The Caenorhabditis elegans larva-to-adult transition is a highly regulated developmental event that involves coordinated behaviors of hypodermal cells: the seam cells, epidermal progenitor cells, exit their self-renewal program and further fuse into a syncytium. Together with the syncytial
hyp7, they secret an adult cuticle, containing a characteristic structure known as alae. Finally, the molting cycle stops. Initiation of the L/A transition at the appropriate time of development is controlled by the heterochronic pathway. The most downstream known gene in this pathway is LIN-29, a Kruppel-family, EGR-type transcription factor. Two
lin-29 isoforms,
lin-29a and
lin-29b have been reported and were proposed to function redundantly based on shared expression patterns and complementation experiments using over-expression. Both isoforms function in a complex with MAB-10, a NAB orthologous co-factor. Here, we revisited the issue of isoform redundancy. Using genome engineering to disrupt the isoforms individually or jointly, we find that different aspects of the L/A transition are controlled by different combinations of LIN-29a, LIN-29b, and MAB-10. Expression analysis using endogenously tagged proteins further reveals that isoforms are expressed in overlapping, but partially unique temporal and spatial patterns. Indeed, we show that differences in expression rather than molecular activity predominantly explain the non-redundant functions of the isoforms. Finally, we identify LIN-41, an RNA-binding protein, and HBL-1, a transcription factor, as factors that shape
lin-29 expression in an isoform-specific manner: whereas LIN-41 prevents precocious LIN-29a and MAB-10 accumulation, HBL-1 does the same for LIN-29b. Taken together, our data demonstrate the existence of two parallel arms of the heterochronic pathway, defined by the two
lin-29 isoforms and the mechanisms that control their temporal expression. We expect that future work will reveal mechanisms that ensure temporally precise, coordinated execution of the different events that constitute the L/A transition, even when they rely on separate pathway arms.