Cell migration is critical in generating complex animal forms during development; misregulation of migration contributes to pathological conditions such as cancer metastasis. Thanks to its easily traceable cell lineages in a transparent body and a compact genome accessible to a wealth of genetic manipulations, the use of the nematode C. elegans as a model system has greatly advanced our understanding of mechanisms governing cell migration conserved through higher organisms. Among several migration processes in C. elegans, sex myoblast (SM) migration is an attractive system that has a simple and well-defined migratory route along the ventral side from the posterior to the precise center of the gonad. A multitude of guidance mechanisms control SM migration, many of which are likely to be conserved in other migratory processes. Similar to vertebrate systems, C. elegans uses Rho family small GTPases to regulate the engine of cell motility, the actin cytoskeleton, in response to guidance cues. The differential utilizations of Rho GTPases in distinct processes in vivo remain a central question in the study of Rho GTPases. I investigated how Rho GTPases regulate different aspects of SM migration, and found that Cdc-42/CDC42 functions in the anteroposterior migration, whereas MIG-2/RhoG and CED-10/Rac1 control ventral restriction independently of FGF and SLIT/Robo signaling. The relative difficulty in perturbing SM migration using constitutively active Rho GTPases compared to other migration processes illustrates the robustness of the mechanisms that control SM migration. On a technical aspect, I established a nematode larval cell culture system that allows access to postembryonic cells. Compared to the flourishing genetic researches in C. elegans, there are few studies of molecules that also extend to the subcellular level in postembryonic development, mainly due to the lack of a larval cell culture system. I developed a novel method combining SDS-DTT presensitization of larval cuticles and subsequent pronase E digestion. My method efficiently isolates both low- and high-abundance cell types from all larval stages. This technical advance will not only facilitate studies such as regulation of actin dynamics with high-resolution microscopy, but is beginning to be used by researchers to tackle cell-type specific questions through profiling methods as gene expression analysis. / Ph. D.

microRNAs are single-stranded small RNAs that function as post-transcriptional regulators of gene expression. We are studying the mir-44 family, specifically mir-44 and mir-45, which have identical sequence. Loss of mir-44 and mir-45 results in defects that suggest that the mir-44 family acts to negatively regulate the MAPK pathway. The MAPK pathway regulates sex myoblast migration, a process which is required for normal egg laying. We hypothesized that the mir-44 family of microRNAs is necessary for normal sex myoblast migration and subsequent formation of the functional egg laying structure in the hermaphrodite. We created a mutant that had mutations in both mir-44 and mir-45 and a transgene that expresses GFP in the sex myoblast cells. Then we observed the migration and division of the sex myoblasts in wild-type and mutant worms using fluorescence microscopy. In all cases, the mutant worms displayed a greater percent difference from average sex myoblast migration and division. However, a two-tailed two-proportions z-test found no significant difference between wild type and mutant sex myoblast migration (p=0.9148), nor in mutant sex myoblast division along the axial (p=0.4205) and sagittal (p=0.3583) planes of the body. This allows us to conclude that mir-44 and mir-45 are unlikely to be responsible for the migration nor division of the sex myoblasts, and the defects are likely due to interference with a different biological mechanism.