Pectoral Fin and Girdle Development in the Basal...

Pectoral Fin and Girdle Development in the BasalActinopterygians Polyodon spathula andAcipenser transmontanusMarcus C. Davis,1* Neil H. Shubin,1 and Allan Force2

1Department of Organismal Biology and Anatomy, University of Chicago, Chicago, Illinois 606372Department of Molecular Genetics, Benaroya Research Institute at Virginia Mason, Seattle, Washington 98101

ABSTRACT The pectoral fins of Acipenseriformes pos-sess endoskeletons with elements homologous to both thefin radials of teleosts and the limb bones of tetrapods.Here we present a study of pectoral fin development in theNorth American paddlefish, Polyodon spathula, and thewhite sturgeon, Acipenser transmontanus, which revealsthat aspects of both teleost and tetrapod endoskeletalpatterning mechanisms are present in Acipenseriformes.Those elements considered homologous to teleost radials,the propterygium and the mesopterygial radials, form viasubdivision of an initially chondrogenic plate of mesenchy-mal cells called the endoskeletal disc. In Acipenseri-formes, elements homologous to the sarcopterygianmetapterygium develop separately from the endoskeletaldisc as an outgrowth of the endoskeletal shoulder girdlethat extends into the posterior margin of the finbud. As intetrapods, the elongating metapterygium and themetapterygial radials form in a proximal to distal order asdiscrete condensations from initially nonchondrogenicmesenchyme. Patterns of variation seen in the Acipens-eriform fin also correlate with putative homology: all vari-ants from the “normal” fin bauplan involved themetapterygium and the metapterygial radials alone. Theprimary factor distinguishing Polyodon and Acipenser findevelopment from each other is the composition of theendoskeletal extracellular matrix. Proteoglycans (visual-ized with Alcian Blue) and Type II collagen (visualized byimmunohistochemistry) are secreted in different placeswithin the mesenchymal anlage of the fin elements andgirdle and at different developmental times. Acipenseri-form pectoral fins differ from the fins of teleosts in therelative contribution of the endoskeleton and dermal rays.The fins of Polyodon and Acipenser possess elaborate en-doskeletons overlapped along their distal margins by der-mal lepidotrichia. In contrast, teleost fins generally pos-sess relatively small endoskeletal radials that articulatewith the dermal fin skeleton terminally, with little or noproximodistal overlap. J. Morphol. 262:608–628, 2004.© 2004 Wiley-Liss, Inc.

KEY WORDS: Polyodon; Acipenser; Acipenseriformes; findevelopment; chondrogenesis; collagen

Understanding the mechanisms underlying verte-brate paired appendage development is a key prob-lem in evolutionary developmental biology. Whileclassical embryological studies in the late 19th andearly 20th century incorporated a diversity of chon-

drichthyans (Mivart, 1879; Balfour, 1881; Goodrich,1906), lobe-finned fishes (Semon, 1898), and ray-finned fishes (Thacher, 1877; Sewertzoff, 1926;Kryzanovsky, 1927), modern molecular studies ofdevelopment have been limited to a few derivedsarcopterygian (Mus, Gallus) and actinopterygian(Danio) model organisms. To make sense of devel-opmental patterns seen in these derived taxa re-quires an understanding of development in phyloge-netically basal taxa. Acipenseriformes (sturgeons,paddlefish, and their extinct relatives) are particu-larly attractive organisms for research on the evolu-tion of development because of their phylogeneticposition near the base of extant actinopterygians.Acipenseriformes are represented by 27 extant spe-cies (Bemis and Grande, 1999) and a fossil recordextending to the Lower Jurassic (Grande et al.,2002). The majority of morphological (Nelson, 1969;Patterson, 1982; Gardiner and Schaeffer, 1989; Be-mis et al., 1997; Coates, 1999) and molecular (Le etal., 1993) phylogenetic hypotheses support Acipens-eriformes as the sister clade to a monophyleticNeopterygii consisting of holosteans (Amia � gar;sensu Gardiner et al., 1996) plus teleosts. However,recent molecular hypotheses based on insertionsand deletions in the coding sequences of nucleargenes (Venkatesh et al., 2001) and mitochondrialgenomic data (Inoue et al., 2003) support an alter-native topology in which Acipenseriformes and ho-losteans form a monophyletic group as the sisterclade to teleosts. In either phylogenetic scenario,Acipenseriformes remain basal to the Teleostei.

The pectoral fin skeleton of Acipenseriformes pro-vides a morphological intermediate between the de-

Contract grant sponsors: University of Chicago, National ScienceFoundation (NSF); Contract grant number: 0207721.

*Current address and correspondence to: Marcus C. Davis, UCSF,Department of Anatomy, Campus Box 2711, San Francisco, CA94143-2711. E-mail: [email protected]

Published online 16 September 2004 inWiley InterScience (www.interscience.wiley.com)DOI: 10.1002/jmor.10264

JOURNAL OF MORPHOLOGY 262:608–628 (2004)

© 2004 WILEY-LISS, INC.

rived pectoral appendages of teleosts and tetrapods.The pectoral fins of sturgeons and paddlefish con-tain elements considered homologous to both the finradials of teleosts and the limb skeleton of tetra-pods. This pectoral fin bauplan, like that of otherbasal actinopterygians and many chondrichthyans,can be divided into three distinct regions. Theseregions were first described by Carl Gegenbaur(1865) and consist of an anterior propterygium, amiddle mesopterygium or series of mesopterygialradials, and a posterior metapterygium. This tri-basal arrangement is also present in fossils of theearliest gnathostomes (Janvier, 1996). Placoderms,acanthodians, and the majority of fossil and extantchondrichthyans possess pectoral skeletal elementscomparable in relative position and shape to thepro-, meso-, and metapterygia of extant taxa (Ørvig,1962; Denison, 1978; Coates, 1994; Shubin, 1995).However, some Paleozoic elasmobranchs possess nu-merous unjointed radials anterior to a metaptery-gium (e.g., Cladoselache; Zangerl, 1981) or possesspectoral fin skeletons consisting only of a metaptery-gium (i.e., the xenacanthid Hagenoselache; Hampeand Heidtke, 1997). Likewise, the pectoral fin skel-etons of holocephalans consist of two basal elementsrather than three (Didier, 1995; Stahl, 1999). Thus,while much of the fossil and comparative evidencesupport the hypothesis that a tribasal pectoral finskeleton is a plesiomorphy of the Gnathostomata, agreater understanding of basal chondrichthyan phy-logeny is needed before alternative evolutionary sce-narios can be discounted.

