Erin J. Cram
Veronica Godoy-Carter, Donald O'Malley, Shashi Murthy, Wendy Smith
Date of Award
Doctor of Philosophy
Department or Academic Unit
College of Science, Department of Biology
biology, developmental biology, molecular biology, cellular biology, Cactin, C. elegans, cell migration, Distal tip cell, Gonad development, Rac GTPase
Biology, general | Cell and Developmental Biology
Cell migration is fundamental to the development and physiological maintenance of organisms. Studying how cell migration is regulated is critical to the understanding of several developmental disorders, and it may help develop novel therapeutic approaches to disease conditions caused by erroneous cell migration, such as metastatic tumors. In the C. elegans nematode, the two specialized distal tip cells (DTCs) migrate long distances during nematode development, and therefore provide an in vivo model system for the study of developmentally regulated cell migration. We identified cacn-1/cactin, a well-conserved, novel regulator of cell migration in a genome-wide RNAi screen for regulators of DTC migration. RNAi depletion experiments and analysis of the hypomorphic allele cacn-1(tm3126) indicate that CACN-1 is required during DTC migration for proper pathfinding and for cessation of DTC migration at the end of larval morphogenesis. Strong expression of CACN-1 in the DTCs, and data from cell-specific RNAi depletion experiments, suggest that CACN-1 is required cell-autonomously to control DTC migration. Importantly, genetic interaction data with Rac GTPase activators and effectors suggest that CACN-1 acts in the mig-2/Rac pathway, and possibly in parallel to ced-10/Rac, to control DTC pathfinding. Rac reporter strain observations, as well as real-time PCR and transcriptome sequencing results, indicate that CACN-1 does not control the Rac GTPase pathway transcriptionally. Analysis of Rac GTPase protein levels in cacn-1 RNAi treated animals suggests that cacn-1 does not regulate MIG-2 or CED-10 protein levels. Our Rac activation assay study, on the other hand, shows increased levels of active MIG-2 and CED-10 Rac GTPases as a result of cacn-1 loss. These results suggest that cacn-1 is required for negative regulation of Rac GTPase activity, thereby allowing the proper level of Rac activation required for normal DTC migration.
In order to gain further insight into the mechanism of action of CACN-1, we next examined its role in reproductive system development and germline differentiation. CACN-1 is expressed in the somatic cells of the hermaphrodite reproductive system, and observations in somatic gonad marker strains treated with cacn-1 RNAi show that cacn-1 is required for the development of the somatic reproductive system. However, our observations in rrf-1 mutant animals, in which the somatic (but not germline) RNAi response is defective, suggest that cacn-1 is also required in the germline for fertility. C. elegans, a self-fertile hermaphrodite, produces sperm late in larval development before switching to oocyte production in adulthood. This switch is regulated by a set of RNA binding proteins and splicing factors upstream of the germline sex determination gene fem-3. In cacn-1 RNAi treated animals, copious sperm, but few, if any, oocytes are produced. DAPI staining experiments support the hypothesis that cacn-1 is required to promote the production of oocytes. Genetic interaction studies indicate that cacn-1 normally functions upstream of the sperm-to-oocyte differentiation decision pathway by repressing the fem-3, fog-1, and fog-3 male-fate promoting genes. Transcriptome sequencing data analysis shows upregulation of terminal male-fate differentiation factors, fog-1 and fog-3, in cacn-1 depleted animals. Increased expression of these genes disrupts the germ cell decision to switch to production of oocytes. Therefore, CACN-1 functions similarly to the known sperm-to-oocyte regulatory RNA binding proteins or splicing factors, which negatively regulate the male fate promoting genes of the fem-3 pathway. Moreover, preliminary results show that cacn-1, like these factors, regulates fem-3 through post-transcriptional 3'UTR-mediated repression. Our results, as well as studies linking human Cactin and Arabidopsis CACTIN to the splicing machinery, suggest that cacn-1 might function in mRNA post-transcriptional regulation.
Tannoury, Hiba, "The conserved gene CACN-1 controls cell migration and germline differentiation in C. Elegans" (2012). Biology Dissertations. Paper 28. http://hdl.handle.net/2047/d20002663
Available for download on Thursday, October 24, 2013
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