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Fig. 1. Macrosyntenic analysis between scallop, sea urchin, amphioxus, and spotted gar. a) Ribbon diagram showing conserved macrosyntenies among FoxP-bearing chromosomes of the 4 species. Horizontal bars represent chromosomes, and pink lines indicate the positions of FoxP orthologs/paralogs. Green arches connected orthologous gene pairs on the chromosome containing FoxP. Gray arches indicate orthologous gene pairs on other corresponding chromosomes among animals. Pairwise comparisons are presented in supplementary fig. S3, Supplementary Material online. b) Cladogram showing the evolutionary history leading to 4 spotted gar FoxP-bearing chromosomes (Loc1, Loc3, Loc5, and Loc8), starting from the proto-vertebrate ALG_E. The evolutionary history of vertebrate chromosomes is based on data from Simakov et al. (2020).
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Fig. 2. Phylogenetic analysis of deuterostome FoxP-encoded proteins. a) A phylogenetic hypothesis for deuterostome FoxP genes based on the 2R-WGD scenario. The 4 gnathostome paralogs arose from the 2R-WGD events. Our JTT + C60 + G ML tree suggests that the cyclostome FoxP paralogs are likely FoxP2/4 descendants, while the foxp1/3 lineage was lost. b) Maximum-likelihood (ML) tree of a set of deuterostome FoxP proteins produced using the site-heterogeneous JTT + C60 + G model. c) ML tree of the same set of genes shown in b) produced by using the site-homogeneous JTT + F + I + G4 model. Note that the lamprey genes are affiliated with the gnathostome FoxP3 in this tree. The scale bars differ between b) and c); branch lengths are underestimated in the tree shown in panel c). For clarity, the bootstrap support values of the internal nodes in the FoxP1-4 clades are omitted. Only those with a bootstrap value under 80 are displayed for deeper nodes. Detailed depictions of tree topologies and bootstrap values are given in supplementary fig. S4, Supplementary Material online.
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Fig. 3. Expression pattern of FoxP in amphioxus embryonic development. WMISH of FoxP in amphioxus B. floridae at different developmental stages, including unfertilized egg a), 64-cell stage b), blastula c), early gastrula d to g), late gastrula h), neurula i and j), and larva k to m). d to h, i, j to m) are viewed from the lateral side. Embryos in h′) and h″) are the blastopore view and dorsal view of the same embryo in h), respectively. i′) shows a cross-section of the plane indicated by the vertical line in i). Asterisks mark the anterior end of the embryo. Dorsal is to the top in d to m). The dark color of the pigment spot (ps) and the frontal eye (fe) in k to m) is endogenous pigmentation, not an in situ hybridization signal. csg, club-shaped gland; cv, cerebral vesicle; en, endoderm; fe, frontal eye; g, gut; gs, gill slit; lhc, left head cavity; me, mesendoderm; nc, notochord; pnp, presumptive neural plate; nt, neural tube; ps, pigment spot; rt, rostral tip; so, somite; tb, tail bud.
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Fig. 4. Transcriptome profiling of FoxP expression in amphioxus and representative gnathostome species. Heatmaps show the relative abundance of transcripts in tissue-specific transcriptomes of the amphioxus (Branchiostoma lanceolatum) FoxP a), the spotted gar (Lepisosteus oculatus) FoxP1-4 b), the catshark (Scyliorhinus torazame) FoxP1, 2 and 4 c), and the mouse (Mus musculus) FoxP1-4 d). Expression levels are shown in transcript per million (TPM).
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Fig. 5. Expression pattern of FoxP in hemichordate P. flava embryos. WMISH of FoxP in hemichordate P. flava at different developmental stages, including unfertilized egg a), blastula b), gastrula c to g), and tornaria larva h). The animal pole is at the top in a to h) and f′–h′). The specimens were viewed from the lateral, oral, or apical side as indicated on the left f to h). The oral side is to the left in f to h) and f″ to h″). hpf, hours postfertilization; ape, apical ectoderm; fg, foregut; hg, hindgut; me, mesoderm; oe, oral ectoderm.
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Fig. 6. Characterization of FoxP in the catenulid worm Stenostomum. a) Amino acid alignment of the region corresponding to the FoxP-specific zinc finger and LZ domains. b to g) Expression pattern of Stenostomum FoxP in a worm undergoing asexual growth. The FoxP-expressing cells in the dorsal posterior region of the pharynx wall and the ventral body wall invagination at the mature fission plane are indicated with red and black arrows, respectively. Expression of FoxP was not detected in the newly developed fission plane (open arrow). The FoxP transcript signals in a pair of unidentified cells at the junction between the pharynx and intestine are denoted by black arrowheads. Lower levels of FoxP expression at the base of cephalic sensory pits (red asterisks) and the bilaterally paired dorsal body wall invaginations at the fission plane (black asterisks) are indicated. b) and e) show the lateral and dorsal views, respectively, of the same specimen. c) and f) are higher magnifications of the pharynx and intestine junction area from the lateral and dorsal view, respectively. d) and g) are higher magnifications of the mature fission plane in the posterior body from the lateral and the ventral view, respectively, focusing on the FoxP-expressing area in the ventral pouch that would give rise to the pharynx. h) Expression pattern of the pan-neuronal marker Elav. ph., pharynx; int., intestine.
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