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Figure S5. Expression of xRARα, xRARβ, and xRARγ in Xenopus embryos
Whole mounts are oriented with anterior toward the right. Expression of RARα and RARγ is detectable from the onset of gastrulation (stage 10) while the first signal for RARβ is detected at the early tailbud stage (stage 25). At mid-gastrula stage (stage 11), RARα is expressed as a narrow ring around the blastopore. As gastrulation proceeds, expression intensifies and the signal around the blastopore widens preferentially on the dorsal side except in the midline, which exhibits a low level of transcripts. During neurulation (from stage 14), transcripts are found predominantly in the neurectoderm, evenly distributed along the anterior-posterior axis, with the exception of a region at the anterior end for which transcripts are largely reduced. At the tailbud stage (stage 30), RARα is predominantly expressed in the spinal cord and the posterior hindbrain, in the eye and the posterior branchial arches. During gastrulation (stage 10), expression of RARγ is more widespread than RARα expression. Transcripts are present in the mesodermal marginal zone as well as in the ectoderm. By the neurula stage (from stage 14), the staining separates into anterior and posterior domains, thus creating a gap with no RARγ transcripts. Expression remains localized to the posterior and anterior ends of the embryo at tailbud stages (stage 30) and is mainly restricted to the branchial arches and the tip of the tailbud. RARβ transcripts are detected at much lower level than RARα and RARγ at the examined stages. The signal is restricted to the caudal part of the hindbrain and the anterior spinal cord. At the late tailbud stage (stage 32), RARβ is strongly expressed in the most posterior branchial arches. d, dorsal views; l, lateral views; f, frontal views.
doi:10.1371/journal.pgen.0020102.sg005
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Figure 5. Schematic Representation of the Expression Territories of RARs
Staining of embryos indicates expression of mRARα (A), mRARβ (B), and mRARγ (C) in mouse embryos at E9; of xRARα (G), xRARβ (H), and xRARγ (I) in stage 30 Xenopus embryos, and of AmphiRAR (M) in 20 h old amphioxus larvae. Schematic representations are shown of the expression territories of mRARs (D�F), xRARs (J�L), and AmphiRAR (N) in mouse, Xenopus, and amphioxus embryos, respectively. Regions with high levels of expression are red and those with lower levels of expression are pink. Arrowheads indicate regions in mouse and Xenopus embryos where the RAR expression cannot be correlated with AmphiRAR expression and can be described as �new expression territories.�
doi:10.1371/journal.pgen.0020102.g005
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Figure 1. Phylogenetic View of Deuterostomes and RARs(A) Current view of deuterostome phylogeny with amphioxus representing the basal chordate [5]. RARs used in the present study are indicated at their respective taxonomic positionsâfor mouse, Xenopus, zebrafish, lamprey, amphioxus, and tunicates. The position of the synthetic ancestral sequence is indicated by a red circle. The two proposed periods of whole genome duplications in vertebrates are indicated as Phase I and Phase II, occurring respectively before and after the divergence of lampreys.(B) Phylogenetic tree showing the placement of the RARs used in this study. Branch length is proportional to evolutionary change (bar = 0.1 substitutions per site); numbers at nodes are bootstrap support, in percent of 1,000 replicates. Branches supported by bootstrap lower than 70% have been polytomised. The tree was rooted by the amphioxus sequence, in agreement with [5]. Species abbreviations and their groups are indicated as follows. Amphioxus: Amphi, Branchiostoma floridae. Tunicates: Pm, Polyandrocarpa misakiensis; Ci, Ciona intestinalis. Lampreys: Lamp, Petromyzon marinus. Teleost fish: Takifugu, Takifugu rubripes; Tetraodon, Tetraodon nigroviridis; and Danio, Danio rerio. Amphibians: Xenopus, Xenopus laevis; Ambystoma, Ambystoma mexicanum; and Notophthalmus, Notophthalmus viridescens. Birds: Gallus, Gallus gallus; and Coturnix, Coturnix coturnix. Mammals: Homo, Homo sapiens; Mus, Mus musculus; and Rattus, Rattus norvegicus.
