An otx cis-regulatory module: a key node in the sea urchin endomesoderm gene regulatory network

An essential node in the gene regulatory network (GRN) for endomesoderm specification in the sea urchin embryo lies within the regulatory system of the otx gene. According to the predictions of the GRN, based on perturbation analysis and expression data, the beta1/2 transcription unit of this gene is engaged during specification in interactions with two other regulatory genes, krox and gatae. It is predicted to be driven into activity by the krox gene, to form a positively reinforcing functional loop with the gatae gene, and to respond to its own output as well. Here we isolate the relevant otx cis-regulatory element, and examine the specific input predictions of the GRN at the level of its genomic DNA sequence. This beta1/2-otx regulatory module performs the necessary functions, as shown by use of expression constructs. It requires gatae, otx, and krox inputs, as predicted, and it operates as an "AND" logic processor in that removal of any one of these inputs essentially destroys activity. The necessary target sites were identified in the module sequence, and mutation of these sites was demonstrated to produce the same respective effects on construct expression as does blocking its regulatory inputs by treatment with morpholino antisense oligonucleotides. For spatial expression in the endoderm, one particular pair of Gata sites is essential and these function synergistically with an adjacent Otx site. We thus demonstrate directly the structure/function relationships of the genomic regulatory code, at this key node of the endomesoderm GRN.