A compact cis-regulatory module defines the exclusive expression of the Hbox12 homeobox-containing gene in the presumptive aboral ectoderm

In the sea urchin embryo, a number of genes encoding transcription factors display territorial restricted expression. Among these, the zygotic Hbox12 homeobox gene is transiently transcribed in a limited number of cells of the animal-lateral half of the early Paracentrotus lividus embryo, whose descendants will constitute part of the ectoderm territory. We have explored the cis-regulatory apparatus of the gene and found that the intergenic region of the tandem Hbox12 repeats drives GFP expression in the presumptive aboral ectoderm and that a 234 bp fragment, defined Aboral Ectoderm Module, accounts for the restricted expression of the transgene. Within this module, a consensus sequence for a Sox factor and the binding of the Otx activator are both required for correct Hbox12 gene expression. Site-directed mutagenesis of the cis-regulatory elements, along with loss of function assays and ChIP analysis, strongly suggested that spatial restriction of the Hbox12 gene to the aboral ectoderm is achieved by a combination of different repressive sequence elements. Among them, we identified a Myb-like consensus, which prevents ectopic expression in the oral ectoderm cells. Further negative sequence elements necessary for repression in the endomesoderm map within the most upstream 60 bp region, while down-regulation at the gastrula stage depends on a multiple GA repeat-containing region. Altogether, these results suggest a role for Hbox12 in aboral ectoderm specification and represent our first attempt in the identification of the gene regulatory circuits involved in this process.