A novel molecule in the Ras signaling pathway, Flightless-1 (FLI-1), affects germ line morphogenesis, muscle development and germ line tumor formation in Caenorhabditis elegans

Abstract

The wild-type C. elegans germ line is a syncytium with multiple nuclei sharing a common cytoplasm. At the distal tips, germ cell nuclei are mitotic and have stem cell character. As the syncytial nuclei move proximally, they enter meiosis and become arranged at the germ cell plasma membrane, forming a single layer of nuclei around the nucleus-free center (the rachis). Fingers of germ cell plasma membrane invaginate around the nuclei but do not completely enclose them. In fli-1 mutants, chains of meiotic germ line nuclei were present in the central rachis. Germ cell plasma membrane and the overlying sheath cell, which normally invaginates into the spaces between germ nuclei, remained associated with the misplaced meiotic nuclei, suggesting excessive invagination of the germ cell plasma membrane and sheath cell. This arrangement is reminiscent of the normal distal tip region, suggesting that fli-1 might control the morphogenetic rearrangement of nuclei accompanying entry into meiosis. A rescuing full-length fli-1::gfp reporter gene was expressed in the germ line, and FLI-1::GFP associated with germ line nuclei.

To dissect molecular mechanisms of FLI-1 activity in the germ line, interactions of fli-1 with other mutations affecting germ line development were analyzed. Loss-of-function and dominant-negative mutations in the let-60 Ras and other Ras pathway components also caused this germ cell phenotype. Constitutively-active LET-60 Ras partially rescued the germ cell defect in both fli-1 alleles, suggesting that FLI-1 and LET-60 Ras control germ cell morphogenesis together. While fli-1 alone caused no defects in mitotic or meiotic differentiation, preliminary results indicate fli-1(ky535) partially suppressed the germ cell tumor phenotype of gld-1(q485). In gld-1(q485) , germ cells re-enter mitosis after beginning meiotic differentiation and proliferate until they fill the gonad and eventually the body cavity, killing the animal. fli-1(ky535);gld-1(q485) animals survived slightly longer than gld-1 alone, and staged animals displayed fewer and larger tumorous germ cells. Experiments are underway to determine how fli-1 mutation suppresses gld-1, but initial analyses indicate that fli-1 might delay reentry into mitosis.

Drosophila flightless 1 mutants display disorganized indirect flight muscle structure. We found that FLI-1 also controls muscle development in C. elegans. In fli-1(ky535) mutants, disorganized body wall muscle myofilament structure was observed by both TEM and birefringence. The fli-1(tm362) caused Pat phenotype (Paralyzed Arrested at Two fold stage), suggesting severe body wall muscle defects.