In contrast to the conserved skeletal patterns ofbasal gnathostomes, much of extant osteichthyandiversity can be characterized by patterns of en-doskeletal loss. The metapterygium was lost in thelineage leading to teleosts. Thus, the radials in theteleost pectoral fin are considered homologous to thepropterygium and mesopterygium of basal taxa.Conversely, sarcopterygians (including tetrapods)have lost the pro- and mesopterygium, retainingonly the metapterygial skeleton. An alternative evo-lutionary scenario proposes that the metapterygialfin skeleton is primitive to gnathostomes and thatthe propterygium and mesopterygium of chondrich-thyans and actinopterygians are convergentlyevolved (Maisey, 1984; Mabee, 2000). This hypothe-sis would seem to contradict much of the paleonto-logical data, particularly the presence of tribasal finendoskeletons in placoderms (e.g., Ctenurella;O� rvig, 1962), acanthodians (e.g., Acanthodes;Coates, 1994), and many basal chondrichthyans.New fossil discoveries of basal gnathostomes andbasal osteichthyans will be necessary to test thesehypotheses. However, the implications of either sce-nario are the same. While the skeletons of teleostpectoral fins and tetrapod forelimbs are homologousat the level of endoskeletal radials, teleosts and tet-rapods do not share homologous skeletal elements atthe level of “individuated” pro-, meso-, and

metapterygia. Among osteichthyans, only basal ac-tinopterygians retain the full complement of ele-ments present in non-osteichthyan gnathostomes.

Teleosts and tetrapods have different mechanismsby which the pectoral endoskeleton is patterned dur-ing development. In tetrapods, secretion of extracel-lular matrix components (collagens and proteogly-cans) begins concomitant with, or subsequent to,condensation of individual elements. In contrast, thepectoral fin endoskeleton of teleosts begins as a sin-gle, plate-like chondrogenic condensation called theendoskeletal disc (Grandel and Schulte-Merker,1998). This endoskeletal disc then subdivides intoindividual radials by decomposition of extracellularmatrix in the inter-radial mesenchyme. Which ofthese patterning mechanisms is representative ofthe primitive gnathostomes condition is equivocaldue to the lack of skeletal homology between teleostsfins and tetrapod limbs. Do the mechanisms of en-doskeletal patterning observed in teleosts and tet-rapods correlate with the homology of the skeletalelements, or do they reflect derived mechanisms spe-cific to teleost and tetrapod development? Only theinvestigation of appendage development in basalgnathostome taxa can test these possibilities.

The following is an investigation of the normaldevelopment of the paired fins in two Acipenseri-formes, the North American paddlefish Polyodonspathula and the white sturgeon Acipensertransmontanus (Fig. 1). This investigation will serveas a basis for comparative studies of gnathostomeappendage development as well as for experimentalstudies utilizing sturgeons and paddlefish as re-search organisms. The natural history and biogeog-raphy of Acipenseriform species has been discussedby previous authors (Grande and Bemis, 1996; Be-mis et al., 1997; Findeis, 1997). Reproduction andgrowth are well documented in sturgeons andpaddlefish, due in part to the economic value of theirroe, which is sold as caviar. This aquaculture data-base, coupled with high fecundity (as many as10,000 eggs per female) make chondrosteans amia-ble to laboratory studies of embryonic and larvalstages. However, sturgeons and paddlefish take sev-eral years to reach sexual maturity and do so at arelatively large body size (Dettlaff et al., 1993), pre-cluding Acipenseriformes from captive breeding un-der normal laboratory settings.

MATERIALS AND METHODSAquaculture and Staging

Fertilized eggs of the North American paddlefish, Polyodonspathula, were acquired at 5 days postfertilization (dpf) fromOsage Catfisheries (Osage Beach, MO). Staging of Polyodon isbased on Ballard and Needham (1964) and Bemis and Grande(1992). Approximately 3,000 Polyodon embryos were hatched in5-L plastic tanks at a density of �100 eggs per liter. Embryoswere raised at a constant temperature of 18.5 � 0.5°C, pH 7.2 �0.7, salinity of 1.0 � 0.2 ppt, and a diurnal 12-h light/dark cycle.Hatchery water was constantly filtered and recirculated with a

609FIN DEVELOPMENT IN POLYODON AND ACIPENSER

30-min replacement time. All water was filtered through acti-vated charcoal and UV irradiated before returning to the tank.Mortality was low (�3 dead per 1,000 embryos per day) until 1week after the onset of feeding (21 dpf) when a sharp increase(�25 dead per 1,000 embryos per day) was observed. This mor-tality spike was primarily due to bites inflicted by other larva.Numerous attacks were observed as well as confirmation of pre-vious reports of larval cannibalism in Polyodon (Mims et al.,1999). These problems were most likely density related, as at thetime of peak mortality density was high (60 embryos per liter).

Polyodon embryos (n � 40) were sampled at random every 12 hbeginning at 6 dpf and continuing until the onset of feeding atstage 46 (15 dpf). At the onset of feeding, larvae (n � 40) weretaken every 24 h until 33 dpf. From 33 dpf until metamorphosis(52 dpf) Polyodon (n � 20) were sampled every fourth day. Meta-morphosis is defined as the point when larvae change feedingmode from the “plucking” of individual prey to filter feeding(sensu Bemis and Grande, 1992). Specimens were labeled bystage with days postfertilization in parentheses until the onset offeeding, after which they were labeled as days poststaging withmean total specimen length in parentheses. Specimen length perstage was determined by taking the mean total length of 10individuals to the nearest millimeter.

