Embryonic and post-embryonic utilization and subcellular localization of the nuclear receptor SpSHR2 in the sea urchin

SpSHR2 (Strongylocentrotus purpuratus steroid hormone receptor 2) is a nuclear receptor, encoded by a maternal RNA in the sea urchin embryo. These maternal SpSHR2 transcripts, which are present in all cells, persist until the blastula stage and then are rapidly turned over. A small fraction of the embryonic SpSHR2 protein is maternal, but the majority of this nuclear receptor in the embryo is the product of new synthesis, presumably from the maternal RNA after fertilization. In agreement with the mRNA distribution, the SpSHR2 protein is also detected in all embryonic cells. Contrary to the RNA though, the SpSHR2 protein persists throughout embryonic development to the pluteus stage, long after the mRNA is depleted. Following fertilization and as soon as the 2-cell stage, the cytoplasmic SpSHR2 protein enters rapidly into the embryonic nuclei where it appears in the form of speckles. During subsequent stages (from fourth cleavage onward), SpSHR2 resides in speckled form in both the nucleus and the cytoplasm of the embryonic cells. The cytoplasmic localization of SpSHR2 differs between polarized and non-polarized cells, maintaining an apical position in the ectoderm and endoderm versus a uniform distribution in mesenchyme cells. Following the end of embryonic development (pluteus stage), the SpSHR2 protein is depleted from all tissues. During the ensuing four weeks of larval development, the SpSHR2 is not detected in either the larval or the rudiment cells which will give rise to the adult. Just prior to metamorphosis, at about 35 days post-fertilization, the protein is detected again but in contrast to the uniform distribution in the early embryo, the larval SpSHR2 is specifically expressed in cells of the mouth epithelium and the epaulettes. In adult ovaries and testes, SpSHR2 is specifically detected in the myoepithelial cells surrounding the ovarioles and the testicular acini. Nuclear SpSHR2 in blastula extracts binds to the C1R hormone response element in the upstream promoter region of the CyIIIb actin gene indicating that the latter may be a target of this nuclear receptor in the sea urchin embryo

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

Intracellular Trafficking of the Nuclear Receptor COUP-TF in Live Sea Urchin Embryos1

COUP-TFs comprise a family of highly conserved transcription factors within the steroid-thyroid-retinoic acid super-family of nuclear receptors. The high degree of conservation among the COUP-TFs suggests that their function among distantly related species might also be conserved. In order to give some insight into this question, we have tested the ability of human COUP-TF to mimic a specific sub-cellular localization pattern characteristic of endogenous SpCOUP-TF in early sea urchin embryos. Similarly, experiments were undertaken to determine if five conserved domains within COUP-TF could act as distinct entities, independent of the complete COUP-TF protein. Fusion constructs were created between the Green Fluorescent Protein (GFP) and the entire human COUP-TF as well as various sub-domains of the sea urchin SpCOUP-TF. Intracellular trafficking of the fusion proteins in live embryos was monitored by confocal fluorescent microscopy. The results of this study suggest that, independent of intact COUP-TFs, the various domains tested are not by themselves sufficient for maintaining the correct subcellular localization pattern. The two intact transcription factors, although they belong to such evolutionarily distant animals, do demonstrate similar intracellular localization in the developing sea urchin embryos, indicating functional conservation between the COUP-TFs

Comparison of <i>Coup-TF's</i> upstream and 5′UTR regions between <i>P.lividus</i> and <i>S.purpuratus</i>.

<p><b><i>A</i></b>: The 5′UTR and the upstream sequence of the PlCoup-TF gene, numbered from the −1930 position. The data were obtained by subcloning and sequencing a 2.5 kb fragment of the λ clone “<i>Φ</i>” insert (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109274#pone-0109274-g002" target="_blank">Fig. 2</a>). The shaded areas correspond to various degrees of homology with the corresponding sequence of <i>SpCoup-TF</i> as revealed by comparisons using the program BLAST. The lighter the shade, the lesser the homology is between the ortholog genes of the two species. Thus, the darkest shade corresponds to the 5′UTR sequence that exhibits the highest homology. The small black arrow denotes the transcription initiation site. Underlined is the upstream sequence of module <i>a</i>, which is not homologous between the two species. <b>B</b>: Graphic comparison of the 5′UTR and 5′ upstream sequences of the two orthologous genes, <i>PlCoup-TF</i> (top) and <i>SpCoup-TF</i> (bottom) using the Family Relations program (32). Crossbars joining the two sequences indicate homology, the thickest of which corresponds to the 5′UTR region.</p

Spatial expression patterns of site-specific mutations into module <i>a</i>.

