Molecular Genetics of Basement Membranes in C Elegans

描述（由申请人提供）： 基底膜是在所有后生动物中发现的专门细胞外基质，参与许多重要的发育过程，包括：细胞粘附、细胞和轴突迁移以及细胞分化。它们还参与某些病理状况，如肿瘤转移、伤口愈合、皮肤起泡疾病和各种形式的肾炎。本申请的广泛目标是利用线虫线虫中可用的遗传工具来阐明基底膜功能。一种主要的保守的基底膜蛋白是巢蛋白，其通过与层粘连蛋白、IV型胶原和串珠素结合，被认为对基底膜组装至关重要。使用秀丽隐杆线虫hid-1巢蛋白基因的缺失突变，我们已经表明，这是不需要的活力或基底膜组装。然而，我们发现它在轴突导向、突触形态发生和神经肌肉协调中具有重要功能。该建议的第一个目的是通过在nid-1缺失动物中转基因表达修饰的nid-1构建体来确定这些不同功能所需的NID-1的哪些结构域。我们还将检查何时何地需要nid-1表达式。第二个目标是鉴定nid-1和与hid-1相互作用或在相同过程中起作用的基因的新等位基因。我们将通过筛选包含大多数C. elegans基因第三个目标是描述nid-1与肌营养不良蛋白聚糖基因dgn-1或转录因子unc-39突变结合时的合成致死性。这些相互作用揭示了nid-1在上皮极性中的潜在功能，而这在单个突变体中并不明显。将在光学和电子显微镜水平上表征致死性潜在的细胞表型，并寻找可以抑制致死性的突变。这些研究将阐明巢蛋白的特征，这些特征对巢蛋白的特定功能至关重要，并确定与巢蛋白相互作用的其他分子。

DESCRIPTION (provided by applicant): Basement membranes are specialized extracellular matrices found in all metazoans and involved in many important developmental processes, including; cell adhesion, cell and axon migration, and cell differentiation. They are also involved in certain pathologic conditions, such as tumor metastasis, wound healing, blistering diseases of the skin, and forms of nephritis. The broad goal of this application is to elucidate basement membrane functions utilizing the genetic tools available in the nematode Caenorhabditis elegans. A major conserved basement membrane protein is nidogen, which, via its binding to laminin, type IV collagen and perlecan, was thought to be critical for basement membrane assembly. Using deletion mutations of the C.elegans hid-1 nidogen gene, we have shown that is not required for viability or basement membrane assembly. However, we find it has significant functions in axonal guidance, synaptic morphogenesis, and neuromuscular coordination. The first aim of this proposal will determine which domains of NID-1 are required for these various functions, by transgenic expression of modified nid-1 constructs in nid-1 null animals. We will also examine when and where nid-1 expression is required. The second aim will identify new alleles of nid-1 and of genes that interact with, or function in the same processes as, hid-1. We will identify genes that become critical in the absence ofnidogen, by screening of an RNAi feeding library encompassing most C. elegans genes. The third aim is to characterize the synthetic lethality that results when nid-1 is combined with mutations in the dystroglycan gene dgn-1 or the transcription factor unc-39. These interactions reveal potential nid-1 functions in epithelial polarity, that are not apparent in the single mutants. The cellular phenotypes underlying lethality will be characterized at the light and electron microscope levels, and mutations that can suppress lethality will be sought. These studies will elucidate features of nidogen that are critical for it specific functions and identify other molecules with which it interacts.