R11: a cis-regulatory node of the sea urchin embryo gene network that controls early expression of SpDelta in micromeres.

A gene regulatory network (GRN) controls the process by which the endomesoderm of the sea urchin embryo is specified. In this GRN, the program of gene expression unique to the skeletogenic micromere lineage is set in train by activation of the pmar1 gene. Through a double repression system, this gene is responsible for localization of expression of downstream regulatory and signaling genes to cells of this lineage. One of these genes, delta, encodes a Notch ligand, and its expression in the right place and time is crucial to the specification of the endomesoderm. Here we report a cis-regulatory element R11 that is responsible for localizing the expression of delta by means of its response to the pmar1 repression system. R11 was identified as an evolutionarily conserved genomic sequence located about 13 kb downstream of the last exon of the delta gene. We demonstrate here that this cis-regulatory element is able to drive the expression of a reporter gene in the same cells and at the same time that the endogenous delta gene is expressed, and that temporally, spatially, and quantitatively it responds to the pmar1 repression system just as predicted for the delta gene in the endomesoderm GRN. This work illustrates the application of cis-regulatory analysis to the validation of predictions of the GRN model. In addition, we introduce new methodological tools for quantitative measurement of the output of expression constructs that promise to be of general value for cis-regulatory analysis in sea urchin embryos.