<p><b>A</b>: Graphic presentation of the wt and mutant sequences of the three elements within module <i>a</i>. The top line depicts the 320 bp region of the wt module <i>a</i>, and the sequences that correspond to the sites RE1 (−453), RE2 (−432) and RE3 (−377) and their respective position. Each additional line shows the nucleotides that substitute for the wt sequence at each site. The bottom line depicts the double mutation, which deletes also the intervening sequences between RE1 and RE2. <b>B</b>: Composite pictures of embryos expressing GFP resulting from injection of wt and mutant module <i>a</i>. The wt module <i>a</i> and mutation −453 show GFP expression specifically in the ciliary band, while mutation −432 shows expression in ciliary band, endoderm and aboral ectoderm. Mutation −377 shows GFP expression in aboral ectoderm and the double mutation −453/−432 in aboral ectoderm, endoderm and ciliary band. All embryos were photographed at pluteus stage.</p

Spatial expression patterns generated by the individual segments a–f fused to the GFP cassette.

<p><b>A</b>: Graphical positioning of the upstream <i>PlCoup-TF</i> segments (a–f) within the 1932 bp upstream sequence, which were individually fused to the <i>EpGFPII</i> reference gene. The numbers surrounding each black bar correspond to the nucleotide borders of each segment. Other designations are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109274#pone-0109274-g004" target="_blank">Figure 4A</a>. <b>B</b>: Composite pictures of embryos resulting from injection of segments a–c. Segment a results in GFP expression specifically in the ciliary band, while segments b and c show GFP expression in the ciliary band, endoderm and mesenchyme cells. All embryos were photographed at pluteus stage. Embryos injected with segments d, e and f did not exhibit GFP expression.</p

Specific binding of embryonic nuclear proteins to elements RE1, RE2 and RE3.

<p>The DNA binding specificity of transcription factors to the elements RE1, RE2 and RE3 was determined by electrophoretic mobility shift assays. NE: Nuclear Extract; SC: Specific Competitor. (−) and (+) denote omission and addition of nuclear extract or specific competitor to each reaction respectively. The arrows point to protein: DNA complexes, which are not formed in the presence of specific competitor.</p

Embryonic cell lineage specific expression of the GFP reference gene for each of the upstream deletions, individual upstream segments and mutations.

<p>Cb: Ciliary band; Cb+: Ciliary band and other cell types; En+: Endoderm and other cell types; Ae+: Aboral ectoderm and other cell types; Me+: Mesenchyme and other cell types.</p><p>Embryonic cell lineage specific expression of the GFP reference gene for each of the upstream deletions, individual upstream segments and mutations.</p

Spatial expression patterns generated by the upstream deletions of the GFP cassette.

<p><b>A</b>: Map of <i>PlCoup-TF's</i> upstream sequence (1930 bp) fused to the <i>EpGFPII</i> reference gene. The bended arrow marks the transcription initiation site. EpAc refers to <i>Endo16′s</i> basal promoter and <i>CyIIa's</i> kozak sequence and ATG. The numbers above the map indicate the starting point of each upstream deletion. <b>B</b>: Composite pictures (GFP epi-fluorescence over bright field image) of embryos resulting from injection of the corresponding deletions −1639 to −232. Constructs −1639, −1398 and −781 show GFP expression in ciliary band and a few mesenchyme cells. Construct −532 shows GFP expression only in ciliary band and construct −232, in the aboral ectoderm. All embryos were photographed at pluteus stage. A picture of an embryo injected with the construct −19 is not shown, since these embryos never exhibited any GFP expression.</p

Spatial expression patterns generated by upstream deletions D-a1 to D-a5 of module <i>a</i>.

<p><b>A</b>: Graphic presentation of the upstream deletions into module a (−532 to −212). Horizontal bars underneath module <i>a</i>, represent the size of each deletion (D-a1 to D-a5) and the numbers above them the corresponding upstream border. The numbers at the right of each bar correspond to the size of each fragment tested. EpAc refers to <i>Endo16′s</i> promoter and <i>CyIIa's</i> kozak sequences as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109274#pone-0109274-g004" target="_blank">figure 4A</a>. The two parallel bars within the black box at the right end of the graph denote that the <i>GFP</i> gene is not depicted on scale. <b>B</b>: Composite pictures of embryos resulting from injection of upstream deletions into module <i>a</i>. The entire module <i>a</i> shows GFP expression specifically in the ciliary band, while deletions D-a1, D-a2 and D-a3 show expression both in ciliary band and endoderm. Deletions D-a4 and D-a5 show expression in endoderm and mesenchyme cells. D-a5 shows GFP expression in skeletogenic mesenchyme cells (see text for non-specific expression caused by random integration of the GFP cassette). All embryos were photographed at pluteus stage.</p