My lab has had a long-standing interest in the molecular mechanisms that regulate organogenesis of the digestive tract. We have chosen a simple organ - the C. elegans pharynx (foregut) - to identify the genes important for organ development and understand how they function. Previously, we discovered the FoxA transcription factor homolog pha-4 and showed it was a critical regulator of pharyngeal development. We also identified additional factors that function combinatorially with PHA-4. These studies have laid a foundation for understanding the transcriptional network that controls foregut development and raised three critical issues that we address in the current proposal:
Aim 1 will determine the consequences of PHA-4 binding to its target promoters. The relative affinity of different DNA binding sites for PHA-4 modulates the onset of target gene expression. We have developed tools to visualize association of PHA-4 with its target promoters in living embryos. We will use this assay to ask i) how does binding site affinity influence PHA-4 association with its target genes and ii) what are the downstream consequences of PHA-4 binding to target promoters? These experiments will address how the affinity of DNA for PHA-4 influences events associated with promoter firing, namely PHA-4 binding, DNA decompaction and association of additional transcription factors with target promoters. These studies will enable us to test the hypothesis that PHA-4 functions as a competence factor for foregut transcription, and by extension, foregut fate.
Aim 2 investigates the regulatory circuitry upstream of pha-4 by analyzing 13 genetic suppressors of partial loss of pha-4 function. We predict these mutants regulate PHA-4 transcriptionally or post- transcriptionally, and we have designed ways to distinguish between these classes. We will characterize the suppressors genetically and initiate a molecular analysis.
Aim 3 will address the processes and molecules that function with PHA-4 for commitment to foregut fate. Our preliminary studies have defined a window in embryogenesis when cells can assume foregut fate in response to PHA-4. Prior to this stage, cells appear developmentally plastic, whereas after this stage cells are committed to become pharyngeal. We have developed assays to measure developmental plasticity, which we will use to explore the mechanisms that control this transition. We have also used microarray to identify genes expressed at this time, and will survey these genes for possible roles to maintain pluripotency or promote the transition to cell fate commitment. These studies will enable us to test the hypothesis that the loss of developmental plasticity is controlled by sequence-specific transcription factors in combination with global transcriptional regulatory mechanisms.
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