Regulation of seamless tube development

生物管是多细胞生物中许多器官的基本结构。它们的功能是长距离输送气体、液体和细胞，以提供生长和发育所必需的基本因素。肾脏、肺和脉管系统几乎完全由管道组成，这些管道以多细胞（上皮）和单细胞（无缝）单位存在。尽管这些管可以以许多不同的方式发育，但它们都有一个共同的特征，即顶表面衬在管腔内。本研究计划的长期目标是了解利用线虫秀丽隐杆线虫的排泄细胞作为模型系统的生物管发育机制。使用蠕虫的主要优点是其强大的遗传学，体内成像和可获得的排泄系统试剂。各种研究表明，内吞运输和肌动蛋白细胞骨架在排泄管延伸和维持膜完整性中起着关键作用。然而，人们对这些过程是如何调节以确保在管延伸过程中协调的膜添加仍然知之甚少。我们最近发现CCM-3蛋白通过小GTPase CDC-42和内吞循环调节管道延伸。我们对脑海绵体畸形和CCM-3蛋白的兴趣是由CIHR资助的一个独立研究分支的主题，在该分支中，我们使用排泄管作为CCM-3活性的读数。

Biological tubes make up fundamental structures of many organs in multicellular organisms. They function to transport gases, fluids and cells over long distances to supply essential factors necessary for growth and development. Kidneys, lungs and vasculature are comprised almost entirely of tubes, and these exist as both multicellular (epithelial) and unicellular (seamless) units. Although these tubes can develop in many different ways they all share the common feature of an apical surface lining the lumen. The long-term goals of this research program are to understand the mechanisms by which biological tubes develop using the excretory cell of the nematode roundworm Caenorhabditis elegans as a model system. The key advantages of using the worm are its powerful genetics, in vivo imaging, and availability of reagents for the excretory system. Various studies have shown critical roles for endocytic trafficking and the actin cytoskeleton in excretory canal extension and maintenance of membrane integrity. However, it remains poorly understood how these processes are regulated to ensure coordinated membrane addition during tube extension. We recently found that the CCM-3 protein regulates canal extension through the small GTPase CDC-42 and endocytic recycling. Our interests in cerebral cavernous malformations and the CCM-3 protein is the subject of a separate branch of research funded by CIHR, where we use the excretory canal as a readout for CCM-3 activity. To understand the basic mechanisms governing excretory canal development we surveyed a list of cytoskeletal and trafficking genes using RNA interference (RNAi) to knock down their expression. From this pilot screen we identified 2 intermediate filament proteins, 3 small GTPase effectors, 2 exocyst proteins, and 1 formin that caused canal truncations when ablated. The short-term goals are to delineate how intermediate filaments and endocytic trafficking regulate tube extension and membrane integrity. Therefore, we will first define their expression patterns and determine if they affect the localization and/or expression of endocytic and cytoskeletal proteins using a panel of fluorescent markers. Using electron microscopy we will determine if these proteins affect the structures of organelles and vesicles required to extend canals. We will also perform epistasis analysis by creating double mutants to define linear and parallel signalling pathways. Finally, we will delineate upstream and downstream components of these pathways by performing a genome-wide RNAi screen for genes that affect canal extension and membrane integrity. This work will uncover conserved mechanisms governing how endocytic trafficking and intermediate filament proteins collaborate to promote seamless tube development in vivo.