Zebrafish organizer development and germ-layer formation require

nodal-related signalsFeldman et al.

Nature 1998

Presented by Maggie Bach and Aparna Vidyasagar

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

Big Idea

• Characterize the Squint gene and its role in development

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

Definitions

• Mesoderm – forms bones, muscles, organs

• Endoderm – digestive and respiratory tract

• Ectoderm – epidermis, nervous system

Fish Embryo before Gastrulation

Scier and Shen, Nature 2000

Organizer

• Region of dorsal mesoderm

• Induced by other signals

• Expresses nodal-related signals

Nodal Signals

• Expressed in dorsal region that includes YSL

• Source of mesoderm-inducing factors

• TGF-β related signals

• Related to Activin and Vg1

gsc

• Goosecoid

• Dorsal mesodermal marker

• Expressed in organizer

Question 1:

What are the different mutant phenotypes?

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

cyc–

• One eye

• Not examined specifically; thought to have the same phenotype as sqt–

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

Feldman et al. Nature 1998

Question 2:

Are the sqt and znr-2 genes one and the same?

Step 1: Genetic mapping

• sqtcz35 mutation maps to linkage group 21 near the zebrafish nodal related-2 (znr 2) gene.

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.

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.

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..

Question 3:

Does sqt induce dorsal mesoderm formation

Step 1: gsc expression

• Embryos injected with sqt RNA.

8-32 cell stage of the blastula

Expanded or ectopic gsc expression

1000 cell stage into YSL

Expanded or ectopic gsc expression

Thus extraembryonic expression of sqt can induce dorsal mesoderm formation.

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.