Ermakova GV et al. (2025), The emergence of chordin-like1 in gnathostomes ...

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Front Cell Dev Biol 2025 Aug 29;13:1649996. doi: 10.3389/fcell.2025.1649996.

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The emergence of chordin-like1 in gnathostomes may have contributed to the evolution of paired appendages.

Ermakova GV , Meyntser IV , Mugue NS , Lyubetsky VA , Zaraisky AG , Bayramov AV .

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Genomic transformations during early vertebrate evolution, including two rounds of whole-genome duplication, laid the groundwork for the emergence of novel morphological features in jawed vertebrates. Among these innovations, paired appendages represent a major evolutionary milestone, whose development and diversification enabled vertebrates to exploit diverse ecological niches in aquatic, terrestrial, and aerial environments. Here, we combined phylogenetic and local genomic synteny analyses to investigate the evolutionary history of chordin-like homologs in vertebrates. Our results indicate that chordin-like1 first appeared in jawed vertebrates, suggesting a possible link between its origin and the emergence of paired appendages. To explore this hypothesis, we examined chordin-like1 expression in representatives of basal jawed vertebrate lineages - cartilaginous fishes (grey catshark, Chiloscyllium griseum) and sturgeons (sterlet, Acipenser ruthenus). We further assessed the expression and functional properties of the chordin-like1 ortholog in the African clawed frog (Xenopus laevis), a representative terrestrial vertebrate with limb morphology that markedly differs from the fins of basal gnathostomes. Together with published data, our findings support a potential role for chordin-like1 in the evolution of paired appendages. In particular, chordin-like1 may have contributed to the development of the metapterygial element and its derivatives, which formed the structural basis for the evolution of tetrapod limbs.

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Species referenced: Xenopus laevis
Genes referenced: chrdl1 chrdl2
GO keywords: Wnt signaling pathway [+]

