Organogenesis of the Pharynx in C.elegans

描述(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--线虫咽(或前肠)--来识别对器官发育至关重要的基因，并了解它们是如何发挥作用的。此前，我们发现FoxA转录因子同系物PHA-4是咽部发育的关键调节因子。然而，PHA-4本身不能解释咽部细胞类型的多样性。我们假设器官同一性的调节因子与控制位置、卵裂球或细胞类型同一性的因素共同作用，以建立咽部细胞内基因表达的模式，从而决定它们的命运。本研究的目的是阐明在线虫咽内结合PHA-4产生不同细胞类型的调控网络。我们的目标是：1)通过分析14个部分丧失PHA-4功能的基因抑制物来研究PHA-4上游的调控电路。我们预测这些突变体可能分为三类，我们已经设计了区分这三类的方法。我们将从基因上确定抑制者的特征，并启动分子分析。Ii)确定控制咽部基因表达的组合密码。我们已经通过微阵列鉴定了338个可能在咽部选择性表达的基因。我们已经对可能在咽部细胞内共同调控的基因进行了分组，并正在使用计算方法来确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，并确定它们的反式作用因子。我们假设这些元件将与PHA-4结合部位一起发挥作用，以产生不同的咽部表达模式。 Iii)利用基于RNA干扰的方法扩展咽部发育的途径。我们将调查与微阵列芯片确定的295个候选咽部基因相关的功能丧失表型。我们的目标是发现在PHA-4下游发挥作用的基因，以确定咽肌的命运。我们将对那些看起来最有前途的候选人进行深入分析。

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.