Embryos of the white sturgeon, Acipenser transmontanus, werecollected in Troutdale, Oregon, by one of us (AF) from a singlemating. Acipenser embryos (n � 50) were sampled every 24 hbetween 8 and 31 dpf. As there are no satisfactory staging datafor Acipenser, specimens were staged based on prelarval develop-ment in the Russian sturgeon Acipenser guldenstadti (Schmal-hausen, 1991; Detlaff et al., 1993). For both species, specimenswere labeled by stage with days postfertilization in parenthesesuntil the onset of feeding, after which they were labeled as dayspoststaging with mean total body length in parentheses. Speci-

men length per stage was determined by taking the mean totallength of 10 individuals to the nearest millimeter.

Fixation

Polyodon were euthanized with a lethal dose of MS-222 (Tri-caine). Specimens were fixed for 24 h in 4% paraformaldehyde,dehydrated stepwise into 100% methanol, and stored at 4°C untiluse. Specimens of Acipenser were fixed in 10% neutral bufferedformalin and stored in 100% methanol at 4°C.

Alcian Blue Staining

Polyodon (Fig. 1A,B) and Acipenser embryos were bleached in6% hydrogen peroxide/25% methanol in PBT and then stained forproteoglycans with Alcian Blue (0.02% Alcian Blue 8GX/30%glacial acetic acid in anhydrous ethanol; pH adjusted to 2.5).Embryos were destained in 30% glacial acetic acid in anhydrousethanol and then cleared through a graded ethanol/glycerol se-ries. Specimens were stored in 100% glycerol at 4°C.

Immunochemistry

Embryos of Polyodon and Acipenser (Fig. 1C,D) were labeledwith an antibody against Type II collagen (II-II6B3; acquiredfrom Hybridoma Bank, University of Iowa) using a modificationof standard zebrafish immunostaining techniques (Schilling andKimmel, 1997; Westerfield, 2000). Modifications included serialwashing with PBT (PBS media plus 0.2% Tween-20) instead ofphosphate buffer, the addition of 0.2% Tween-20 to the blockingsolutions, and the use of a biotinylated avidin complex amplifica-

Fig. 1. Survey of Polyodon spathula (A,B) and Acipenser transmontanus (C,D) embryos representing developmentally early(A,C) and late (B,D) stages used in this study. A: Stage 42 P. spathula stained with Alcian Blue in left lateral (top) and ventral (bottom)view. B: Metamorphic (52 days poststaging) P. spathula stained with Alcian Blue and Alizarin Red in left lateral (top) and ventral(bottom) view. C: Stage 42 A. transmontanus immunostained for Type II collagen in left lateral (top) and ventral (bottom) view. D: 13days poststaging A. transmontanus immunostained for Type II collagen in left lateral (top) and ventral (bottom) view. Embryos not toscale.

610 M.C. DAVIS ET AL.

tion step (Vectastain ABC Elite Kit, Vector Laboratories, Burlin-game, CA) prior to color development. Signals were developed bydiaminobenzidine reaction plus nickel chloride. Stained speci-mens were graded through an ethanol/glycerol series and storedin 100% glycerol. A minimum of six specimens was immuno-stained for each developmental stage for both Polyodon and Aci-penser. To support correct interpretation of specimens that lackedstaining in the pectoral fins, only specimens that stained posi-tively for Type II collagen in other parts of the embryo wereanalyzed. Likewise, both whole mount and dissected pectoral finswere immunostained to ensure that antibody penetrance was notan issue.

Histology

Embryos of Acipenser were embedded in paraplast followinginfiltration in xylene and paraplast. Sections (10 �m thickness)were cut on a Microm HM330 rotary microtome. Sections werestained either with hematoxylin and eosin (H&E) or with atrichrome stain that combined H&E and Alcian Blue. For thetrichrome stain, slides were soaked in 2 mg/ml 8GX Alcian Bluein glacial acetic acid (pH � 2.5) for 1 min following eosin stainingbut prior to clearing with xylene. Meyer’s H&E were utilized forboth staining techniques. Stain slides were preserved with Per-mount.

Imaging

Specimens were photographed in 100% glycerol using a NikonD1X digital camera attached to either a Leitz Laborlux S com-pound microscope for dissected material and sections or a WildM3C dissecting microscope for whole mounts. Images were editedusing Adobe PhotoShop 7.0 (San Jose, CA) for OS X and stan-dardized to absolute white using the “Auto Levels” option.

Terminology

Terminology follows that of Gegenbaur (1865) and Jessen(1972). Additional details of the scapulocoracoid follow Findeis(1997). The dermal skeleton of the pectoral girdle and fin havebeen described in detail for Polyodon (Grande and Bemis, 1991)and for Acipenser sp. (Jessen, 1972; Jollie, 1980; Findeis, 1997)and will be discussed only with respect to the girdle and finendoskeleton.

RESULTSPolyodon spathula: Pectoral Girdle and FinDevelopment

Embryos and larva of Polyodon spathula werestained with Alcian Blue and immunostained withType II collagen to investigate pectoral fin and girdledevelopment. Type II collagen immunostaining pro-duced negative results with respect to the pectoralfin and girdle (data not shown). Possible explana-tions for this result are discussed in the section onvariation in the extracellular matrix (below). Figure1 depicts late embryo (Fig. 1A; Stage 42) and meta-morphic (Fig. 1B, total length (TL) � 45 mm) P.spathula used in this study. The specific stages usedin this study were selected based on the presence ofkey developmental events not present in previousstages. The pectoral girdles and fins of P. spathulawere positioned caudoventral and slightly medial tothe elongate opercular flaps (Fig. 1A,B). As a result,visualization of the pectoral fins is obscured in whole

mount, necessitating dissection of the pectoral girdleand fin prior to imaging (Fig. 2).