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	FIGURE 1. Maximum likelihood (ML) phylogenetic tree of Chordin-like proteins in chordates. Chordin-like proteins from C. griseum, A. ruthenus, and Xenopus tropicalis are highlighted in red. Bootstrap support values greater than 60 are indicated. Branch lengths represent the number of substitutions per site, as shown by the scale bar. Most analyzed representatives of Gnathostomes possess two paralogs, Chordin-like1 and Chordin-like2, which can be confidently distinguished, with bootstrap support values of 100 and 92, respectively. In Chondrichthyes, only Chordin-like1 is present. In the basally divergent lineages of Teleostei (Elopomorpha and Osteoglossomorpha), both Chordin-like paralogs are retained, whereas in the majority of the examined Teleostei species, only Chordin-like2 is detected. AcRuAcipenser ruthenus, AmCaAmia calva, AnAn - Anguilla anguilla, AnCaAnolis carolinensis, AnSt - Antennarius striatus, BrFlBranchiostoma floridae, CaCa - Carcharodon carcharias, CaMi - Callorhinchus milii, ChCh - Chanos chanos, ChGr - Chiloscyllium griseum, ChPu - Chiloscyllium punctatum, ChPlChiloscyllium plagiosum, CiInCiona intestinalis, ClGa - Clarias gariepinus, DaReDanio rerio, DeCl - Denticeps clupeoides, EsLu - Esox lucius, GaGa - Gallus gallus, GaMo - Gadus morhua, HeAk - Hemitrygon akajei, HeOc - Hemiscyllium ocellatum, HePe - Heptranchias perlo, HoSaHomo sapiens, LaChLatimeria chalumnae, LeErLeucoraja erinacea, LeOcLepisosteus oculatus, MeCyMegalops cyprinoides, MoBu - Mobula birostris, MuMuMus musculus, PeFl - Perca flavescens, PeMaPetromyzon marinus, PoSePolypterus senegalus, PrAnProtopterus annectens, PrJa - Pristiophorus japonicas, PrPe - Pristis pectinata, RhTy - Rhincodon typus, SaKoSaccoglossus kowalevskii, ScCa - Scyliorhinus canicula, ScFo - Scleropages formosus, ScTo - Scyliorhinus torazame, StTi - Stegostoma tigrinum, TaRuTakifugu rubripes, XeTrXenopus tropicalis.
	FIGURE 2. Analysis of local genomic synteny of chordin-like genes in chordates and some invertebrates. The chordin-like2 gene has a neighboring polD3 gene in the genomes of vertebrates and invertebrates. The chordin-like1 gene with its characteristic neighboring genes is found only in gnathostomes. The ammecr and tmem164 genes are present in the vicinity of the gnathostome chordin-like1 genes and the lamprey (P. marinus) chordin-like2 genes. AcRuAcipenser ruthenus, AmRaAmblyraja radiate, AsRuAsterias rubens, BrFlBranchiostoma floridae, CaCa - Carcharodon carcharias, CaMi - Callorhinchus milii, ChPlChiloscyllium plagiosum, CrSe - Cryptotermes secundus, DaReDanio rerio, DeCl - Denticeps clupeoides, EpBuEptatretus burgeri, EsLu - Esox lucius, GaGa - Gallus gallus, HoSaHomo sapiens, LaChLatimeria chalumnae, LeOcLepisosteus oculatus, LiAn - Lingula anatine, MeCyMegalops cyprinoides, PeMaPetromyzon marinus, PrPe - Pristis pectinata, SaKoSaccoglossus kowalevskii, ScCa - Scyliorhinus canicula, ScFo - Scleropages formosus, XeTrXenopus tropicalis. * sequence of E. burgeri chordin-like (Eptbu0006070.t1) was found in Squalomix sequence server (https://treethinkers.nig.ac.jp/squalomix/blast/).
	FIGURE 3. Analysis of the spatial expression of the chordin-like1 during early developmental stages of the grey catshark C. griseum. Staging was performed according to Ballard et al. (1993). At stage 24 (A, B), chordin-like1 expression is observed in the primordia of the mandibular, hyoid, and branchial arches. By stage 25, in addition to expression in these arches (C, D), chordin-like1 expression also appears in the early primordia of the dorsal (J, N) and pelvic (U) fins. From stage 26 onwards (EI) in addition to branchial arches, chordin-like1 expression is detected in the posterior regions of the developing primordia of both paired (pectoral and pelvic) (RT; VX) and unpaired dorsal fins (KM; OQ). From stage 27 onwards, expression is detected in the anal fin (Y, Z). The photo of the 2nd dorsal fin in panel (Q) is the same as that in Figure 4K, and the photo of the pelvic fin in panel (X) is the same as that in Figure 4G. aanterior, afanal fin, babranchial arches, ddorsal, dfdorsal fin, disdistal, hyhyoid arch, mdmandibular arch, pposterior, pcfpectoral fin, plfpelvic fin, prproximal.
	FIGURE 4. Chordin-like1 expression in C. griseum embryos in the primordia of the branchial arches (A), dorsal (B), and pelvic (C) fins, shown on transverse sections. In the branchial arches, chordin-like1 is expressed in the mesodermal core of the primordia. (DK) Comparison of BMP signaling activity (assessed via phosphorylated Smad1/5 immunolabeling, arrowheads) and chordin-like1 expression domains in the fin primordia of C. griseum at stages 2829. In both paired and unpaired fins, BMP activity is observed in the distal anterior and posterior regions of the primordia, whereas chordin-like1 expression is detected only in the posterior region, (L, M) BMP signaling activity in caudal denticles. The photo of the pelvic fin in panel (G) is the same as that in Figure 3X, and the photo of 2nd dorsal fin in panel (K) is the same as that in Figure 3Q. aanterior, dfdorsal fin, disdistal, chchord, ectectoderm, endendoderm, mshmesenchyme, mdmmesoderm, ntneural tube, pposterior, plfpelvic fin, prproximal.
	FIGURE 5. Spatial expression analysis of the chordin-like1 gene in prelarval stages of the sterlet Acipenser ruthenus. (AG) Chordin-like1 expression in whole-mount prelarvae. (HM) Expression of chordin-like1 in the pectoral fins is observed in a broad domain corresponding to the formation of endoskeletal elements and displays a gradient pattern with a maximum in the posterior region of the fin (HK). As fin development progresses, chordin-like1 expression becomes restricted to the posterior region, spatially corresponding to the location of the future metapterygial element (L,M). (NP) Expression of chordin-like1 in the pelvic fins is observed in the domain of endoskeletal elements formation, with a maximum in the posterior region. (QS) Expression of chordin-like1 in the dorsal fin is detected uniformly throughout the region of endoskeletal elements development. (T, U) Expression of chordin-like1 in the caudal fin is observed in the region of the hypuralia. (V) Expression of chordin-like1 is detected in somites. (WZ1) Expression of chordin-like1 is detected in jaw structures.
	FIGURE 6. Expression and functional analysis of the Chordin-like1 in Xenopus laevis. Stages of Xenopus laevis are indicated according to Nieuwkoop and Faber (1994). (A) Analysis of chordin-like1 expression dynamics in Xenopus laevis in comparison to chordin expression. A significant increase in chordin-like1 expression is observed in tadpoles at stages 4347, while the maximum expression of chordin occurs at significantly earlier stages: 10.518. (BG) The spatial expression pattern of chordin-like1 in the developing Xenopus laevis limb is first observed at stage 51 (B) in the marginal regions of the early limb bud. At later stages (5354), expression becomes localized to the proximal-posterior region and the marginal zones of the digit primordia (DG). Additionally, chordin-like1 is expressed in the developing eye structures (C). (H) Effects of chordin-like1 mRNA injection on the activity of reporters for endogenous intracellular signaling pathways in luciferase assay compared with chordin mRNA effects. Compared to Chordin, Chordin-like1 exhibits a weaker inhibitory effect on the endogenous BMP pathway but a stronger inhibitory effect on the canonical Wnt/-catenin pathway. Additionally, Chordin-like1 markedly activates the Wnt/PCP pathway. (I) Modulation of reporter activity for intracellular signaling cascades by chordin-like1 in the context of their experimental activation by ligand co-injection in luciferase assay.
	FIGURE 7. Comparison of gnathostome groups possessing a metapterygial element (or its derivatives) in the fins/limbs and the presence of the chordin-like1 gene in their genomes.
	chrdl1( chordin like 1) gene expression in X. laevis tadpole, NF stage 51 hindlimb bud, assayed via in situ hybridization, anterior up, posterior down.
	chrdl1( chordin like 1) gene expression in X. laevis tadpole, NF stage 53 eye, assayed via in situ hybridization.
	chrdl1( chordin like 1) gene expression in X. laevis tadpole, NF stage 53 hindlimb (left) and forelimb (right) , assayed via in situ hybridization.
	chrdl1(chordin like 1) gene expression in X. laevis tadpole, NF stage 54 hindlimb (left) and forelimb (right), assayed via in situ hybridization.