................................................................................... 109
7. References .......................................................................................................................... 110
8. Appendix ...............................................................................................