Prechondrogenic Stages

The pectoral finbud first appears at Stage 37 (com-parable to Ballard and Needham, 1964) as a swell-ing of the ectoderm on the dorsolateral surface of theyolk sac posterior to the pronephros (data notshown). At Stage 37, the anteroposterior axis of thefinbud is parallel to the anteroposterior axis of thebody. In subsequent stages the pectoral fins moveanteroventrally to a position close to the posteriormargin of the opercular flaps. This movement isprimarily due to a change in the relative position ofthe ectoderm ventral to the hypaxial musculature asthe yolk sac is depleted rather than the actual mi-gration of the finbud across the ectoderm. At Stages38 and 39 the finbud consists of the mesenchymalanlage of the shoulder girdle and fin endoskeletonoverlaid by the basal stratum and peridermal cells ofthe ectoderm. During Stages 39 and 40 the apicalectodermal ridge begins to form as a thickening ofthe basal stratum along the distal margin of the fin.

Stage 42

At Stage 42 (13 days postfertilization, mean TL � 13mm), Alcian Blue staining first appears in the commonanlage of the pectoral fin and girdle (Fig. 2A). Extra-cellular proteoglycans are present in the chondrogeniccondensations of the longitudinal plate of the scapulo-coracoid, the presumptive glenoid ridge, and the mostproximal regions of the scapular process andmetapterygium. The metapterygium is continuouswith the shoulder girdle anlage and is a distinct con-densation consisting of stacked chondrocytes sur-rounded by thickened extracellular matrix. In con-trast, the finbud mesenchyme anterior and distal tothe metapterygium is chondrogenic (stains diffuselywith Alcian Blue), but consists of a flattened disc ofcells lacking any discernable condensations. This flat-tened disc of cells averages one or two cells thick dor-soventrally and is histologically comparable to the en-doskeletal disc described for Danio rerio (Grandel andSchulte-Merker, 1998). Collagenous actinotrichia arenot yet visible within the elongating finfold. Globularsecretory granules within the epidermis can be seendotting the fin surface, particularly in the finfold re-gion. Similar vesicles have been described within thepelvic fin epidermis of the rainbow trout Salmo gaird-neri (Geraudie and Landis, 1982).

Stage 44

At Stage 44 (14 days postfertilization, mean TL �14 mm), Alcian Blue is visible in the coracoid processwith relatively weak staining of the extracellularmatrix connecting the coracoid process to the centralportion of the scapulocoracoid (arrowhead in Fig.


2B). This central portion consists of a longitudinalplate (“mittelstuck” of Jessen, 1972) connecting thescapular and coracoid processes. The posterior mar-gin of this plate, the glenoid ridge, articulates withthe radials in the adult pectoral fin. At Stage 44, the

presumptive glenoid ridge area is visible as a smallinvagination of cells at the base of the metaptery-gium (Fig. 2B). The scapular process begins toproject from the medial margin of the glenoid ridgeand the metapterygium continues to elongate pos-

Fig. 2. Developmental series of pectoral fins and girdles of Polyodon spathula. Alcian Blue. Fins are shown in dorsal view withgirdle in oblique medial view to avoid distortion. Anterior to the left. A: Stage 42 (TL � 13 mm). B: Stage 44 (TL � 14 mm). Arrowheaddenotes connection between the longitudinal plate and the coracoid process. C: Stage 45 (TL � 15 mm). D: Stage 46 (TL � 17 mm).Arrowheads mark the path of the marginal artery of the fin. E: 4 days poststaging (TL � 17 mm). F: 10 days poststaging (TL � 18mm). *Initial subdivision of the common anlage of the propterygium and mesopterygial radial 1. G: 16 days poststaging (TL � 19 mm).H: 37 days poststaging (TL � 32 mm). I: Metamorphosis (52 days poststaging; TL � 45 mm). Scale bars � 100 �m. ac, actinotrichia;app, anterior process of the propterygium; cop, coracoid process; dr, distal radials; ed, endoskeletal disc; gl, glenoid ridge; lep,lepidotrichia; lp, longitudinal plate; lsc, lateral scapular process; ma, marginal artery; me1-2, mesopterygial radials 1 and 2; mpg,metapterygial process of the glenoid; mt, metapterygium; mtr1-4, metapterygial radials 1 through 4; pr, propterygium; pr/me1,common anlage of the propterygium and mesopterygial radial 1, prb, propterygial bridge; scp, scapular process; sg, secretory granules.


terodistally within the finbud. Finbud mesenchymeof the endoskeletal disc stains more intensely thanat Stage 42, although condensations are still notpresent. Stain is absent or weak among cells at thejunction between the endoskeletal disc and the gle-noid ridge and proximal metapterygium. Acti-notrichia are now visible within the finfold (Figs. 2B,3A). The proximal ends of the actinotrichia overlap

the distal margins of the endoskeletal disc (arrow inFig. 3A). Distally, actinotrichia extend to within 100�m (arrowhead in Fig. 3A) of the fin margin.

Stage 45

At Stage 45 (15 days postfertilization, mean TL �15 mm), the scapular and coracoid processes and the

Fig. 3. Development of the dermal fin skeleton in Polyodon spathula. Alcian Blue. All specimens shown in dorsal view with anteriorto the left. A: Stage 44 (TL � 14 mm). Actinotrichia within the finfold can be seen overlapping the distal portion of the endoskeletaldisc (arrow) and extending to within 100 �m of the finfold margin (arrowhead). B: Stage 46 (TL � 17 mm). Close-up of the anteriorradials showing overlap of the actinotrichia (arrows). C: 16 days poststaging (TL � 19 mm). Close-up of the anterior radials showingoverlap of the actinotrichia (arrows). Muscle fibers (arrowheads) can be seen extending distally to the finbud/finfold junction (openarrowheads). D: Metamorphosis (52 days poststaging; TL � 45 mm). Distal radials can be seen within the proximal finfold, overlappeddorsally and ventrally by lepidotrichia. Chondrogenic mesenchyme medial to each hemi-ray stain with Alcian Blue (arrowhead).E: Close-up of boxed region in D showing asymmetry of overlap of the dorsal and ventral lepidotrichia. F: Close-up of boxed region inD. Actinotrichia are still present between and medial to the lepidotrichia. Scale bars � 100 �m for A–D, 200 �m for E, and 400 �mfor F. dr, distal radial; ed, endoskeletal disc; dlp, dorsal lepidotrichia; ff, finfold; me1-2, mesopterygial radial 1; pr, propterygium;pr/me1, common anlage of the propterygium and mesopterygial radial 1; sg, secretory granule, vlp, ventral lepidotrichia.


