Formery L et al. (2025), Antero-posterior patterning in the brittle star...

ECB-ART-53929

Evodevo 2025 May 31;161:7. doi: 10.1186/s13227-025-00244-8.

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Antero-posterior patterning in the brittle star Amphipholis squamata and the evolution of echinoderm body plans.

Formery L , Peluso P , Rank DR , Rokhsar DS , Lowe CJ .

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Although the adult pentaradial body plan of echinoderms evolved from a bilateral ancestor, identifying axial homologies between the morphologically divergent echinoderms and their bilaterian relatives has been an enduring problem in zoology. The expression of conserved bilaterian patterning genes in echinoderms provides a molecular framework for resolving this puzzle. Recent studies in juvenile asteroids suggest that the bilaterian antero-posterior axis maps onto the medio-lateral axis of the arms, perpendicular to the proximo-distal axis of each of the five rays of the pentaradial body plan. Here, we test this hypothesis in another echinoderm class, the ophiuroids, using the cosmopolitan brittle star Amphipholis squamata. Our results show that the general principles of axial patterning are similar to those described in asteroids, and comparisons with existing molecular data from other echinoderm taxa support the idea that medio-lateral deployment of the bilaterian AP patterning program across the rays predates the evolution of the asterozoans, and likely the echinoderm crown-group. Our data also reveal expression differences between A. squamata and asteroids, which we attribute to secondary modifications specific to ophiuroids. Together, this work provides important comparative data to reconstruct the evolution of axial properties in echinoderm body plans.

