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 i s 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.
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