metapterygium continue to elongate distally (Fig.2C). The endoskeletal disc stains more intenselythan at Stage 44, but staining is no longer uniformthroughout the disc. Proximodistally oriented zonesof weakly stained cells/matrix within the endoskel-etal disc mark sites of extracellular proteoglycansdecomposition (arrowheads in Fig. 2C). The loss ofchondrogenic character within these zones results insubdivision of the endoskeletal disc into individualradials. The first cleared zones to form divide thedisc into three chondrogenic regions. The two ante-rior regions are continuous with the glenoid ridgeregion of the scapulocoracoid, while cells of the pos-teriormost region are continuous with the anteriormargin of the metapterygium. Diffuse Alcian Bluestain distal to the endoskeletal disc marks chondro-genic mesenchyme adjacent to and within the prox-imal finfold.

Stage 46

At Stage 46 (16 days postfertilization, mean TL �17 mm), decomposition of the extracellular matrixwithin the endoskeletal disc continues (Fig. 2D). Thefirst element to form within the disc, the secondmesopterygial radial, is separated from the rest ofthe endoskeletal disc by widened zones of nonchon-drogenic mesenchyme. The anterior mesenchymalanlage will form the propterygium and first me-sopterygial radial (Figs. 2D, 3B). The posterior an-lage, still continuous with the anterior margin of themetapterygium, will form the first metapterygialradial. The zones of matrix decomposition do notextend into the chondrogenic mesenchyme along thedistal periphery of the disc. This distal mesenchymeextends further into the finfold than in previousstages. As a result, the actinotrichia appear to over-lap a greater percentage of the developing fin en-doskeleton than at earlier stages (compare Fig.3A,B). The path of the marginal artery can be seenwithin the finfold and along the anterior margin ofthe endoskeletal disc (arrowheads in Fig. 2D).

Four Days Poststaging

By 4 days after the onset of feeding (20 days post-fertilization, mean TL � 17 mm), decomposition ofthe chondrogenic extracellular matrix componentssurrounding the mesopterygial radial 2 is complete(Fig. 2E). At this stage, matrix decomposition at thecenter of the common anlagen of the propterygiumand mesopterygial radial 1 begins. The firstmetapterygial radial forms by decomposition of ma-trix between the posterior margin of the developingradial and the anterior margin of the metaptery-gium. Metapterygial radial 1 remains connected tothe metapterygium proximally. Likewise, the devel-oping propterygium, mesopterygial radials, andmetapterygium remain connected to the pectoralgirdle anlagen at the glenoid ridge. The metaptery-

gium now extends distally to the arch of diffuselystained mesenchyme along the distal periphery ofthe radials. Stained mesenchyme along the anteriormargin of the scapular process marks the formationof the lateral scapular process (Fig. 2E).

Ten Days Poststaging

By 10 days after the onset of feeding (26 dayspostfertilization, mean TL � 18 mm), the proptery-gium and first mesopterygial radial are separated bya strip of nonchondrogenic cells (see * in Fig. 2F).Metapterygial radial 2 begins to condense within thezone of nonchondrogenic mesenchyme separatingmetapterygial radial 1 and the anterior margin ofthe metapterygium. The first signs of joint formationbetween the glenoid ridge and the radials appear at10 days poststaging, as the extracellular matrix inthe presumptive joint region stains weakly. Despitethe breakdown of extracellular proteoglycans in thejoint region, cells in this area retain the stackedmorphology of chondrogenic condensations. The me-dial margin of the glenoid ridge, still continuouswith the metapterygium at this stage, expands pos-terodistally into a prominent metapterygial processof the glenoid (Fig. 2F). The scapular and coracoidprocesses continue to elongate and increase in over-all size. The lateral scapular process begins to elon-gate toward the anterior margin of the scapulocora-coid plate, which has broadened anteroposteriorlyrelative to previous stages.

Sixteen Days Poststaging

By 16 days after the onset of feeding (32 dayspostfertilization, mean TL � 19 mm), metapterygialradial 2 begin to separate from the anterior marginof the metapterygium (Fig. 2G). At this stage,metapterygial radial 1 remains connected to themetapterygium. The propterygium, mesopterygialradials 1 and 2, and the metapterygium remain at-tached to the glenoid ridge by partially chondrogeniccells. However, the proximal ends of the radials aremore rounded, consisting of compact chondrocyteswith fewer nonchondrogenic cells connecting the ra-dials to the glenoid ridge. Ventral elongation of thelateral scapular process continues towards the an-terior margin of the scapulocoracoid plate. Stainintensity increases in the extracellular matrix alongthe distal periphery of the finbud, particularlyaround the distal tips of the radials. Within thischondrogenic band, the distal radials will condenseat later stages, concomitant with formation of thelepidotrichia. Actinotrichia (arrows in Fig. 3C) over-lap the distal margin of the radials (open arrow-heads in Fig. 3C). Under oblique lighting, musclefibers are visible extending to the distal tips of theradials (arrowheads in Fig. 3C), but are not seen inthe presumptive distal radial mesenchyme withinthe proximal finfold.


