Award details

Control of the cavefish eyeless phenotype by alteration of the Spemann's organiser and midline signalling

Reference	BB/C517041/1
Principal Investigator / Supervisor	Dr Yoshiyuki Yamamoto
Co-Investigators / Co-Supervisors
Institution	University College London
Department	Cell and Developmental Biology
Funding type	Research
Value (£)	256,804
Status	Completed
Type	Research Grant
Start date	01/09/2005
End date	30/11/2008
Duration	39 months

Abstract

Astyanax mexicanus cavefish embryos, Shh has an expanded expression pattern in the anterior head region. This correlates eye degeneration, wider jaws and more taste buds. In the project, I will investigate the mechanisms of expanded Shh expression during cavefish development. Objective 1. Have the regulatory elements of Shh changed during cavefish evolution? The Shh promoter and introns will be obtained by genomic library screening in surface and cavefish populations. The Shh promoter and introns will be compared between surface fish and cavefish VISTA (visualisation and alignment software). To test whether differences in shh regulatory elements are important for the expansion of Shh expression in cavefish, the surface fish Shh promoter and introns with Green Fluorescein Protein (GFP) gene as a reporter will be injected into cavefish embryos, and the GFP expression pattern will be compared with normal cavefish Shh expression at tail bud stage (GFP antibody staining with shh in situ hybridisation). The opposite experiment (injection of cavefish shh promoter and introns with GFP into surface fish embryos) will be performed. These experiments will reveal whether differences in Shh regulatory elements are important for the expanded expression of Shh. Objective 2. Has the expression of Shh-regulating genes changed during cavefish evolution? The full length of Nodal related Cyclops which is one of the Shh upstream genes will be obtained by cDNA library screening, and its expression pattern in the anterior midline will be compared between surface and cavefish embryos. After in situ hybridisation, the embryos will be sectioned and counterstained with nuclear fast red. The number of nuclei stained with both NBT/BCIP and nuclear fast red will be counted. These experiments will reveal whether the expression of the Shh regulating, nodal-related gene Cyclops is different between surface fish and cavefish. Objective 3. Has Spemann¿s organiser (presumptive prechordal plate and notochord) behaviour changed during cavefish evolution? The number and distribution of cells expressing the prechordal plate marker goosecoid will be compared beween surface and cavefish. This will reveal differences between the two forms and suggest whether Spemann¿s organiser is important for expansion of Shh expression and cavefish eye degeneration. Reciprocal transplants of the organiser will be done between surface fish and cavefish, comparisons made of cell proliferation and movements and the eye phenotype recorded. At shield stage (early gastrula stage), a donor fluorescein-labelled organiser will be transplanted into a host after removal of the host organiser. Grafted embryos will be filmed by confocal microscopy to examine cell proliferation and movements of the labelled cells during gastrulation. They will be fixed at various stages to examine the expression of Shh, eye phenotype (eye size and lens programmed cell death), jaw size and taste bud numbers. These transplant experiments will enable us to understand whether Spemann¿s organiser activity has changed during cavefish evolution.

Summary

unavailable

Committee	Closed Committee - Genes & Developmental Biology (GDB)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

I accept the terms and conditions of use (opens in new window)

export PDF file

back to list new search