This project utilizes a genetic approach to identify and characterize novel genes which determine pattern formation in the early vertebrate embryo. Nearly a century of experimental vertebrate embryology has provided us with deep insights into the cellular mechanisms of vertebrate development, from primary embryonic induction to patterning of the nervous system. Still, one of the major deficits in vertebrate developmental biology is our limited knowledge on the genetic basis of the functional interactions that underlie the determination of embryonic pattern and organogenesis. Chemical saturation mutagenesis has been instrumental for the major advances in understanding the genetic control of development in Drosophila. We will apply the Drosophila way of doing genetics to vertebrates and perform extensive chemical mutagenesis screens in zebrafish (Brachydanio rerio). Among all vertebrates, the small size, short generation time and high fertility make zebrafish the organism of choice for large scale genetic screens. Embryos are transparent, develop outside the mother and hatch after only 48 hours - optimal prerequisites for phenotypic analysis. Specifically, conditions for chemical mutagenesis will be optimized to induce two embryonic lethal mutations per gamete. Using an F2 genetic screen, within the proposed five year period we will be able to analyze 10,000 to 20,000 lethal mutations and thus identify 70-90% of all genes that can be mutated to a lethal embryonic phenotype. The screen will provide a rich source of mutations for us and other laboratories. In continuation of my previous research interests, we will focus on those mutations which appear to affect early pattern formation in the embryo: generation of the germlayers and patterning of the anterior-posterior and dorsal-ventral body axes. Applying the ratio of patterning genes to total number of embryonic lethal mutations found in Drosophila, we expect to identify mutations in 100 to 500 genes involved in the inductive events during gastrulation and neurulation in zebrafish. Results from classic embryological experiments in Xenopus, chicken and mice tell us what phenotypes to expect and will help to interpret and classify mutant phenotypes. New mutations will be genetically characterized (complementation analysis, allelic strength, genetic mapping). Mutant phenotypes will be analyzed (time and region with requirement for gene activity, cell autonomy of gene action) and epistatic relationships established to determine hierarchies of genetic interactions. This project is one initial step in a long genetic analysis of vertebrate development, leading towards the elucidation of all the regulatory interactions and genetic components that participate in the generation of the basic vertebrate body plan. It will complement other current approaches, because it allows to identify genes which are not readily accessible by molecular or biochemical methods. A detailed knowledge of the genetic control of vertebrate ontogeny will help us to understand human development and genetic disorders.
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