Organogenesis of the Pharynx in C.elegans

描述（由申请人提供）：我的实验室长期以来一直对调节消化道器官发生的分子机制感兴趣。我们选择了一个简单的器官——秀丽隐杆线虫的咽（或前肠）——来识别对器官发育重要的基因，并了解它们是如何起作用的。此前，我们发现FoxA转录因子同源物pha-4是咽发育的关键调节因子。然而，单独的PHA-4不能解释咽内细胞类型的多样性。我们假设器官身份的调节因子与控制位置、卵裂球或细胞类型身份的因素联合作用，以建立咽细胞内基因表达的模式，从而指定它们的命运。本研究的目的是阐明线虫咽部与PHA-4结合产生不同细胞类型的调控网络。我们的目的是：i)通过分析14个导致pha-4功能部分丧失的基因抑制因子来研究pha-4上游的调控回路。我们预测这些突变体可以分为三类，我们已经设计了区分这些类别的方法。我们将描述抑制基因和启动分子分析。Ii)确定控制咽内基因表达的组合代码。我们已经通过微阵列鉴定出338个可能在咽中选择性表达的基因。我们对咽部细胞内可能共调控的基因进行了分组，并使用计算方法来鉴定这些基因的顺式作用调控位点。我们的目标是确定这些位点在体内的功能，并确定它们的交互作用因子。我们假设这些元件将与PHA-4结合位点联合起作用，从而产生不同的咽表达模式。

DESCRIPTION (provided by applicant): 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 (or foregut) - to identify the genes important for organ development and to understand how they function. Previously, we discovered the FoxA transcription factor homolog pha-4 as a critical regulator of pharyngeal development. However, PHA-4 alone cannot account for the diverse array of cell types within the pharynx. We hypothesize that regulators of organ identity act combinatorially with factors that control positional, blastomere, or cell type identity to establish patterns of gene expression within pharyngeal cells and thereby specify their fate. The goal of this study is to elucidate the regulatory network that generates different cell types within the C. elegans pharynx in combination with PHA-4. Our Aims are i) to investigate the regulatory circuitry upstream of pha-4 by analyzing 14 genetic suppressors of partial loss of pha-4 function. We predict these mutants could fall into three classes and we have designed ways to distinguish between these classes. We will characterize the suppressors genetically and initiate a molecular analysis. ii) to determine the combinatorial code that .governs gene expression within the pharynx. We have identified 338 genes by microarray that are likely to be selectively expressed in the pharynx. We have grouped genes that are likely to be co-regulated within pharyngeal cells and are using computational approaches to identify cis-acting regulatory sites for these genes. Our goal is to determine the function of these sites in vivo and identify their trans-acting factors. We hypothesize that these elements will function combinatorially with PHA-4 binding sites to generate the diverse patterns of pharyngeal expression. iii) to extend the pathway for pharynx development using an RNA interference-based approach. We will survey the loss-of-function phenotypes associated with 295 candidate pharyngeal genes identified by microarray chip. Our goal is to discover genes that function downstream of PHA-4 to specify the fate of pharyngeal muscles. We will perform an in-depth analysis of those candidates that appear most promising.