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, 106 (14), 5720-4

Shared Developmental Mechanisms Pattern the Vertebrate Gill Arch and Paired Fin Skeletons

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Shared Developmental Mechanisms Pattern the Vertebrate Gill Arch and Paired Fin Skeletons

J Andrew Gillis et al. Proc Natl Acad Sci U S A.

Abstract

Here, we describe the molecular patterning of chondrichthyan branchial rays (gill rays) and reveal profound developmental similarities between gill rays and vertebrate appendages. Sonic hedgehog (Shh) and fibroblast growth factor 8 (Fgf8) regulate the outgrowth and patterning of the chondrichthyan gill arch skeleton, in an interdependent manner similar to their roles in gnathostome paired appendages. Additionally, we demonstrate that paired appendages and branchial rays share other conserved developmental features, including Shh-mediated mirror-image duplications of the endoskeleton after exposure to retinoic acid, and Fgf8 expression by a pseudostratified distal epithelial ridge directing endoskeletal outgrowth. These data suggest that the skeletal patterning role of the retinoic acid/Shh/Fgf8 regulatory circuit has a deep evolutionary origin predating vertebrate paired appendages and may have functioned initially in patterning pharyngeal structures in a deuterostome ancestor of vertebrates.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Gill arch anatomy and gene expression in the embryonic skate. The 7 pharyngeal arches of embryonic skates (A) give rise to the serially homologous segments of the adult branchial skeleton (B and C; lateral and ventral views, respectively). The anteriormost mandibular arch (ma) forms the palatoquadrate (pq) and Meckel's cartilage (mk), whereas the 6 posterior arches develop into the pseudohyal (ph) and 5 branchial arches (ba1–5). The pseudohyal and the 4 anterior branchial arches possess branchial ray cartilages (br) that support the gill flaps; the fifth branchial arch possesses no rays. Shh is normally expressed by the distal ectoderm of each developing gill flap, shown in lateral (D) and dorsal (E) views, correlating with expression of the Shh target gene Ptc2 in subjacent mesoderm (F). Fgf8 is coexpressed by the leading edge of the gill flap epithelium (G, black arrows). RA treatment induces ectopic Shh expression (red arrows) in anterior branchial arch ectoderm (H and I; dorsal and lateral views, respectively). Shh and Fgf8 function in an interdependent feedback loop: Fgf8 is locally down-regulated by beads loaded with the SHH inhibitor cyclopamine (J, black arrowhead), and Shh expression is extinguished adjacent to beads loaded with the FGF inhibitor SU5402 (K, white arrow). Anterior is to the left except in C, where anterior is to the top. (Scale bars: A, 500 μm; B and C, 3 mm; D–K, 300 μm.)
Fig. 2.
Fig. 2.
RA, SHH, and FGF8 pattern the skate branchial arch skeleton. (A–D) Each gill arch in the skate is composed of a dorsal epibranchial (eb) and ventral ceratobranchial (cb) element that line the pharynx; branchial rays (br) articulate with the posterior border of the epi- and ceratobranchials, projecting laterally and curving caudally at the distal tip. (E–H) RA-treated arches exhibit mirror-image duplications of the branchial rays (red arrows); the supernumerary complement articulates with the anterior border of the epibranchial, with the distal tips curving rostrally. (I–K) Exogenous SHH protein can mimic RA-mediated duplications of branchial rays. (I) Untreated third branchial arches invariably have 5 rays articulating with the posterior margin of the epibranchial cartilage; SHH-loaded beads induce a single ectopic branchial ray (J and K), articulating with the anterior epibranchial and curving rostrally (black arrows). (L–N) SHH and FGF signaling are required for ray specification and outgrowth. Beads loaded with the SHH inhibitor cyclopamine (red asterisks) before ray condensation results in the complete deletion of neighboring rays (L), whereas postcondensation treatment results in distal ray truncations (M). Early treatment with the FGF antagonist SU5402 (blue asterisk) similarly results in deletion of ray cartilages. (Scale bars: A and E, 3 mm; B–D, F–H, 1.25 mm; I–N, 1.25 mm.)
Fig. 3.
Fig. 3.
Putatively homologous mechanisms regulate gnathostome appendage outgrowth. Comparative analyses of the fins and limbs of skates and chicks, and the gill arch of chondrichthyans, reveal that shared developmental signaling pathways regulate skeletal outgrowth and patterning. Shh expression is posteriorly restricted in the fin buds and gill arches of chondrichthyans such as the skate (A and B), as well as the limb buds and pharyngeal arches of tetrapods such as the chick (E and F). Similarly, Fgf8 is expressed in the apical ectoderm of skate fin buds and gill arches (C and D), as well as in chick limb buds and pharyngeal arches (G and H). The structure of Fgf8-expressing epithelia is also conserved. Histological sections reveal that specialized pseudostratified ectodermal ridges define the distal edge of outgrowing skate gill arches (I), skate fin buds (J), and chick limb buds (K), and direct proximodistally complete skeletal formation (L–N, respectively). (O) The functional and regulatory conservation of molecular pathways in patterning structural outgrowths is consistent with Gegenbaur's “Archipterygium” hypothesis, in which he proposed that the vertebrate paired fin and gill arch skeletons are transformational homologues (a and e in O, respectively). pr, propterygium; mt, metapterygium; rad, radials; hu, humerus; ra, radius; ul, ulna. (Scale bars: A–D, 300 μm; E–H, 250 μm; I–K, 5 μm; L, 1.5 mm; M, 2 mm.)

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