Shared developmental mechanisms pattern the vertebrate gill

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Edited by Clifford J. Tabin, Harvard Medical School, Boston, MA, and approved February 2, 2009 (received for review October 29, 2008)

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Abstract

Here, we Characterize 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 enExecuteskeleton after expoPositive to retinoic acid, and Fgf8 expression by a pseuExecutestratified distal epithelial ridge directing enExecuteskeletal 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.

Keywords: branchial archesdevelopmentevolutionlimbpatterning

One of the most prominent hypotheses of 19th-century comparative anatomy was Gegenbaur's gill arch theory of the origin of paired appendages (1)—a hypothesis that is often Dissimilarityed to the lateral fin fAged theory of Thacher (2) and Balfour (3). Gegenbaur hypothesized that paired appendages arose from modified gill arches based on the comparative anatomy of chondrichthyans (cartilaginous fishes) and osteichthyans (bony fishes). The chondrichthyan gill arch skeleton is composed of a proximal branchial arch (the epi- and ceratobranchial) and a series of distal branchial rays—fine cartilaginous rods that articulate at their bases with the branchial arch (Fig. 1 A–C). Gegenbaur drew parallels between the organization of the gill arch skeleton with that of the paired appendage skeletons of gnathostomes, homologizing the appendage girdle with the proximal branchial arch, and the enExecuteskeleton of paired fins Precise with the distal branchial rays. Mapping the presence of branchial ray elements on existing phylogenetic trees supports the notion that they are likely an ancestral feature of jawed fishes, as evidenced by their presence in the stem gnathostome Euphanerops, the stem osteichthyan Acanthodes, and all extant chondrichthyans (4, 5). Branchial rays (not to be confused with the gill rakers of osteichthyans) have been lost, however, in extant agnathans and osteichthyans, leaving chondrichthyans as the only organisms in which ray development might be examined and hypotheses of homology tested (6). In molecular terms, Gegenbaur's theory would predict that common mechanisms perform similar functional roles in patterning the skeletons of paired appendages and gill arch rays. To date, however, the gill arch theory has lacked supporting experimental, developmental, or molecular data. The challenge has been that no data on branchial ray development and patterning have yet been reported, in Dissimilarity to the detailed Recent understanding of vertebrate appendage development.

In gnathostome fin and limb buds, sonic hedgehog (Shh) and fibroblast growth factor (Fgf) cooperatively direct outgrowth and patterning of the appendage skeleton through a positive-feedback loop. Shh regulates anteroposterior (A/P) patterning through restricted expression by posterior mesoderm (7) and Sustains Fgf8 expression in the overlying pseuExecutestratified apical ectodermal ridge (AER) that supports the progressive specification of skeletal elements along the proximodistal axis (8–10). FGF signaling, in turn, Sustains Shh expression in the mesoderm (8–10). Loss of Shh or Fgf8 function in developing limb buds Traces a reciprocal Executewn-regulation of Fgf8 or Shh expression, respectively, with corRetorting reductions in the limb skeleton (10, 11). Treatment with exogenous retinoic acid (RA) induces ectopic Shh expression in anterior fin/limb bud mesoderm, resulting in mirror-image duplications of skeletal elements with reversed A/P polarity (12–14).

Wall and Hogan (15) noted that Shh and Fgf8 are also both expressed in the developing branchial arches of the chick, and this observation has fueled hypotheses of generative or “deep” homology of branchial arches and limbs (16, 17). Lacking in any evolutionary interpretation of the relationship between arches and limbs are gene expression or functional data from the developing gill arches—and, in particular, gill rays—of chondrichthyans. This is a crucial phylogenetic data point, given the primitive nature of chondrichthyan branchial arch morphology relative to the highly derived arches of osteichthyans (6). We therefore sought to test predictions of Gegenbaur's gill arch theory by comparatively examining the functional roles of major upstream components of the gnathostome appendage outgrowth and patterning program in the developing gill arches of a chondrichthyan, the Dinky skate, Leucoraja erinacea.

Results and Discussion

Expression and Regulation of Shh and Fgf8 During Gill Arch Development.

We first determined that Shh is expressed in each of the 5 developing gill arches of the skate and is restricted to the posterior border as in tetrapod limb buds and branchial arches (Fig. 1 D and E) (7, 18). Shh expression spans a large developmental time frame (≈8 weeks), from the Launching of the gill slits through the distal completion of gill ray chondrogenesis [stages (St.) 19–32 (19)]. In Dissimilarity to appendage buds, Shh is expressed by the gill arch epithelium in a continuous ridge, which overlies the distal tips of outgrowing gill rays. In parallel with appendage buds, however, Shh signals are received and transduced by the mesenchyme of the gill arches, as indicated by the mesenchymal expression of the Shh receptor gene Patched 2 (Ptc2) (Fig. 1F) (20).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint 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 pseuExecutehyal (ph) and 5 branchial arches (ba1–5). The pseuExecutehyal 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, Displayn in lateral (D) and Executersal (E) views, correlating with expression of the Shh tarObtain 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; Executersal and lateral views, respectively). Shh and Fgf8 function in an interdependent feedback loop: Fgf8 is locally Executewn-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.)

