A fundamental question of developmental biology is how initially equivalent cells specify into the many cell types present in multicellular organisms. We study four cells that initially have the potential to be the Anchor Cell (AC) of the C. elegans gonad (Seydoux et al. 1990): two proximal "alpha" cells (Z1.ppp and Z4.aaa) flanked by their distal sisters, the "beta" cells (Z1.ppa and Z4.aap). The beta cells lose AC competence soon after they are born and commit to becoming Ventral Uterine precursor cells (VUs). Their fate is influenced by, but does not require,
lin-12/Notch activity. The fates of the alpha cells are naturally variable (Kimble & Hirsh 1979); they signal to each other via the LIN-12/Notch signaling pathway to determine which becomes the AC and which becomes a VU. Unlike the beta cells, the alpha cells require
lin-12/Notchactivity to adopt the VU fate (Greenwald et al. 1983; Seydoux & Greenwald 1989; Sallee et al. 2015). The transcription factor HLH-2 is crucial to AC specification-it is required to confer AC competence to the alpha and beta cells, to drive transcription of
lag-2 (a LIN-12 ligand) during the AC/VU decision, and for the differentiated functions of the AC (Karp & Greenwald 2003 & 2004). HLH-2 is initially present in the four alpha and beta cells, but is post-translationally degraded in the three VUs by a proteasome-dependent mechanism (Sallee & Greenwald 2015). In addition to the established post-translational regulation of HLH-2, we have now observed that
hlh-2 is transcriptionally downregulated specifically in beta cells, potentially accounting for their early adoption of the VU fate. In contrast, we hypothesize that the post-translational mechanism is necessary for an alpha cell to adopt the VU fate. To elucidate this mechanism, we used CRISPR/Cas9 to engineer an allele of
hlh-2 that encodes GFP-HLH-2 and conducted an RNAi screen of 175 conserved ubiquitin-related genes (Du et al. 2015; Kim et al. 2018). We identified 10 E3 ubiquitin ligases that caused ectopic stabilization of GFP-HLH-2: 4 of these are generally associated with cullin-containing complexes, 3 are monomeric HECT-domain ligases, and 3 are RING-finger ligases. We are currently analyzing these genes for their roles in gonadogenesis and regulation by/of
lin-12.