Thirty-seven Days Poststaging

At 37 days after the onset of feeding (53 dayspostfertilization, mean TL � 32 mm), joint forma-tion between the radials and the glenoid ridge iscomplete or nearly complete (Fig. 2H). The proptery-gium remains connected by a small number of par-tially chondrogenic cells to the lateral margin of theglenoid ridge. The metapterygium remains con-nected to the metapterygial process of the glenoid in60% of 37 dps specimens analyzed (n � 15).Metapterygial radial 3 has condensed within thenonchondrogenic mesenchyme posterior to the sec-ond metapterygial radial. The lateral scapular pro-cess has fused to the anterior margin of the scapu-locoracoid plate forming the propterygial bridge onwhich the propterygium pivots. The fusion of thelateral scapular process with the scapulocoracoidplate forms a large foramen bordered by the scapu-lar processes and the glenoid ridge. This foramenaccommodates much of the adductor musculature ofthe pectoral fin (Jessen, 1972) and is likely homolo-gous to the supraglenoid foramen of sarcopterygians(Janvier, 1996). Within the band of Alcian Blue-staining cells distal to the radials, and medial to theproximal margin of the developing fin rays, the dis-tal radials begin to condense. Each distal radialforms as a separate condensation foci distal orslightly posterodistal to an underlying radial. Underoblique lighting, the collagenous prototypes of thelepidotrichia are visible along the anterior margin ofthe finfold (data not shown).

Metamorphosis (52 Days Poststaging)

At 52 days after the onset of feeding (68 dayspostfertilization, mean TL � 45 mm), larva reachmetamorphosis. By this stage in development thepectoral fin endoskeleton has reached the adult mor-phology, with a full complement of radials and distalradials (Fig. 2I). The number of metapterygial radi-als can vary, but the usual complement is four (seediscussion regarding variation). The posteriormostmetapterygial radial generally articulates with adistal facing process on the metapterygium. Distalradials are condensed, although some chondrogenicmesenchyme remains surrounding the condensa-tions. An anterior process is present on the proptery-gium that articulates with the lepidotrichia thatcomprise the leading edge of the finfold. Lepi-dotrichia overlap the distal radials and the distalends of the proximal radials (Fig. 3D). The proximo-distal overlap of the dermal rays and endoskeletalradials is equal to 6–8% of total fin length at meta-morphosis (n � 10). The degree of proximodistaloverlap between the lepidotrichia and the radials inadulthood is equivalent to the degree of overlap be-tween actinotrichia and the endoskeletal disc atearly stages of pectoral fin development. However,the total amount of endoskeletal/dermal overlap has

increased due to the condensation of distal radialswithin the proximal finfold. There is some asymme-try between dorsal and ventral sets of lepidotrichia,with ventral lepidotrichia overlapping more proxi-mally than do the dorsal lepidotrichia (Fig. 3E).Overlap of the endoskeleton by the lepidotrichia fol-lows the orientation of the actinotrichia (Fig. 3F),with decreasing overlap of more posterior rays(Fig. 3D).

Acipenser transmontanus: Pectoral Girdleand Pectoral Fin Endoskeleton

Embryos and larva of Acipenser transmontanuswere stained with Alcian Blue and immunostainedwith Type II collagen to investigate pectoral fin andgirdle development. Figure 1 depicts the earlieststage to stain for Type II collagen in the pectoralgirdle (Fig. 1C; Stage 42) and the latest larval stageavailable for this study (Fig. 1D, TL � 20 mm). Thespecific stages used in this study were selected basedon the presence of key developmental events notpresent in previous stages. The pectoral fins of A.transmontanus project laterally from the body andare not obscured by the operculum as in Polyodon.This condition allows for unobstructed viewing andimaging of the pectoral girdle and fins in wholemount (Fig. 4).

Prechondrogenic Stages

Pectoral fin development is first visible betweenStages 37 and 38 (comparable to Acipenser gulden-stadti in Schmalhausen, 1991) as a swelling of theectoderm on the dorsal surface of the yolk sac (datanot shown). This swelling is due to local mesenchy-mal proliferation that will form the common anlagenof the pectoral girdle and pectoral fin endoskeleton.At these early stages the pectoral finbuds are ori-ented such that the anteroposterior axis of the fin isparallel to the anteroposterior axis of the body. Atthis point in development the epidermis of the fin-bud consists of a superficial peridermal layer over-lying cells of the basal stratum. By Stage 38, theapical ectodermal ridge is visible as an anteroposte-riorly oriented thickening of the basal stratum alongthe distal margin of the finbud. Between Stages 38and 39, this apical ridge elongates distally to formthe finfold.

Stage 40

At Stage 40 (14 days postfertilization, mean TL �14 mm), the first chondrogenic condensations areseen in the common anlage of the pectoral girdle andfin (Fig. 4A). The endoskeletal disc has begun todifferentiate into separate radials, as evidenced byfaint strips of varying stain intensity (arrowheads inFig. 4A). At Stage 40, the anlage of the scapulocora-coid is continuous with the finbud mesenchyme.


Figure 4


Cells along the dorsal and ventral margins of thelongitudinal plate region of the scapulocoracoid be-gin to condense to form the scapular and coracoidprocesses, respectively (Fig. 4B). Transverse sec-tions of a Stage 40 fin show that the mesenchyme inthe anterior portion of the fin is loosely packed, withonly cells with the endoskeletal disc exhibiting thestacked morphology of typical chondrogenic conden-sations (Fig. 5A). Transverse sections through thepresumptive metapterygium show a more com-pacted and dorsoventrally compressed fin mesen-chyme (Fig. 5B). The primary dorsal and ventralmusculature of the fin are differentiated by Stage 40(Fig. 5A,B). Immunostaining detected no Type IIcollagen in the fin/girdle extracellular matrix atStages 40 (Fig. 4C,D). Under oblique lighting, col-lagenous actinotrichia can be seen within the elon-gating finfold (Fig. 4A).

