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(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，我们发现了FoxA转录因子同源基因pha-4，并表明它是咽部发育的关键调节因子。我们还确定了与PHA-4结合起作用的其他因子。这些研究为理解控制前肠发育的转录网络奠定了基础，并提出了我们在当前提案中解决的三个关键问题：目标1将确定PHA-4与其目标启动子结合的后果。不同DNA结合部位对PHA-4的相对亲和力调节靶基因表达的开始。我们已经开发了工具来可视化PHA-4与其在活胚胎中的目标启动子的关联。我们将利用这一实验来问：i)结合位点亲和力如何影响PHA-4与其靶基因的结合；ii)PHA-4与靶启动子结合的下游后果是什么？这些实验将讨论DNA与PHA-4的亲和力如何影响与启动子激活相关的事件，即PHA-4结合、DNA分解以及额外的转录因子与目标启动子的关联。这些研究将使我们能够检验PHA-4作为前肠转录的能力因子，进而影响前肠命运的假设。目的2通过分析13个PHA-4功能部分丧失的基因抑制子，研究PHA-4上游的调控电路。我们预测这些突变体在转录或转录后调节PHA-4，我们已经设计了区分这些类别的方法。我们将从基因上确定抑制者的特征，并启动分子分析。目标3将阐述与PHA-4一起发挥作用的过程和分子，以承诺放弃命运。我们的初步研究已经在胚胎发生中定义了一个窗口，当细胞可以对PHA-4做出反应而承担前奏命运时。在此阶段之前，细胞呈现发育可塑性，而在此阶段之后，细胞致力于成为咽部。我们已经开发了测量发育可塑性的方法，我们将用它来探索控制这种转变的机制。我们还使用微阵列来鉴定此时表达的基因，并将调查这些基因在维持多能性或促进向细胞命运承诺的过渡中可能发挥的作用。这些研究将使我们能够检验这样的假设，即发育可塑性的丧失是由序列特异性转录因子与全球转录调控机制相结合控制的。

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 (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.