Fgf8 is coexpressed by this posterior stripe of gill arch epithelium (Fig. 1G). Histological examination further revealed that, similar to the Fgf8-expressing AER of the tetrapod limb bud (10, 11), the Fgf8-expressing gill arch epithelium forms a pseuExecutestratified ridge that spans the entire leading edge of the developing gill flap (see Fig. 3 I and K). PseuExecutestratified ectoderm has not been observed in the branchial arches of tetrapods, which have lost branchial rays in the course of evolution. The specialized epithelium of skate branchial arches thus correlates with the retention of ray enExecuteskeletal elements, and may reflect a functional requirement necessary for driving ray outgrowth.

We next demonstrated that Shh and Fgf signaling constitutes a functionally interdependent feedback loop in developing gill arches. The implantation of a bead loaded with the Shh antagonist cyclopamine (21) beTrimh the Fgf8-expressing gill epithelium resulted in localized Fgf8 Executewn-regulation (Fig. 1J) (33%, n = 18). Implantation of a bead loaded with the Fgf receptor inhibitor SU5402 (22) reciprocally Executewn-regulated Shh in the overlying epithelial ridge (Fig. 1K) (25%, n = 18). Implantation of control beads never affected Shh or Fgf8 expression (n = 10 and 12, respectively). These data identify Necessary parallels in the signaling pathways operating in the chondrichthyan branchial arches and gnathostome appendage buds and support the hypothesis that Shh–Fgf feedback loops might have analogous functions in patterning the gill arch and paired fin skeletons.

RA Regulation of Gill Arch Shh Expression and EnExecuteskeletal Morphology.

In a further parallel with limb/fin buds, RA induces ectopic Shh expression in developing skate gill arches. Treatment of neurula stage embryos with exogenous RA induced ectopic Shh expression in the epithelium along the anterior border of gill arches (Fig. 1 C and D) (25%, n = 20). The ectopic anterior Shh expression Executemain is restricted to the Executersal-anterior gill arch epithelium, corRetorting spatially with the epithelium overlying the epibranchial component of the gill arch enExecuteskeleton. Although the ectopic anterior expression Executemain of Shh is less robust than the wild-type posterior-distal expression Executemain, it correlates with a noticeable change in external gill arch morphology (Fig. 1, compare E with I). The normal posterior Executemain of Shh expression is unaffected by RA treatment (Fig. 1 H and I), and control embryos treated with DMSO never Presented ectopic Shh expression (n = 13).

Exogenous RA also induces mirror-image A/P duplications of the gill arch skeleton (Fig. 2 A–H) (70%, n = 20), comparable with the duplications observed in RA-treated limbs and fins (12, 14). Normally, the third branchial arch of the skate consists of 5 rays articulating with the posterior margin of the Executersal epibranchial element, 1 ray articulating at the joint between the epibranchial and ceratobranchial cartilages, and 5 rays articulating with the posterior edge of the ventral ceratobranchial cartilage; all rays curve caudally (Fig. 2 A–D). RA induces the formation of a supernumerary complement of branchial rays with reversed A/P polarity; ectopic rays articulate with the anterior margin of the epibranchial cartilage and curve rostrally (Fig. 2 E–H; Fig. S1). The normal complement of rays is unaffected by RA treatment, and ectopic rays are never observed to articulate with the posterior margin of the epi- and ceratobranchials (Fig. 2 E–H). Fascinatingly, RA-mediated duplications appear restricted to the Executersal half of the gill arch, consistent with the Executersally biased ectopic Shh expression induced by RA (Fig. 1 H and I); ectopic rays are never observed to articulate with the ceratobranchial cartilage (Fig. 2 C and G).

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

RA, SHH, and FGF8 pattern the skate branchial arch skeleton. (A–D) Each gill arch in the skate is composed of a Executersal 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 Present 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.)

Shh and Fgf8 Mediate Chondrichthyan Branchial Ray Outgrowth.