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	Fig. 1. Morphological development of Amphipholis squamata. A Adult Amphipholis squamata, viewed from the oral side. B Schematic representation of the life cycle in A. squamata. C, C’ Images of a dissected adult showing four distinct juveniles (arrowheads and asterisk) developing within the bursa (C), with the largest (asterisk) being manually extracted (C’). D–L, selected developmental stages of A. squamata: cleavage (D), blastula (E), gastrula (F), early larva (G), late larva (H), pentagon (I, I’), early juvenile (J), mid-juvenile (K), late juvenile (L). The pentagon stage is shown in aboral (I) and oral (I’) views, other juvenile stages are shown in aboral views (J–L). butf: buccal tube foot, brtf: brachial tube foot, es: esophagus, g: gut, h: hydrocoel, lr: larval skeletal rod, prp: primary radial plate, ps: proximal segment, rc: right coelom, sp: spine, sts: sub-terminal segment, ts: terminal segment. Scale bars: 2 mm (A), 50 µm (D–L).
	Fig. 2. Anatomy of Amphipholis squamata juveniles. Z-projections of calcein stainings (A), single HCRs for MHC (B) and zic (C) and HCRs for elav combined with immunostainings against FMRF-amide-like neuropeptide (D) showing the anatomy of the endoskeleton (A), muscles (B), water vascular system (C) and nervous system (D) in Amphipholis squamata whole-mount early (left panel), mid- (middle panel) and late (right panel) juveniles viewed from the oral side (Aa, Ba, Ca, Da), and in detailed oral (Ab, Bb, Cb, Db), lateral (Ac, Bc, Cc, Dc) and transversal (Ad, Bd, Cd, Dd) views of brachial segments at the late juvenile stage. Transversal views show sections through the proximal (left panel) and distal (right panel) regions of a brachial segment. All samples are counterstained with DAPI (grey) to mark cell nuclei. In (Db–Dd), white dotted lines outline the ectoneural part of the radial nerve cords, and white asterisks indicate scattered neurons in the epidermis and the spines. aim: aboral intervertebral muscle, asp: aboral shield plate, bg: brachial ganglion, brtf: brachial tube foot, butf: buccal tube foot, cnr: circumoral nerve ring, eim: external interradial muscle, hyp: hyponeural neuroepithelium, ig: interradial ganglion, iim; internal interradial muscle, jp: jaw plates, lsp: lateral shield plate, oim: oral intervertebral muscle, osp: oral shield plate, pl: plexus, ptf: primary tube foot, rc: radial canal, rg: radial ganglion, rm: radial muscle, sp: spine, t: tooth, tfg; tube foot ganglion, v: vertebra. Scale bars: 100 µm
	Fig. 3. Expression of anterior head markers. Z-projections of single HCRs for hedgehog (A), sfrp1/5, (B), fzd5/8 (C), six3/6 (D), nkx2.1 (E) and double HCRs for nkx2.1 + elav (F) in Amphipholis squamata whole-mount early (left panel), mid- (middle panel) and late (right panel) juveniles viewed from the oral side (Aa, Ba, Ca, Da, Ea, Fa), and in detailed oral (Ab, Bb, Cb, Db, Eb, Fb), lateral (Ac, Bc, Cc, Dc, Ec, Fc) and transversal (Ad, Bd, Cd, Dd, Ed, Fd) views of brachial segments at the late juvenile stage. Transversal views show sections through the proximal regions of a brachial segment. All samples are counterstained with DAPI (grey) to mark cell nuclei. In (Ab–Ad, Bb–Bd, Cb–Cd, Db–Dd, Eb–Ed), white dotted lines outline the ectoneural part of the radial nerve cord, yellow asterisks indicate expression at the base of the tube feet epidermis, white arrowheads highlight expression in the brachial ganglia, white asterisks highlight sfrp1/5 positive cells on the sides of the midline in the brachial ganglia, purple asterisk indicates expression in the radial canal, and blue asterisks indicate expression in the aboral coeloms. In (Ba, Ca), purple dotted lines outline expression in the region of the ring canal. bg: brachial ganglion, ig: interradial ganglion, rg: radial ganglion; rnc: radial nerve cord. Scale bars: 100 µm
	Fig. 4. Expression of posterior head markers. Z-projections of single HCRs for irx (A), dmbx, (B), otx (C), barH (D), pax6 (E) and double HCRs for otx + nkx2.1 (F) and otx + pax6 (G) in Amphipholis squamata whole-mount early (left panel), mid- (middle panel) and late (right panel) juveniles viewed from the oral side (Aa, Ba, Ca, Da, Ea, Fa, Ga), and in detailed oral (Ab, Bb, Cb, Db, Eb, Fb, Gb), lateral (Ac, Bc, Cc, Dc, Ec, Fc, Gc) and transversal (Ad, Bd, Cd, Dd, Ed, Fd, Gd) views of brachial segments. Transversal views show sections through the distal regions of a brachial segment. All samples are counterstained with DAPI (grey) to mark cell nuclei. In (Aa), white dotted lines in the insets outline interradial ganglia. In (Ab–Ad, Bb–Bd, Cb–Cd, Db–Dd, Eb–Ed), white dotted lines outline the ectoneural part of the radial nerve cord, yellow asterisks indicate expression at the base of the tube feet epidermis. In (Ac, Ad), purple asterisks indicate expression in the ring canal, blue asterisks indicate expression in the aboral coelom. In (Ad), blue arrows indicate expression in the spines. brtf: brachial tube feet, butf: buccal tube feet, cnr: circumoral nerve ring, ig: interradial ganglion, rnc: radial nerve cord. Note that staining in the gut for dmbx, otx and pax6 HCRs results from aspecific autofluorescence. Scale bars: 100 µm
