Coup-TF: A maternal factor essential for differentiation along the embryonic axes in the sea urchin Paracentrotus lividus.

Coup-TF, a member of the nuclear receptor super-family, is present in the pool of maternal mRNAs and proteins in the sea urchin egg. The presence of this protein seems to be essential for the execution of the early developmental program, leading to all three embryonic layers. Our results demonstrate that Pl-Coup-TF morphants, i.e. Pl-Coup-TF morpholino knockdown embryos, resemble blastulae that lack archenteron at 24 hpf (hours post fertilization), a stage at which normal embryos reach the end of gastrulation in Paracentrotus lividus. At 48 hpf, when normal embryos reach the pluteus larva stage, the morphants are seemingly underdeveloped and lack the characteristic skeletal rods. Nevertheless, the morphant embryos express vegetal endomesodermal marker genes, such as Pl-Blimp1, Pl-Endo16, Pl-Alx1 and Pl-Tbr as judged by in situ hybridization experiments. The anterior neuroectoderm genes, Pl-FoxQ2, Pl-Six3 and Pl-Pax6, are also expressed in the morphant embryos, but Pl-Hbn and Pl-Fez mRNAs, which encode proteins significant for the differentiation of serotonergic neurons, are not detected. Consequently, Pl-Coup-TF morphants at 48 hpf lack serotonergic neurons, whereas normal 48 hpf plutei exhibit the formation of two bilateral pairs of such neurons in the apical organ. Furthermore, genes indicative of the ciliary band formation, Pl-Hnf6, Pl-Dri, Pl-FoxG and Pl-Otx, are not expressed in Pl-Coup-TF morphants, suggesting the disruption of this neurogenic territory as well. In addition, the Pl-SynB gene, a marker of differentiated neurons, is silent leading to the hypothesis that Pl-Coup-TF morphants might lack all types of neurons. On the contrary, the genes expressing signaling molecules, which establish the ventral/dorsal axis, Pl-Nodal and Pl-Lefty show the characteristic ventral lateral expression pattern, Pl-Bmp2/4, which activates the dorsal ectoderm GRN is down-regulated and Pl-Chordin is aberrantly over-expressed in the entire ectoderm. The identity of ectodermal cells in Pl-Coup-TF morphant embryos, was probed for expression of the ventral marker Pl-Gsc which was over-expressed and dorsal markers, Pl-IrxA and Pl-Hox7, which were silent. Therefore, we propose that maternal Pl-Coup-TF is essential for correct dissemination of the early embryonic signaling along both animal/vegetal and ventral/dorsal axes. Limiting Pl-Coup-TF's quantity, results in an embryo without digestive and nervous systems, skeleton and ciliary band that cannot survive past the initial 48 h of development.

Cis-regulatory control of the nuclear receptor Coup-TF gene in the sea urchin Paracentrotus lividus embryo.

Coup-TF, an orphan member of the nuclear receptor super family, has a fundamental role in the development of metazoan embryos. The study of the gene's regulatory circuit in the sea urchin embryo will facilitate the placement of this transcription factor in the well-studied embryonic Gene Regulatory Network (GRN). The Paracentrotus lividus Coup-TF gene (PlCoup-TF) is expressed throughout embryonic development preferentially in the oral ectoderm of the gastrula and the ciliary band of the pluteus stage. Two overlapping λ genomic clones, containing three exons and upstream sequences of PlCoup-TF, were isolated from a genomic library. The transcription initiation site was determined and 5' deletions and individual segments of a 1930 bp upstream region were placed ahead of a GFP reporter cassette and injected into fertilized P.lividus eggs. Module a (-532 to -232), was necessary and sufficient to confer ciliary band expression to the reporter. Comparison of P.lividus and Strongylocentrotus purpuratus upstream Coup-TF sequences, revealed considerable conservation, but none within module a. 5' and internal deletions into module a, defined a smaller region that confers ciliary band specific expression. Putative regulatory cis-acting elements (RE1, RE2 and RE3) within module a, were specifically bound by proteins in sea urchin embryonic nuclear extracts. Site-specific mutagenesis of these elements resulted in loss of reporter activity (RE1) or ectopic expression (RE2, RE3). It is proposed that sea urchin transcription factors, which bind these three regulatory sites, are necessary for spatial and quantitative regulation of the PlCoup-TF gene at pluteus stage sea urchin embryos. These findings lead to the future identification of these factors and to the hierarchical positioning of PlCoup-TF within the embryonic GRN.