zebrafish organizer development and germ-layer formation require nodal-related signals
DESCRIPTION
Zebrafish organizer development and germ-layer formation require nodal-related signals Feldman 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? - PowerPoint PPT PresentationTRANSCRIPT
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Zebrafish organizer development and germ-layer formation require
nodal-related signalsFeldman et al.
Nature 1998
Presented by Maggie Bach and Aparna Vidyasagar
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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
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Big Idea
• Characterize the Squint gene and its role in development
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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
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Definitions
• Mesoderm – forms bones, muscles, organs
• Endoderm – digestive and respiratory tract
• Ectoderm – epidermis, nervous system
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Fish Embryo before Gastrulation
Scier and Shen, Nature 2000
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Organizer
• Region of dorsal mesoderm
• Induced by other signals
• Expresses nodal-related signals
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Nodal Signals
• Expressed in dorsal region that includes YSL
• Source of mesoderm-inducing factors
• TGF-β related signals
• Related to Activin and Vg1
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gsc
• Goosecoid
• Dorsal mesodermal marker
• Expressed in organizer
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Question 1:
What are the different mutant phenotypes?
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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
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cyc–
• One eye
• Not examined specifically; thought to have the same phenotype as sqt–
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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
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Feldman et al. Nature 1998
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Question 2:
Are the sqt and znr-2 genes one and the same?
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Step 1: Genetic mapping
• sqtcz35 mutation maps to linkage group 21 near the zebrafish nodal related-2 (znr 2) gene.
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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.
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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.
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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..
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Question 3:
Does sqt induce dorsal mesoderm formation
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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.
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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.