The genes and mechanisms that drive foregut development are largely unknown and represent a new area of developmental biology. We are studying foregut development by investigating pharynx formation in the nematode Caenorhabditis elegans. Previously, we identified a locus, pha-4, that is required to establish the pharynx and rectum primordia: animals bearing pha-4 mutations arrest as larvae that lack a pharynx and a rectum. We have recently cloned pha-4 and shown that it encodes a homolog of the Drosophila gene fork head. This result, coupled with our phenotypic analyses, suggest that foregut (pharynx) and hindgut (rectum) development have been conserved between worms and flies for over 500 million years. Strikingly, imperforate anus coupled with esophageal atresia is a common human birth defect, suggesting that similar developmental pathways may exist in humans. This proposal will extend our studies of pharynx formation in three ways.
Aim 1. Pharynx development depends on cells from different cell lineages assembling into an integrated organ. The pha-4 gene marks the convergence of these different cell lineages both by phenotype (i.e. pharynx cells from all cell lineages are lost in pha-4 mutants) and by expression (i.e. a pha-4::GFP reporter is expressed in pharynx cells from all lineages). To understand how the convergence of multiple cell lineages into one organ primordium is achieved, we will analyze how pha-4 expression is established in pharynx cells. We will use a combination of deletion analysis, reporter constructs, different mutant backgrounds, and mobility shift assays to address this issue.
Aim 2. We will investigate whether PHA-4 directly activates two candidate pharynx genes to distinguish between two models of pha-4 function: 1) pha-4 could act early to initiate a hierarchy of downstream regulators 2) pha-4 could be an obligate co-factor for many or all pharynx genes, early and late.
Aim 3. Few genes required for pharynx development have been identified. To discover new loci that are necessary to form the pharynx, we will phenotypically characterize homozygous deficiencies that remove genes needed for pharynx development. These fall into three phenotypic classes: i) bifurcated pharynx ii) small pharynx iii) arrested pharynx primordium. On the basis of our initial survey, we will choose the most promising candidates for further analysis.
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