	Fig. 5. Expression of head-trunk boundary markers. Z-projections of single HCRs for gbx (A), hox1, (B), pax2/5/8 (C) and double HCRs for gbx + six3/6 (D), gbx + hox1 (E), and hox1 + nkx2.1 (F) in Amphipholis squamata whole-mount early (left panel), mid- (middle panel) and late (right panel) juveniles viewed from the oral side (Aa, Ba, Ca, Da, Ea, Fa), and in detailed oral (Ab, Bb, Cb, Db, Eb, Fb), lateral (Ac, Bc, Cc, Dc, Ec, Fc) and transversal (Ad, Bd, Cd, Dd, Ed, Fd) views of brachial segments at the late juvenile stage. Transversal views show sections through the proximal regions of a brachial segment. All samples are counterstained with DAPI (grey) to mark cell nuclei. In (Aa), white arrows indicate expression at the edge of the interradial ganglia. In (Ab–Ad, Bb–Bd, Cb–Cd), white dotted lines outline the ectoneural part of the radial nerve cord. In (Cb, Cc), blue arrows indicate expression in the developing spines. bg: brachial ganglion, sp: spine. Scale bars: 100 µm
	Fig. 6. Expression of other Hox genes. Z-projections of HCRs for hox2 (A), hox4, (B), hox5 (C), hox7 (D), hox8 (E) and hox9/10 (F) in Amphipholis squamata whole-mount early (left panel), mid- (middle panel) and late (right panel) juveniles viewed from the oral side (Aa, Ba, Ca, Da, Ea, Fa), and in detailed oral (Ab, Bb, Cb, Db, Eb, Fb), lateral (Ac, Bc, Cc, Dc, Ec, Fc) and transversal (Ad, Bd, Cd, Dd, Ed, Fd) views of brachial segments at the late juvenile stage. Transversal views show sections through the distal (Ad, Bd, Fd) or proximal (Bd, Cd, Ed) regions of a brachial segment. All samples are counterstained with DAPI (grey) to mark cell nuclei. In (Ab–Ad), blue asterisks indicate expression in the aboral coeloms. In (Ba–Bd), white arrowheads indicate expression in the brachial epidermis. In (Cb–Cd, Db–Dd), white dotted lines outline the ectoneural part of the radial nerve cord. In (Cb, Cc, Db), blue arrows indicate expression in the developing spines. In (Eb–Ed), orange arrowheads indicate expression in the oral intervertebral muscle. In (Fb–Fd), blue dotted lines outline the region of the aboral coelom expressing hox9/10. ac: aboral coelom, bg: brachial ganglion, ric: ring canal, rm: radial muscle. Note that staining in the gut for hox2, hox4 and hox9/10 HCRs results from aspecific autofluorescence. Scale bars: 100 µm
	Fig. 7. Evolution of axial patterning in echinoderms. A Comparison of the AP patterning genes analyzed in this study in Amphipholis squamata juveniles with published datasets from other echinoderm species, including Metacrinus rotundus [51], Anneissia japonica [93], Strongylocentrotus purpuratus [6, 7], Heliocidaris erythrogramma [19, 90], Holopneustes purpurescens [86–88], Peronella japonica [1, 125], Paracentrotus lividus [94, 95], Apostichopus japonicus [63], Parvulastra exigua [20, 26], Patiria miniata [44] and Amphiura filiformis [30]. Only genes for which expression was investigated during the development of the adult body plan are reported and expression in embryonic or larval tissues was not considered. Squares indicate genes that have been surveyed, and are colored according to their expression domain (dark blue: medial ambulacral ectoderm, light blue: tube feet epidermis lateral, green: ambulacral ectoderm boundary, yellow: internal germ layers, grey: interradial region or non-ambulacral epidermis). Black outlines indicate genes for which the expression pattern does not match the prediction of the anterior-ambulacral model, either for missing important expression domains (e.g. six3/6 absent from the radial nerve cords in P. lividus), or being expressed in unexpected territories (e.g. hox4, hox5 and hox7 expression in ectoderm derivatives in A. squamata). Absence of expression for posterior Hox genes is considered as fitting the ambulacral-anterior model. B Simplified conceptual representation of the deployment of the AP patterning program in bilateral animals, and in two echinoderm classes (asteroids and ophiuroids) following the ambulacral-anterior model. Only expression in ectoderm derivatives is represented. Note that these schematics only represent a general trend and individual gene expression may vary. Cri: crinoids, Ech: echinoids, Hol: holothuroids