Stage 42

At Stage 42 (16 days postfertilization, mean TL �15 mm), the finbud mesenchyme continues to differ-entiate into separate radials (Figs. 4E,F, 6A). Thecells of the endoskeletal are more compacted than atStage 40 (compare Fig. 5A,C), but are still sur-rounded by loose mesenchyme. The common anlageof the scapulocoracoid and the metapterygium isvisible in anteroposterior (Fig. 5D) and proximodis-tal section (Fig. 6B,C). The proximal metapterygiumis continuous with the glenoid ridge (Fig. 6C,D). Thecoracoid foramen can be seen piercing the proximalcoracoid process just ventral to the glenoid ridge(Fig. 6B–D). As in Polyodon, the anterior most con-densation consists of the common anlage of the prop-terygium and mesopterygial radial 1. Mesopterygialradials 2 and 3, the metapterygium, and the firstmetapterygial radial are also visible in Alcian Blue-stained specimens (Fig. 4E,F) and in section (Fig.6E,F). The scapulocoracoid is mediolaterally thickerthan in previous stages and the scapular and cora-coid processes continue to elongate distally (Fig. 4F).By Stage 42, Type II collagen secretion in the extra-

cellular matrix of the shoulder girdle begins (Fig.4G,H). Stain just dorsal and ventral to the fin basemarks the chondrogenic condensations of the scap-ular and coracoid processes respectively (Fig. 4H).Type II collagen is also present within the longitu-dinal plate of the scapulocoracoid. However, Type IIcollagen is absent from the proximal portions of thescapular and coracoid processes where they connectto the longitudinal plate (Fig. 4H). The first Type IIcollagen secretion in the fin mesenchyme appearsbetween Stages 42 and 43 within the condensationof the metapterygium (Fig. 4H). The finfold andactinotrichia continue to elongate distally.

Stage 44

At Stage 44 (18 days postfertilization, mean TL �16 mm) the anteriormost anlagen within the fin hasdivided into the propterygium and mesopterygialradial 1. These two elements remain attached prox-imally to the anterolateral margin of the glenoidridge, whereas mesopterygial radials 2 and 3 haveseparated, as evidenced by the loss of Alcian Bluestaining in this area (Fig. 4I). Metapterygial radial 2is now present and the metapterygium continues toelongate distally. The lateral scapular process be-gins to condense, connecting the anterior margin ofthe scapular process (the medial scapular process ofJessen, 1972) to the anterior longitudinal plate (Fig.4J). Type II collagen is present in the propterygium,mesopterygial radials 1 through 3, and themetapterygial radial 1. At Stage 44, Type II collagenis confined to the core of each developing radial (Fig.4K). In contrast, extracellular proteoglycans arepresent throughout the entire condensation (Fig.4I). Type II collagen and Alcian Blue stain similarportions of the scapular, lateral scapular, and cora-coid processes. However, Type II collagen is absent(or present at very low concentrations) from thelongitudinal plate and the proximal margins of thescapular and coracoid processes (Fig. 4L).

Two Days Poststaging

Two days after the onset of feeding (20 days post-fertilization, mean TL � 18 mm) the mesopterygialradials have separated from the glenoid ridge, whilethe propterygium remains weakly connected (Fig.4M). The metapterygium, which is generally the lastelement to separate from the girdle anlagen, is stillpartially connected in most specimens at this stage.The metapterygium continues to condense and elon-gate distally and has developed the distinctive pos-teromedially oriented bend present in mature spec-imens of Acipenser transmontanus (Fig. 4M).Metapterygial radial 2 continues to condense, al-though Type II collagen has yet to appear in the coreof the condensation (Fig. 4O). Differences in thepattern of extracellular proteoglycans and Type IIcollagen in the pectoral girdle can be clearly seen in

Fig. 4. Developmental series of pectoral fins and girdles ofAcipenser transmontanus. Alcian Blue (A,B,E,F,I,J,M,N,Q,R,U,V) and immunostained for Type II collagen (C,D,G,H,K,L,O,P,S,T,W,X). Whole-mount fins and girdles are shown in ven-tral (first and third columns) and lateral (second and fourthcolumns) view. Anterior is to the left. A–D: Stage 40 (TL � 14mm). E–H: Stage 42 (TL � 15 mm). I–L: Stage 44 (TL � 16 mm).M–P: 2 days poststaging (TL � 18 mm). Q–T: 4 days poststaging(TL � 18 mm). U–X: 13 days poststaging (TL � 20 mm). Scalebars � 100 �m. ac, actinotrichia; cop, coracoid process; ed, en-doskeletal disc; ff, finfold; gl, glenoid ridge; lsc, lateral scapularprocess; me1-3, mesopterygial radials one and three; mt,metapterygium; mtr1-3, metapterygial radials1-3; pr, proptery-gium; pr/me1, common anlage of the propterygium and me-sopterygial radial 1, prb, propterygial bridge; scp, scapular pro-cess; scl, supracleithral cartilage.


Fig. 5. Pectoral fin and girdle development in Acipenser transmontanus at Stage 40 (A,B) and Stage 42 (C,D) in anteroposteriorsection. Insets denote plain of section for A–D. Hematoxylin-eosin and Alcian Blue trichrome stain. A: Larval Stage 40 section throughthe anterior half of the pectoral fin/girdle at the level of the presumptive propterygium/mesopterygial radial 1. Fin and girdlemesenchyme are loosely packed. B: Stage 40 section through the posterior margin of the finbud. Stacked chondrocytes of themetapterygium are visible. C: Stage 42 section through the anterior half of the pectoral fin/girdle at the level of the presumptivepropterygium/mesopterygial radial 1. Stacked chondrocytes of the endoskeletal disc are visible. D: Stage 42 section through theposterior half of the pectoral fin/girdle including the metapterygium showing the common anlage of the scapulocoracoid andmetapterygium. Scale bars � 100 �m. All sections 10 �m thick. bm, basement membrane; bs, basal stratum; cmf, chondrogenicmesenchyme in the finfold; cop, coracoid process; dm, dorsal musculature; ed, endoskeletal disc; ff, finfold; li, liver; mt, metapterygium;pe, periderm; pn, pronephros; scp, scapular process; vm, ventral musculature; yo, yolk.

ventrolateral view (Fig. 4N,P). Whereas Alcian Bluestaining demonstrates that proteoglycans are ubiq-uitous throughout the entire girdle anlagen, Type IIcollagen is limited to the scapular and coracoid pro-cesses and is absent from the scapulocoracoid. Like-wise, Type II collagen is absent from the proximalportion of the fin radials but is present distally.

