The C. elegans pharynx consists of 5 different cell types - muscles, neurons, epithelia, glands, and marginal cells. Pharyngeal cells are derived from the ABa and MS blastomeres, which form anterior and posterior pharynx, respectively. We are functionally characterizing the T-box gene,
tbx-2, and its role in pharyngeal development. We found
tbx-2 loss resulted in a lack of ABa-derived pharyngeal muscle and L1 arrest. In comparison,
tbx-2 loss did not eliminate ABa-derived marginal cells or MS-derived pharynx. Both a full-length
tbx-2::gfp translational fusion and a
tbx-2 promoter::gfp fusion were expressed in 12 likely ABa-derived pharyngeal precursors near the 200 cell stage. Full-length
tbx-2::gfp expression was transient and disappeared prior to pharyngeal formation, but
tbx-2 promoter::gfp perdured until hatching and was detectable in a subset of pharyngeal muscles which included all ABa-derived pharyngeal muscle types. In an extensive yeast two-hybrid screen, we found TBX-2 interacted specifically with a small number of proteins, which included UBC-9, GEI-17, and UNC-37. UBC-9 and GEI-17 are components of the SUMO-conjugating pathway, suggesting TBX-2 activity is regulated by sumoylation, and indeed, TBX-2 contains 2 consensus sumoylation sites.
ubc-9(RNAi) resulted in a loss of ABa-derived pharyngeal muscles and altered localization of full-length TBX-2::GFP fusion protein to sub-nuclear puncta.
gei-17(RNAi) resulted in partially penetrant larval lethality and pharyngeal defects similar to those of mildly affected
tbx-2(RNAi) animals. The
gei-17(RNAi) lethality and pharyngeal defects were enhanced in a weak
tbx-2 mutant strain. UNC-37 is the C. elegans Groucho/TLE corepressor, and TBX-2 contains a potential
eh1 motif that may mediate interaction with UNC-37.
unc-37 null mutants were previously shown to exhibit sterility and maternal effect lethality, and
unc-37(RNAi) produced elongation defects and embryonic lethality. We are currently examining the pharyngeal phenotype of
unc-37(RNAi) and
unc-37 null mutants to determine the role of this gene in pharyngeal development. Both sumoylation and interaction with Groucho/TLE proteins are usually associated with transcriptional repression, and we hypothesize TBX-2 is a repressor whose activity depends on SUMO and UNC-37.
tbx-2 is the sole C. elegans member of the phylogenetically conserved Tbx2 sub-family, and our work with
tbx-2 may reveal functions conserved in other Tbx2 sub-family members.