Precise cis-regulatory control of spatial and temporal expression of the alx-1 gene in the skeletogenic lineage of s. purpuratus

Deployment of the gene-regulatory network (GRN) responsible for skeletogenesis in the embryo of the sea urchin Strongylocentrotus purpuratus is restricted to the large micromere lineage by a double negative regulatory gate. The gate consists of a GRN subcircuit composed of the pmar1 and hesC genes, which encode repressors and are wired in tandem, plus a set of target regulatory genes under hesC control. The skeletogenic cell state is specified initially by micromere-specific expression of these regulatory genes, viz. alx1, ets1, tbrain and tel, plus the gene encoding the Notch ligand Delta. Here we use a recently developed high throughput methodology for experimental cis-regulatory analysis to elucidate the genomic regulatory system controlling alx1 expression in time and embryonic space. The results entirely confirm the double negative gate control system at the cis-regulatory level, including definition of the functional HesC target sites, and add the crucial new information that the drivers of alx1 expression are initially Ets1, and then Alx1 itself plus Ets1. Cis-regulatory analysis demonstrates that these inputs quantitatively account for the magnitude of alx1 expression. Furthermore, the Alx1 gene product not only performs an auto-regulatory role, promoting a fast rise in alx1 expression, but also, when at high levels, it behaves as an auto-repressor. A synthetic experiment indicates that this behavior is probably due to dimerization. In summary, the results we report provide the sequence level basis for control of alx1 spatial expression by the double negative gate GRN architecture, and explain the rising, then falling temporal expression profile of the alx1 gene in terms of its auto-regulatory genetic wiring.