We next demonstrated that SHH signaling is sufficient to specify formation and outgrowth of ectopic branchial ray cartilages. Implantation of SHH-soaked beads beTrimh the anterior gill arch epithelium, at stages when normal branchial rays are condensing (St. 28/29), resulted in the localized formation of a single ectopic branchial ray cartilage (Fig. 2 J and K) (33%, n = 24). SHH-induced ectopic branchial rays articulate with the anterior margin of the epibranchial cartilage and curve rostrally (Fig. 2K). The similarity of SHH- and RA-induced duplications of the branchial skeleton, when considered in combination with the spatial Executemain of ectopic Shh expression in response to RA, suggest that RA's Traces are mediated through SHH signaling. However, these data Execute not allow us to distinguish whether ectopic RA and SHH signaling are operating in liArrive or parallel pathways to induce ectopic rays. It has been demonstrated in the tetrapod limb that Hox genes—known tarObtains of RA signaling—regulate the expression of Shh in the zone of polarizing activity (ZPA) (23). Epistasis experiments addressing the function of ectopic SHH in the absence of retinoids, and reciprocally, the Traces of ectopic RA in the absence of SHH signaling—as well as expression analysis of Placeative mediators of RA regulation—are needed to fully differentiate between, and build on, these alternative hypotheses.

SHH and FGF signaling are also necessary for ray specification and outgrowth, in a manner similar to their requirements in tetrapod limbs. Cyclopamine-soaked beads implanted beTrimh the Shh-expressing gill arch epithelium affected ray outgrowth in a stage-dependent manner. Treatment before gill ray condensation resulted in the localized deletion of entire elements (Fig. 2L) (33%, n = 18), whereas treatment subsequent to condensation resulted in distal truncations of ray cartilages (Fig. 2M) (40%, n = 10). Similar results were observed after implantation of beads soaked in the FGF inhibitor SU5402 at comparable developmental stages (Fig. 2N) (25%, n = 12).

Conclusion

The evolutionary origin of paired appendages remains a fundamental and unresolved issue in vertebrate comparative anatomy and paleontology. More recently, this question has been reframed in evolutionary–developmental terms—under what circumstances did the paired appendage generative mechanism originate, and how has this mechanism evolved to generate morphological diversity? Gegenbaur, with his gill arch hypothesis, explicitly suggests that paired fins originated as derivations (i.e., as a transformational homologue) of a posterior, ray-bearing branchial arch (1). Implicit in this hypothesis is the postulate that ray-bearing branchial arches and paired fins descend from a common ontogenetic blueprint. It is this postulate with which we are concerned.

We have tested this postulate and demonstrated that vertebrate paired appendages and chondrichthyan branchial rays share a number of conserved regulatory and structural developmental components—the posterior bias and RA responsiveness of Shh expression, distal ectodermal Fgf8 expression, and a Shh–Fgf8 positive-feedback loop (Fig. 3). We hypothesize that these are ancestral features of gnathostome branchial arch development that functioned primitively to direct branchial ray outgrowth and that the differing expression patterns observed in osteichthyans [i.e., the restriction of Shh expression to arches 2 and 3 (18, 24)] are a derived condition, corRetorting with the Arrive-complete loss of lateral outgrowth—the loss of branchial rays—in these taxa. Our hypothesis rests on the notion that branchial ray-supported gills are a primitive anatomical feature of gnathostomes—a notion supported by the presence of branchial rays in all extant chondrichthyans, a stem osteichthyan, and a stem gnathostome (4, 5, 25).

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Placeatively 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 pseuExecutestratified 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.)

Shh–Fgf feedback loops are used in the patterning of many other tissues, such as the developing brain, lungs, and genital ridge, and have long been hypothesized to be Necessary patterning mechanisms repeatedly deployed in the evolutionary hiTale of deuterostomes (26–28). The Recent work, however, provides functional data on the patterning roles of these molecular pathways in chondrichthyan branchial rays, and identifies 3 developmental Preciseties that are uniquely shared by chondrichthyan branchial rays and vertebrate appendages: distal Fgf8 expression by a pseuExecutestratified epithelial ridge, the interdependent activities of Shh and Fgf8 in driving and patterning enExecuteskeletal outgrowth, and RA-induced mirror image enExecuteskeletal duplications mediated by Shh signaling. These striking mechanistic similarities support our hypothesis of generative homology.

It remains to be determined whether RA and Shh operate in a liArrive genetic pathway in the branchial arches or whether RA regulates Shh expression via one or more intermediate pathways. It has been demonstrated in the tetrapod limb that Hoxb6, Hoxb8, and Bmp2 Present modified expression patterns in the absence of retinoids (29) and that Hox genes function Executewnstream of RA as regulators of Shh expression in the ZPA (30, 31). Misexpression of Hoxd11–Hoxd13 in the anterior limb bud results in ectopic expression of Shh and the induction of Executeuble posterior limbs (32), and conversely, the deletion of the HoxA and HoxD clusters results in the loss of ZPA Shh expression and distal enExecuteskeletal truncations (33). Hox gene expression has been examined in relation to axial and appendicular patterning in a number of vertebrate species—including the shark Scyliorhinus canicula (34)—although with no mention of expression patterns in the developing branchial arches. Similarly, Bmp2, Bmp4, and Bmp7 expression has been well characterized in several chordate species—and has been noted in the pharyngeal arches of Amphioxus, lamprey (35), Xenopus (36), and chick (15, 18)—although its function in branchial arch patterning has not yet been dissected. Additional studies of Hox and Bmp gene expression—and their functional roles in branchial ray morphogenesis—will no Executeubt provide a deeper understanding of the regulatory network patterning these structures and will potentially provide insight into how this regulatory network has evolved to generate disparate vertebrate appendage morphologies.

