Tinya Fleming et al. (2003) International Worm Meeting "Structure-function analysis of unc-34, the C. elegans Enabled homolog"

Megan Gavin, Megan Dell, Gian Garriga, Wayne Forrester, Tinya Fleming

[International Worm Meeting, 2003]

As a member of the highly conserved Ena/VASP family, UNC-34 possesses an N-terminal EVH1 (Ena Vasp Homology) domain, a central proline-rich domain, and a C-terminal EVH2 domain. unc-34 loss-of-function mutants display defects in cell migration and in axon guidance. unc-34 mutants also show a synthetic embryonic lethality with a was-1 (the WASp ortholog) mutant. To determine how unc-34 is functioning, we are characterizing the domain requirements for UNC-34 activity in cell migration, axon outgrowth, and embryogenesis.Western blots using an antibody directed against the C-terminal EVH2 domain of UNC-34 reveal that most unc-34 alleles lack detectable protein. Currently, we have identified six alleles of unc-34 that produce UNC-34 protein, and will therefore provide information about UNC-34 domains. One of the alleles, unc-34(gm114), is a missense mutation in the EVH1 domain. unc-34(gm114) mutants display the same cell migration and axon outgrowth defects as genetic nulls, indicating that the EVH1 domain is required for these UNC-34 functions. Another allele, unc-34(e315), is a nonsense mutation just upstream of the EVH2 domain. While we detect no protein in unc-34(e315) mutants with the EVH2 antibody, we believe the mutants produce a functional truncated protein because they display minor cell migration and axon outgrowth defects at 15 degrees C. This observation indicates that the EVH2 domain does not play a major role in cell migration and axon outgrowth. Both alleles, however, show complete embryonic lethality with a was-1 mutant, suggesting that both EVH1 and EVH2 domains are essential for this function. We are currently analyzing the other four alleles of unc-34.We are also interested in determining which domains are required for UNC-34 localization. Antibody staining shows that UNC-34 localizes to adherens junctions and axons. In unc-34(gm114) mutants, UNC-34 fails to localize to adherens junctions and axons but is visible in neuronal cell bodies, suggesting that the EVH1 domain is required for localization. UNC-34 localization in other unc-34 mutants is currently being analyzed. We also plan to create unc-34 domain fusions to GFP that are driven by hypodermal and neuronal promoters to test which UNC-34 domain/s are sufficient for localization.

Carlos Carvalho et al. (2005) International Worm Meeting "Where are the Fems? Suppressors of C. briggsae tra-2 are not required for the onset of hermaphrodite spermatogenesis"

Robin C Hill, Eric S Haag, Carlos Carvalho, John Salogiannis, David B Pilgrim

[International Worm Meeting, 2005]

Recent phylogenetic evidence suggests that hermaphroditism in C. elegans and C. briggsae evolved independently (1). Accordingly, the molecular mechanisms controlling germline sex determination in these two species appear to differ. In the C. elegans germline, the transitory inhibition of tra-2 mRNA translation by the GLD-1/FOG-2 complex allows entry in spermatogenesis through activity of the FEM (FEM-1, FEM-2, FEM-3), FOG-1, and FOG-3 proteins. Loss-of-function mutations that disrupt inhibition of tra-2 mRNA translation or the downstream activity of the FEM and FOG proteins result in female worms whose germline only produce oocytes. Interestingly, C. briggsae lacks a fog-2 orthologue and Cb-gld-1 appears to promote female germline fate instead of spermatogenesis (2). Thus, the molecular control that generates a sperm/oocyte-producing germline in C. briggsae remains unknown. In C. elegans, alleles of fem-1 to fem-3 were isolated as suppressors of tra-2 (3), which feminize both XX and XO animals. To search for Fem alleles in C. briggsae, we isolated suppressors of a Cb-tra-2 allele (see poster by Kelleher et al.). 54 independent sup strains were isolated using two different tra-2(ts) alleles. The strains showed different degrees of suppression of the somatic masculinization phenotype. Surprisingly, no female worms were ever seen and all tra-2;sup doubly-mutant strains consist of self-fertile hermaphrodites, suggesting that the C. briggsae orthologues of the Fem genes may not be required for the onset of hermaphrodite spermatogenesis. Complementation experiments revealed the presence of at least three such Fem-like genes in C. briggsae. In addition, double mutant analysis using confirmed or likely C. briggsae alleles of tra-2, tra-3, and fem-2, as well as an unmapped suppressor, indicates that the epistatic relationships in the soma are similar to those in the C. elegans sex determination pathway. Further characterization of the role of Cb-fog genes should help to clarify the mechanism through which C. briggsae XX worms regulate the dual germline fate. 1 Kiontke K, Gavin NP, Raynes Y, Roehrig C, Piano F, Fitch DHA (2004). PNAS 101:9003 2 Nayak S, Goree J, Schedl T (2005) PloS 3: 1 3 Hodgkin J. (1986). Genetics 114:15