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Figure 2. Protein Sequence Alignment of Selected Gnathostome RARsRARs are represented from lamprey (LampRAR, Petromyzon marinus), amphioxus (AmphiRAR, Branchiostoma floridae), tunicate (RAR_POLM1, Polyandrocarpa misakiensis), and the synthetic predicted ancestral RAR (Ancestor). The position of the 12 helices is indicated above the alignment (H1âH12). Residues implicated in direct contacts with the ligand are numbered from 1 to 25 below the alignment. The three divergent residues within the LBP between vertebrate RARs are within vertical rectangles in helices 3, 5, and 11. Gnathostome and Polyandrocarpa sequences are named with the nomenclature code used in the nuclear receptor database NUREBASE (http://www.ens-lyon.fr/LBMC/laudet/nurebase/nurebase.html) [39].
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Figure 3. Transcriptional Activity and Binding Selectivity of Vertebrate RARsTranscriptional activity is shown in (AâC), (GâI), (M), and (N), and corresponding binding selectivity in (DâF), (JâL), (O), and (P). Identities of the vertebrate RARs for each activity-selectivity pair are indicated above each bar graph. In each case, a chimera comprising the RAR LBD fused to the GAL4 DNA-binding domain (GAL-RAR(LBD)) has been used. The analysis of transcriptional activity in (AâC), (GâI), (M), and (N) shows transient transactivation assays in Cos1 cells with the indicated GAL-RAR(LBD) expression vector and the cognate (17m)5x-G-luc reporter plasmid, in the presence of increasing concentrations (10â10 to 10â6 M) of ATRA (red bars), BMS753 (yellow bars), BMS641 (light green bars), and BMS961 (dark green bars) respectively. The black bars indicate transactivation in the absence of hormone. Partial proteolysis maps of different in vitro-translated RARs are shown in (DâF), (JâL), (O), and (P). For each proteolysis gel lane 1 represents the undigested protein, lane 2 shows digestion of the receptor in the absence of ligand, lanes 3 and 4 show digestion of the receptor in the presence of ATRA (10â4 to 10â5 M), lanes 5 and 6 show digestion in the presence of BMS753 (10â4 to 10â5 M), lanes 7 and 8 show digestion in the presence of BMS641 (10â4 to 10â5 M), and lanes 9 and 10 show digestion in the presence of BMS961 (10â4 to 10â5 M). Protected bands in the presence of BMS641 are indicated by an asterisk, and slightly protected bands are indicated by arrowheads.
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Figure 4. Transcriptional Activity and Binding Selectivity of Chordate RARsTranscriptional activity is shown in (AâD) and (IâL), and corresponding binding selectivity in (EâH) and (MâP). Identities of the chordate RARs for each activity-selectivity pair are indicated above each bar graph. Transcriptional activity is shown in (AâD) for LampRAR, AmphiRAR, PmRAR, and AncRAR, and that of AmphiRAR mutants is shown in (IâL). Partial proteolysis maps of the different in vitro-translated RARs are shown in (EâH) and (MâP). Chimeric GAL-RAR(LBD) transactivation methods, colour code of the transactivation figures, and contents of each proteolysis gel are as in Figure 3. Protected bands in the presence of BMS641 are indicated by an asterisk, and slightly protected bands are indicated by arrowheads.
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Figure 6. Representation of the Transactivation and Binding Capacities of the RARs Used in the Present StudyThe three synthetic retinoids are shown as α, BMS753; β, BMS641; and γ, BMS961. The phylogenetic relationships between the RARs have been schematized by a phylogenetic tree (the tunicate and amphioxus RARs have been polytomised, LampRAR is also polytomised with the vertebrate RARs). The putative position in the tree of the ancestral sequence is indicated by a dashed branch in red.
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