Cis-regulatory analysis of the sea urchin delta gene : validating the architecture of the gene regulatory network model for micromere lineage specification

Abstract

During the specification of the endomesoderm of the sea urchin embryo, mesodermal and endodermal cell types derive from common progenitors. The Delta signal, a ligand of the Notch receptor, serves as the spatial cue that triggers the segregation between these two fates. Expression of the delta gene exclusively in the micromere lineage early in development is essential for Delta to be able to correctly serve this role. According to a model of the gene regulatory network (GRN) underlying this process, the mechanism by which the micromere lineage is specified as a distinct domain, and by which the delta gene is expressed exclusively there, depends on a double repression system. A gene encoding a transcriptional repressor, pmar1, is activated specifically in the micromeres, where it represses transcription of a second repressor that is otherwise active globally. Zygotic expression of delta and micromere specific control genes depends on ubiquitous activators, and localization in the micromere lineage depends on repression by the second repressor everywhere else. In this model the second repressor is an unidentified gene, the existence of which is implied by numerous experiments. The work presented in this thesis experimentally validates the double repression architecture for micromere lineage specification and localization of delta expression. To prove the existence of the double repression system a genomic screen was devised to identify the gene playing the role of the second repressor. hesC, a transcription factor of the HES family, was found to be this gene. It is expressed at the right time and place, and its function is to repress micromere specific regulatory genes. To show that expression of delta in the micromere lineage depends on ubiquitous activators and HesC-dependent repression, the relevant cis-regulatory module (CRM) was recovered. This CRM, named R11, is shown to be able to drive the expression of a reporter gene exclusively in the micromere lineage at the right time. Dissection of R11 and its response to blockade of hesC expression show that R11 expression depends on ubiquitously present activators, and on HesC-dependent repression everywhere except the micromere lineage.