GENETIC ANALYSIS OF CHEMOSENSATION IN C ELEGANS

这个正在进行的研究项目的主要目标是使用C。elegans dauer形成作为化学感觉的遗传学易处理模型。Dauer幼虫是一种交替的第三幼虫阶段，其形成受化学感受信号和营养状态的组合调节。以前的工作定义了大量的Daf-C（dauer形成组成型和Daf-d（缺陷型）基因，调节dauer形成，并定义了它们参与一组复杂的遗传途径。这些途径在分子和细胞上是多样的。化学感觉神经元使用cGMP介导的初级感觉转导过程来控制dauer形成，部分通过释放TGF相关配体。下游步骤包括TGF反应途径和与感觉途径整合的胰岛素信号传导途径。这些途径的主要dauer调节输出可能是孤儿daf-12核激素受体的配体，其启动dauer分化。我们对dauer形成的遗传学方法使我们研究了这一途径的各种要素。在这里，我们建议从遗传学上分析一组新的daf基因，我们已经确定，其中大部分似乎在胰岛素信号通路中发挥作用，并从分子上研究这些基因的选择。我们将类似地分析我们新发现的两个基因，它们可能在TGF-β通路的下游步骤起作用。在dauer途径的胰岛素信号分支中，最后一个已知的步骤是翼状螺旋转录因子daf-16。在两个目标中，我们将研究daf-16附近或下游的基因。在遗传学方法中，我们将分离蛋白激酶pdk-1的Daf-c等位基因的抑制子，该等位基因被认为在daf-16的上游起作用。在生物化学方法中，我们将使用结合位点选择、基因组搜索和基于DNA阵列的转录分析的组合来直接搜索daf-16的转录靶点。最后，我们最近发现，daf-19，一个长期以来被认为是调节感觉神经元发育的基因，编码一个RFX型转录因子，控制核心感觉纤毛蛋白的表达。我们将使用基因组搜索，转基因表达测试和DNA阵列分析的组合来进一步研究daf-19的作用。这里研究的所有途径都对应于人类的重要调控途径，许多基因在各种遗传性疾病中受到影响。

The broad objective of this ongoing research project is to use C. elegans dauer formation as a genetically tractable model for chemosensation. The dauer larva is a dispensable alternative third larval stage whose formation is regulated by a combination of chemosensory signals and nutritional status. Previous work defined a large number of Daf-C (dauer formation constitutive and Daf-d (defective) genes that regulate dauer formation, and defined their participation in a complex set of genetic pathways. These pathways are molecularly and cellularly diverse. Chemosensory neurons use a cGMP mediated primary sensory transduction process to control dauer formation, partly through release of a TGF-related ligand. Downstream steps include the TGF-response pathway and an insulin- signaling pathway that integrates with the sensory pathways. It is likely that the main dauer-regulating output of these pathways is the ligand for the orphan daf-12 nuclear hormone receptor, which initiates dauer- regulating output of these pathways is the ligand for the orphan daf-12 nuclear hormone receptor, which initiates dauer differentiation. Our genetic approach to dauer formation has lead us to study diverse elements of this pathway. Here, we propose to genetically analyze a set of new daf genes that we have identified, most of which appear to function in the insulin-signaling pathway, and to molecularly study a select few of these genes. We will similarly analyze two genes that we have newly identified that may act at a downstream step in the TGF-beta pathway. The last known step in the insulin-signaling branch of the dauer pathway is the winged-helix transcription factor daf-16. In two aims, we will study genes that act close to or downstream of daf-16. In a genetic approach, we will isolate suppressors of a Daf-c allele of the protein kinase pdk-1, thought to act just upstream of daf-16. In a biochemical approach, we will use a combination of binding site selection, genome searches, and DNA-array based transcript analysis to search directly for transcriptional targets for daf-16. Finally, we have recently found that daf-19, a gene long thought to regulate sensory neuron development, encodes an RFX-type transcription factor that controls expression of proteins that comprise the core sensory cilium. We will use a combination of genome searches, transgenic expression tests, and DNA-array analysis to further investigate the role of daf-19. All of the pathways under study here correspond to important regulatory pathways in humans, and many of the genes are affected in various inherited disorders.