Zebrafish organizer development and germ-layer formation require nodal-related signals

1. Zebrafish organizer development and germ-layer formation require nodal-related signals Feldman et al. Nature 1998 Presented by Maggie Bach and Aparna Vidyasagar

2. Outline • Introduction and Background • Questions • What are the mutant phenotypes? • Are the sqt and znr-2 genes one and the same? • Can sqt induce dorsal mesoderm formation? • Conclusion

3. Big Idea • Characterize the Squint gene and its role in development

4. Definitions • Blastula - a liquid-filled sphere whose wall is composed of a single layer of cells • Gastrula - double-walled stage of the embryo resulting from invagination of the blastula; outer layer of cells is the ectoderm and the inner layer differentiates into the mesoderm and endoderm

5. Definitions • Mesoderm – forms bones, muscles, organs • Endoderm – digestive and respiratory tract • Ectoderm – epidermis, nervous system

7. Fish Embryo before Gastrulation Scier and Shen, Nature 2000

8. Organizer • Region of dorsal mesoderm • Induced by other signals • Expresses nodal-related signals

9. Nodal Signals • Expressed in dorsal region that includes YSL • Source of mesoderm-inducing factors • TGF-β related signals • Related to Activin and Vg1

10. gsc • Goosecoid • Dorsal mesodermal marker • Expressed in organizer

11. Question 1: What are the different mutant phenotypes?

12. sqt– • One eye • Expression of gsc reduced at blastula stages, but gsc expression increases as gastrulation progresses • Defects in prechordal plate and ventral nervous system • No Zebrafish organizer region

13. cyc– • One eye • Not examined specifically; thought to have the same phenotype as sqt–

14. sqt– cyc– • No mesoderm development • Muscle differentiation can occur in tail • Lack of gsc expression • No Zebrafish organizer region • Morphogenesis of mesoderm and endoderm is abnormal

15. Feldman et al. Nature 1998

16. Question 2:Are the sqt and znr-2 genes one and the same?

17. Step 1: Genetic mapping • sqtcz35 mutation maps to linkage group 21 near the zebrafish nodal related-2 (znr 2) gene.

19. Step 2: DNA libraries • DNA probe derived from a partial znr-2 sequence identified a full length znr-2 cDNA clone in a library of blastula and gastrula stages. • Clone had an ORF of 1,176 bp. • Conceptual Translation revealed a 392 amino acid long protein similar to proteins belonging to the TGF-ß subfamily.

20. Step 3: Molecular lesions in sqtcz35 • Molecular lesions showed: A 1.9 kb insertion after codon 30 in the ORF. • Assaying sqt crosses with this insertion showed that znr-2 was closely linked to sqt. • 0 recombinants among 111 meioses. Thus znr-2 is tightly linked to sqt.

21. Step 4: Evidence from the goosecoid gene • sqt/cyc double mutants lack the dorsal mesodermal marker goosecoid (gsc). • Microinjection of synthetic znr-2 RNA into sqt mutants leads to the restoration of gsc expression. Thus znr-2 is the sqt gene. .

23. Question 3:Does sqt induce dorsal mesoderm formation

24. Step 1: gsc expression • Embryos injected with sqt RNA. Thus extraembryonic expression of sqt can induce dorsal mesoderm formation.

25. Conclusions • Zebrafish nodal related signals are essential in mesoderm and endoderm development. • Nodal related signals also necessary for induction of dorsal mesoderm and organizer formation.