Morphogenesis of multicellular organisms requires the synthesis and assembly of complex extracellular structures. Little is known about how such structures are formed. The goals of the research proposed here are to understand, at the molecular level, how extracellular molecules interact to build complex structures, and how these extracellular structures are involved in organismal morphogenesis. Studies will be performed with the nematode, Caenorhabditis elegans, because its normal morphogenes is well characterized, a large number of morphogenesis defective mutants have been described, and it is possible to study these mutants at a molecular level. Many C. elegans morphogenesis mutants have defects in the structure of the cuticle. For example, mutations in the sqt-1 and rol-6 genes transform the normally linear cuticle into a helically twisted structure. The cuticle is a complex, multi-layered extracellular structure that is composed of collagens. Collagens are the major class of extracellular (connective tissue) molecules in all multicellular animals. Several human connective tissue disorders have been shown to result from defects in collagens, however, the molecular bases for most connective tissue disorders are not known. Studies of the collagens in a simple system, such as the cuticle of C. elegans, will provide a better understanding of how collagens assemble into complex extracellular structures. Wild type and mutant alleles of the C. elegans morphogenesis genes sqt-1 and rol-6 will be cloned and analyzed. Previous studies suggest that these genes encode collagens, however, they could also encode proteins that are involved in the assembly of collagens into the cuticle structure. DNA sequence analysis of the clones will determine their identities. Comparisons between the wild type and mutant alleles will identify the nature of the mutational changes that cause severe morphological abnormalities, such as helical twisting. Mutant alleles will be transformed back into C. elegans strains in which the normal sqt-1 or rol-6 genes have been deleted. Successful transformation will produce helically twisted progeny animals. It will then be possible to mutagenize genes in vitro, transform them back into animals, and determine the affects on cuticle structure and morphogenesis. These studies will provide insights into the molecular mechanisms that control assembly of collagens into complex extracellular structures.
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