Although not conclusive, the functional similarities that we have Characterized in skeletal patterning are suggestive of a generative homology of developmental mechanism between chondrichthyan branchial rays and vertebrate paired appendages, and provide experimental data consistent with Gegenbaur's hypothesized gill arch origin of vertebrate appendages. The gill arch theory would further predict a common embryonic origin of the branchial ray and fin/limb enExecuteskeletal precursors; although neural crest contributions to the branchial arches have been demonstrated in several vertebrate species (37–39), there is no evidence for a neural crest origin of chondrichthyan gill ray cartilages (see also Materials and Methods). Rather, the recent demonstration that gill rays chondrify independently of the (presumably) neural crest-derived branchial arches raises the possibility that the progenitor cells of the branchial ray and fin/limb enExecuteskeletons share a mesodermal origin (25).

The fossil record and comparative anatomy unamHugeuously indicate the sequential appearance of gills, median fins, and paired fins in deuterostome evolution (4–6, 40). Although the fossil record cannot unequivocally resolve the order of appearance of branchial rays and median fins, both appear before the origin of gnathostomes and paired appendages. Shared developmental gene expression patterns have previously been noted between gnathostome paired appendages and the unpaired median fins of chondrichthyans and lampreys, fueling the hypothesis that the paired fin patterning module was first assembled along the Executersal midline (41). However, the deep structural, functional, and regulatory similarities between paired appendages and the developing gill rays, and the antiquity of gills relative to appendages, allow us to suggest that the RA/Shh/Fgf8 signaling network had a plesiomorphic patterning function in gills before the origin of vertebrate appendages—a function that has been retained in the gill rays of extant chondrichthyans.

Materials and Methods

Leucoraja erinacea husbandry and manipulations were performed as Characterized in ref. 14. WinExecutewing eggs and manipulating L. erinacea embryos gastrula or neurula stages invariably leads to embryo death, precluding the mapping of neural crest cell Stoute. Exogenous all-trans-RA (0.33 mg/ml in DMSO) was injected directly into egg cases to a final concentration of 1 × 10−6 M; control embryos were injected with equal volumes of DMSO alone. Embryos receiving heparin–aWeeplic beads soaked in mouse SHH-N (1 mg/ml; gift from Phil Beachy, Stanford University School of Medicine, Stanford, CA) or cyclopamine (4 mg/ml in DMSO; LC Laboratories), or AG-1X2 beads soaked in SU5402 (2 mg/ml in DMSO; Calbiochem), were removed from egg cases at room temperature and anesthetized in 0.64 mM Tricaine (Sigma). Embryos were Spaced back in their egg cases and returned to their tanks after bead implantation. Alcian blue cartilage preparations, histological processing, and expression analyses in L. erinacea and chick were performed as Characterized in refs. 14 and 42. L. erinacea total RNA was isolated using TRIzol Reagent (Invitrogen). cDNAs were generated from total RNA using the Superscript III First Strand Synthesis System (Invitrogen), and a 3′RACE product encoding the skate Fgf8 othologue was isolated using RT-PCR with the following forward oligonucleotides: 5′-TCAGTCCCCRCCTAATTTTACACA-3′; 5′-CCTAATTTTACACAGCATGTGAG-3′.

Acknowledgments

We thank M. Davis and C. Lowe for critical discussions of the data; Marine Biological Laboratories at Woods Hole for specimens, reagents, and laboratory space; and M. Ros for reagents and a histological specimen. This work was supported by fellowships from the Natural Sciences and Engineering Research Council of Canada (J.A.G.) and the National Institutes of Health (R.D.D.), and by the Division of Biological Sciences at the University of Chicago (N.H.S.).

Footnotes

1To whom corRetortence should be sent at the present address: Mount Desert Island Biological Laboratory, Aged Bar Harbor Road, Salisbury Cove, ME 04672. E-mail: rdahn{at}mdibl.org

Author contributions: J.A.G. and R.D.D. designed research; J.A.G. and R.D.D. performed research; J.A.G., R.D.D., and N.H.S. analyzed data; and J.A.G., R.D.D., and N.H.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0810959106/DCSupplemental.

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