Genes Regulating Valve Formation
One goal of the lab is to identify the genes needed for cushion and valve formation in zebrafish, under the assumption that mutations in these genes may be causative of congenital heart malformations in infants.
One approach to identify these molecules is to start with a known gene and then to use multiple techniques to find upstream and downstream players in the pathway (for example, promoter analysis and gel-shift assays, yeast-two-hybrid screens, etc.). These approaches are used commonly in other species. However, the zebrafish gives a more powerful approach to identify genes in previously unrecognized pathways in an unbiased manner. This technique is the forward genetic screen, and has been very effective at finding new important molecules for morphogenesis.
The result of a forward genetic screen is the identification of fish which carry a mutation in an unknown gene affecting a specific developmental process of interest – cushion and valve formation in our case. Then, by a (sometimes laborious) process of positional cloning, the mutation is identified. Our lab is currently positionally cloning two genes required for cushion development. In addition, we recognize that the forward genetic screens performed to date have not been particularly specific or sensitive for finding mutants with errors in cushion or valve formation. Therefore, one of our goals is to create novel transgenic lines with cushion and valve fluorescence to serve as the basis for a new screen as well as to allow for better analysis of the role of known genes and agents in cushion and valve formation. The work on our valve morphogenesis project will drive the selection of appropriate promoters to use in these transgenic lines. These transgenic lines will also allow us to select fluorescent cells from developing cushions and valves for microarray analysis. Finally, we expect to find new genes regulating cushion/valve formation through our work on heart function and form.
