Kalampoki LG , Flytzanis CN .

	Figure 1. Spatial expression pattern of the PlCoup-TF gene.In situ hybridization of P.lividus embryos with antisense and sense digoxygenin labeled PlCoup-TF probes. a–f: antisense; g–l: sense probe hybridization. The maternal PlCoup-TF mRNA seems evenly distributed in the egg and at the 16-cell stage embryo. Zygotic transcripts are expressed in the presumptive oral ectoderm at blastula and gastrula stages and in the ciliary band at prism and pluteus stages. E: Unfertilized egg; 16c: 16-cell stage embryo; B: Hatching blastula; G: Gastrula; P: Prism and Pl: Pluteus.
	Figure 2. Restriction digest map of the overlapping inserts of genomic clones “A” and “Φ”.The arrow marked by +1 symbolizes the initiation site and the direction of transcription. ‘atg’, marks the site of translation initiation. The scale (1 kb) is shown by a small bar. The enzymes used for mapping are: B: BamHI; E: EcoRI; H: HindIII; K: KpnI; S: SalI; Sc: SacI.
	Figure 3. Identification of PlCoup-TF transcription initiation sites. A: Electrophoretic analysis of the 5'-RACE products. The two arrows point to the major DNA bands produced by the nested PCR (lane 1). The 100 bp ladder (NEB) was used as DNA length reference (lane M). B: Sequence of the proximal PlCoup-TF promoter and the positions of the multiple transcription initiation sites (capital and lower case bold characters). The most upstream site was designated as +1. A putative CCAAT box is underlined at position −65. The translation initiation site is located at +544.
	Figure 4. Spatial expression patterns generated by the upstream deletions of the GFP cassette. A: Map of PlCoup-TF's upstream sequence (1930 bp) fused to the EpGFPII reference gene. The bended arrow marks the transcription initiation site. EpAc refers to Endo16′s basal promoter and CyIIa's kozak sequence and ATG. The numbers above the map indicate the starting point of each upstream deletion. 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.
	Figure 5. Spatial expression patterns generated by the individual segments a–f fused to the GFP cassette. A: Graphical positioning of the upstream PlCoup-TF segments (a–f) within the 1932 bp upstream sequence, which were individually fused to the EpGFPII reference gene. The numbers surrounding each black bar correspond to the nucleotide borders of each segment. Other designations are as in Figure 4A. 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.
	Figure 6. Comparison of Coup-TF's upstream and 5′UTR regions between P.lividus and S.purpuratus. A: 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 “Φ” insert (Fig. 2). The shaded areas correspond to various degrees of homology with the corresponding sequence of SpCoup-TF 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 a, which is not homologous between the two species. B: Graphic comparison of the 5′UTR and 5′ upstream sequences of the two orthologous genes, PlCoup-TF (top) and SpCoup-TF (bottom) using the Family Relations program (32). Crossbars joining the two sequences indicate homology, the thickest of which corresponds to the 5′UTR region.
	Figure 7. Spatial expression patterns generated by upstream deletions D-a1 to D-a5 of module a. A: Graphic presentation of the upstream deletions into module a (−532 to −212). Horizontal bars underneath module a, 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 Endo16′s promoter and CyIIa's kozak sequences as in figure 4A. The two parallel bars within the black box at the right end of the graph denote that the GFP gene is not depicted on scale. B: Composite pictures of embryos resulting from injection of upstream deletions into module a. The entire module a 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.
	Figure 8. Spatial expression patterns generated internal deletions D1–D4 into module a. A: Map of the internal deletions. Black boxes correspond to the deleted regions D1–D4. The numbers surrounding each box mark the borders of each deletion. EpAc refers to Endo16′s promoter and CyIIa's kozak sequences as in figure 4A. The two parallel bars within the black box at the right end of the graph denote that the GFP gene is not depicted on scale. B: Composite pictures of embryos resulting from injection of the internal deletions D1–D4. The entire module a shows GFP expression specifically in the ciliary band, deletion D1 in ciliary band, endoderm and aboral ectoderm and D2 in aboral ectoderm. D3 shows expression in ciliary band and endoderm while D4 shows expression in mesenchyme cells. All embryos were photographed at pluteus stage.
	Figure 9. Specific binding of embryonic nuclear proteins to elements RE1, RE2 and RE3.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.
	Figure 10. Spatial expression patterns of site-specific mutations into module a. A: Graphic presentation of the wt and mutant sequences of the three elements within module a. The top line depicts the 320 bp region of the wt module a, 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: Composite pictures of embryos expressing GFP resulting from injection of wt and mutant module a. The wt module a 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.

