Accurate adoption of cell fates during development requires transcription of developmentally important genes at the requisite time and abundance. In C. elegans, the number of cells, their division timing and lineage history is uniform, with only minor variations between animals. The need for temporal coordination is especially important in developmental contexts such as the C. elegans embryo, where rapid cell division occurs simultaneously with complex patterns of cell fate decisions, and where temporal misregulation could significantly alter the final pattern of cell fates. The E lineage provides an excellent test case for the study of temporal mechanisms because it is specified early, and clonally produces all intestinal cells. This requires a group of GATA transcription factors, which are expressed at different times in the E lineage. These include
end-1,
end-3 (transcribed beginning in E),
elt-7 (transcribed beginning in E2) and
elt-2 (transcribed beginning in E4, and maintained through the life of the animal by autoregulation). These factors share a common DNA binding motif, but the difference in timing of transcription of the E-specific genes during development raises the question of how this temporal specificity is achieved. We mined RNA-seq data from the Yanai lab to identify hundreds of genes specifically expressed either early (prior to E4) or late (after E8) in isolated E cell progeny. In the pharynx, temporal control is achieved by motif strength; early expressed genes have higher affinity for the pharynx-specific transcription factor PHA-4, allowing them to be bound and regulated earlier when the PHA-4 concentration is lower. In contrast, early E lineage genes actually have fewer promoter GATA motifs and less GATA factor binding in yeast one-hybrid data compared with late E lineage genes. This suggests that other factors may regulate early E lineage expression. Consistent with this, we identified several candidate transcription factors (TFs) with motifs enriched upstream of early genes including motifs for the E2F, Atf, Myb and Ets family TFs. All of these are expressed at the appropriate stage and thus are candidate temporal regulators. Intriguingly, a similar analysis of early vs. late genes expressed broadly across lineages identifies a different set of motifs, suggesting that dissimilar mechanisms may control their expression timing. To test the role of these predicted temporal regulators, we are examining the effect of their loss of function on the timely expression of E lineage genes using automated lineage tracing methods. This will be followed up by mutagenizing the motifs in the promoter regions of affected genes, and testing for TF binding to these motifs in vitro by electrophoretic mobility shift assays. Our results will help understand how the integration of lineage and cell division specific transcription codes direct expression timing and developmental robustness.