cis-Regulatory sequences driving the expression of the Hbox12 homeobox-containing gene in the presumptive aboral ectoderm territory of the Paracentrotus lividus sea urchin embryo

Abstract

Embryonic development is coordinated by networks of evolutionary conserved regulatory genes encoding transcription factors and components of cell signalling pathways. 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. To obtain insights on the regulation of Hbox12 expression, we have explored the cis-regulatory apparatus of the gene. In this paper, we show 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 (AE) 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. Spatial restriction to the aboral ectoderm is achieved by a combination of different repressive sequence elements. Negative sequence elements necessary for repression in the endomesoderm map within the most upstream 60 bp region and nearby the Sox binding site. Strikingly, a Myb-like consensus is necessary for repression in the oral ectoderm, while down-regulation at the gastrula stage depends on a GA-rich region. 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.

Structure–function correlation of micro1 for micromere specification in sea urchin embryos

“…Micro1/pmar1-related genes, PlHbox12, Hpmicro1, Sppmar1, Acmicro1, and Lvpmar1, have been isolated, respectively, from five sea urchins belonging to the order Echinoida: Paracentrotus lividus, Hemicentrotus pulcherrimus, Strongylocentrotus purpuratus, Bernardo et al, 1995;Kitamura et al, 2002;Oliveri et al, 2002;Nishimura et al, 2004;Wu and McClay, 2007). Micro1/pmar1 represents a multicopy gene with polymorphic loci clustered along the Echinoida genome (Nishimura et al, 2004;Sea Urchin Genome Sequencing Consortium, 2006;Ettensohn et al, 2007;Cavalieri et al, 2008).…”

Symmetry Breaking and Establishment of Dorsal/Ventral Polarity in the Early Sea Urchin Embryo

“…In this regard, we have shown in P. lividus that the Hbox12 homeodomain repressor is expressed by prospective dorsal ectoderm cells, spatially facing and preceding the onset of nodal transcription, where it acts preventing the ectopic activation of nodal expression [51][52][53][54]. Functional analysis revealed that expression of hbox12 and nodal genes are mutually exclusive and both required for DV polarization.…”

“…Although the spatial distribution of Myb in the early embryo has not yet been determined, Myb is a redox-sensitive factor [59,60] known to act as a repressor in ventral territories of the sea urchin embryo [61]. This evidence has been provided by the cis-regulatory analysis of the CyIIIa and hbox12 genes, both beginning to be specifically transcribed in the presumptive dorsal ectoderm during early embryogenesis [52,61]. Surprisingly, the cis-regulatory apparatus of the nodal gene also contains a Myb-like consensus binding sequence that, when mutated in gene transfer assays, leads to increased reporter expression [39].…”

Specific expression of a TRIM-containing factor in ectoderm cells affects the skeletal morphogenetic program of the sea urchin embryo

“…Reverse-transcription and Q-PCR analysis were performed as described previously (Cavalieri et al, 2008;Cavalieri et al, 2009a). The oligonucleotide sequences, length, and amplicon size are described in Table S1 in the supplementary material.…”