Achatz, Functional domains of the human orphan receptor ARP-1/COUP-TFII involved in active repression and transrepression. 1997, Pubmed

Achatz, Functional domains of the human orphan receptor ARP-1/COUP-TFII involved in active repression and transrepression. 1997, Pubmed
Arenas-Mena, Spatial expression of Hox cluster genes in the ontogeny of a sea urchin. 2000, Pubmed , Echinobase
Arnone, Green Fluorescent Protein in the sea urchin: new experimental approaches to transcriptional regulatory analysis in embryos and larvae. 1997, Pubmed , Echinobase
Arnone, Cis-regulation downstream of cell type specification: a single compact element controls the complex expression of the CyIIa gene in sea urchin embryos. 1998, Pubmed , Echinobase
Brown, Paircomp, FamilyRelationsII and Cartwheel: tools for interspecific sequence comparison. 2005, Pubmed
Brown, New computational approaches for analysis of cis-regulatory networks. 2002, Pubmed , Echinobase
Calzone, Developmental appearance of factors that bind specifically to cis-regulatory sequences of a gene expressed in the sea urchin embryo. 1988, Pubmed , Echinobase
Cameron, cis-Regulatory activity of randomly chosen genomic fragments from the sea urchin. 2004, Pubmed , Echinobase
Chan, SpCOUP-TF: a sea urchin member of the steroid/thyroid hormone receptor family. 1992, Pubmed , Echinobase
Consales, Functional characterization of Ets-binding sites in the sea urchin embryo: three base pair conversions redirect expression from mesoderm to ectoderm and endoderm. 2002, Pubmed , Echinobase
Cooney, Multiple mechanisms of chicken ovalbumin upstream promoter transcription factor-dependent repression of transactivation by the vitamin D, thyroid hormone, and retinoic acid receptors. 1993, Pubmed
Fjose, Functional conservation of vertebrate seven-up related genes in neurogenesis and eye development. 1993, Pubmed
Fjose, A novel vertebrate svp-related nuclear receptor is expressed as a step gradient in developing rhombomeres and is affected by retinoic acid. 1995, Pubmed
Flytzanis, Persistence and integration of cloned DNA in postembryonic sea urchins. 1985, Pubmed , Echinobase
Hiromi, Ectopic expression of seven-up causes cell fate changes during ommatidial assembly. 1993, Pubmed
Jonk, Cloning and expression during development of three murine members of the COUP family of nuclear orphan receptors. 1994, Pubmed
Krishnan, Mediation of Sonic hedgehog-induced expression of COUP-TFII by a protein phosphatase. 1997, Pubmed
Kruse, Identification of COUP-TFII orphan nuclear receptor as a retinoic acid-activated receptor. 2008, Pubmed
Ladias, Regulation of the apolipoprotein AI gene by ARP-1, a novel member of the steroid receptor superfamily. 1991, Pubmed
Laudet, Evolution of the nuclear receptor gene superfamily. 1992, Pubmed
Laudet, Evolution of the nuclear receptor superfamily: early diversification from an ancestral orphan receptor. 1997, Pubmed
Lutz, Developmental regulation of the orphan receptor COUP-TF II gene in spinal motor neurons. 1994, Pubmed
McMahon, Introduction of cloned DNA into sea urchin egg cytoplasm: replication and persistence during embryogenesis. 1985, Pubmed , Echinobase
Mlodzik, The Drosophila seven-up gene, a member of the steroid receptor gene superfamily, controls photoreceptor cell fates. 1990, Pubmed
Oliveri, Gene regulatory network controlling embryonic specification in the sea urchin. 2004, Pubmed , Echinobase
Pereira, The orphan nuclear receptor COUP-TFII is required for angiogenesis and heart development. 1999, Pubmed
Pereira, Chicken ovalbumin upstream promoter transcription factor (COUP-TF): expression during mouse embryogenesis. 1995, Pubmed
Qiu, Null mutation of mCOUP-TFI results in defects in morphogenesis of the glossopharyngeal ganglion, axonal projection, and arborization. 1997, Pubmed
Qiu, Spatiotemporal expression patterns of chicken ovalbumin upstream promoter-transcription factors in the developing mouse central nervous system: evidence for a role in segmental patterning of the diencephalon. 1994, Pubmed
Rizzo, Identification and developmental expression of the ets gene family in the sea urchin (Strongylocentrotus purpuratus). 2006, Pubmed , Echinobase
Salas, Induction of chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI ) gene expression is mediated by ETS factor binding sites. 2002, Pubmed
Saudemont, Ancestral regulatory circuits governing ectoderm patterning downstream of Nodal and BMP2/4 revealed by gene regulatory network analysis in an echinoderm. 2010, Pubmed , Echinobase
Shan, Copulation in C. elegans males requires a nuclear hormone receptor. 2008, Pubmed
Su, A perturbation model of the gene regulatory network for oral and aboral ectoderm specification in the sea urchin embryo. 2009, Pubmed , Echinobase
Tsai, Chick ovalbumin upstream promoter-transcription factors (COUP-TFs): coming of age. 1997, Pubmed
Vlahou, Maternal mRNA encoding the orphan steroid receptor SpCOUP-TF is localized in sea urchin eggs. 1996, Pubmed , Echinobase
Vlahou, Subcellular trafficking of the nuclear receptor COUP-TF in the early embryonic cell cycle. 2000, Pubmed , Echinobase
Walton, Genomics and expression profiles of the Hedgehog and Notch signaling pathways in sea urchin development. 2006, Pubmed , Echinobase
Wang, COUP transcription factor is a member of the steroid receptor superfamily. 1989, Pubmed
Xu, Distal cis-acting elements restrict expression of the CyIIIb actin gene in the aboral ectoderm of the sea urchin embryo. 1996, Pubmed , Echinobase
Zhou, UNC-55, an orphan nuclear hormone receptor, orchestrates synaptic specificity among two classes of motor neurons in Caenorhabditis elegans. 1998, Pubmed
van der Wees, Developmental expression and differential regulation by retinoic acid of Xenopus COUP-TF-A and COUP-TF-B. 1996, Pubmed