Highlights d LIN-12/Notch resolves a stochastic cell fate decision of two equipotential a cells d Two stochastic events bias their fates: relative birth order and HLH-2 expression d Onset of HLH-2 expression is linked to the birth of the parents of the a cells d LIN-12 expression requires HLH-2: a potential deterministic decision mechanism

The Caenorhabditis elegans E protein ortholog HLH-2 is required for the specification and function of the anchor cell (AC), a unique, terminally differentiated somatic gonad cell that organizes uterine and vulval development. Initially, four cells—two α cells and their sisters, the β cells—have the potential to be the AC. The β cells rapidly lose AC potential and invariably become ventral uterine precursor cells (VUs), while the two α cells interact via LIN-12/Notch in the “AC/VU decision” to resolve which will be the AC and which will become another VU. HLH-2 protein stability is dynamically regulated in cells with AC potential; initially present in all four cells, HLH-2 is degraded in presumptive VUs while remaining stable in the sole AC. Here, we demonstrate that stability of HLH-2 protein is regulated by the activity of lin-12/Notch in both α and β cells, providing evidence that activation of LIN-12 in an α cell promotes degradation of HLH-2 as part of a negative feedback loop during the AC/VU decision in α cells, and that ectopic stabilization of HLH-2 promotes AC fate in β cells. We also performed an RNAi screen of 232 ubiquitin-related genes and identified seven genes that contribute to HLH-2 degradation in VUs. When HLH-2 is stabilized by RNAi-depletion of ubiquitin ligases in a lin-12(+) background, there are no supernumerary ACs, suggesting that LIN-12 activation does not oppose hlh-2 activity solely by causing HLH-2 protein degradation. Furthermore, we provide evidence that hlh-2 transcription is negatively regulated in β cells independent of lin-12 activity. Together, our results indicate that hlh-2 activity is regulated at multiple levels to restrict the AC fate to a single cell.

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