Four Days Poststaging

By 4 days after the onset of feeding (22 days post-fertilization, mean TL � 18 mm) jointing betweenthe fin endoskeleton and the shoulder girdle is com-plete (Fig. 4Q). The lateral scapular process and theanterolateral margin of the longitudinal plate meetto form the propterygial bridge (Fig. 4Q). In Aci-penser the propterygial bridge separates the glenoidridge from the medial surface of the cleithrum, open-ing a fossa to accommodate the pectoral muscula-ture that rotates the propterygium and dermal finspine forward (Findeis, 1997). By 4 days poststag-ing, Type II collagen is present in the extracellularmatrix of all fin radials (Fig. 4S). Type II collagenhas expanded beyond the core cells of each radial,but is still absent from the most peripheral cells(compare radial widths in Fig. 4Q,S). The proptery-gium now stains more intensely for Type II collagenthan do the mesopterygial radials or the metaptery-gium (Fig. 4S), a condition that persists throughoutall later stages of Acipenser transmontanus avail-able for this study. The pectoral girdle also shows anincrease in the intensity of Type II collagen staining(Fig. 4T). However, Type II collagen remains absentin the glenoid ridge, propterygial bridge, and proxi-malmost regions of the scapular and coracoid pro-cesses.

Thirteen Days Poststaging

At 13 days after the onset of feeding (31 dayspostfertilization, mean TL � 20 mm), the radials ofthe fin endoskeleton are discrete condensations withdefined articulations with the glenoid ridge or ante-rior margin of the metapterygium (Fig. 4U). In con-trast to earlier stages, Type II collagen is presentthroughout the extracellular matrix of the radials(Fig. 4W). As in previous stages, Type II collagen isabsent in the glenoid ridge and longitudinal plate,the proximal portions of the scapular and coracoidprocesses adjacent to the longitudinal plate, and theproximal region of the metapterygium (Fig. 3X). Astrip of Type II collagen staining extending an-terodorsally from the anterior margin of the scapu-lar process marks the position of the supracleithralcartilage (Fig. 3X), a unique element found in stur-geons (Acipenseridae, sensu Findeis, 1997) but ab-sent in Polyodon and other basal actinopterygians.At 13 days poststaging, lepidotrichial formation hasnot yet begun, but is likely temporally correlated

with distal radial condensation as in Polyodonspathula (see above).

DISCUSSIONSkeletal Homology

Phylogenetic analyses and fossil evidence supportthe hypothesis that a tribasal fin endoskeleton con-sisting of one or more anterior radials (homologousto the propterygium and/or mesopterygium) and ametapterygium is the primitive condition for gna-thostomes (Janvier, 1996; but see Maisey, 1984, foran alternative hypothesis). There are two extantclades of crown-group gnathostomes, chondrichthy-ans (elasmobranchs � holocephalans) and osteich-thyans (sarcopterygians � actinopterygians), andtwo major extinct clades, acanthodians and placo-derms. The phylogenetic relationships of acantho-dians and placoderms to the extant clades are un-certain at best (Coates, 1994), although a number ofmorphological characters support the hypothesisthat acanthodians may be the sister-group to Os-teichthyes (Maisey, 1986). Acanthodians possess aperichondrally ossified endoskeletal pectoral girdleand mineralized actinotrichia (Denison, 1979). Thefin endoskeleton is poorly known in acanthodiansand likely remained cartilaginous or possessed athin perichondrum. In the Permian acanthodian Ac-anthodes, three proximal radials are present(Coates, 1994). The posteriormost radial, presum-ably the metapterygium, supports two radials artic-ulating with its distal margin. Placoderms wereheavily armored gnathostomes mostly endemic tothe Devonian period. Like acanthodians, the fin ra-dials of placoderms are poorly known and likelyremained cartilaginous or were weakly ossified. TheLower Devonian Pseudopetalichthys possesses twoproximal radials in the pectoral fin, although theposterior radial appears to consist of two fused ele-ments (Gross, 1962). The pectoral fin endoskeletonof the Upper Devonian Ctenurella consists of threeproximal elements, the posteriormost being the larg-est of the three (Ørvig, 1962). The majority of extantand fossil elasmobranch chondrichthyans possesstribasal pectoral fin endoskeletons (Zangerl, 1981;Janvier, 1996), while holocephalans possess dibasalfins (Didier, 1995; Stahl, 1999). Notable exceptionsinclude the multiple unjointed anterior radials ofCladoselache, symmoriids, and stethacanthids(Zangerl, 1981), and the monobasal pectoral fins ofxenacanthids elasmobranchs and chondrenchelyidholocephalans (Stahl, 1999). In the latter case, thecondition likely evolved convergently in xenacan-thids and chondrenchelyids in conjunction with aspecialized mode of life (Stahl, 1999). A series ofanterior radials and a posterior metapterygium arepresent in stem actinopterygians (e.g., Mimia andMoythomasia, Gardiner, 1984; Palaeoniscus, Jes-sen, 1972), as well as extant nonteleost actinoptery-gians. The loss of the metapterygium appears to be


Figure 6


correlated with the origin of teleosts, although theosteoglossomorph Hiodon possesses radials that bi-furcate distally (Hilton, 2002) and are similar inmorphology to the metapterygium of the nonteleostactinopterygian Lepisosteus (MCD, pers. obs.). Thetotal number of radial elements in most teleost pec-toral fins rarely exceeds four (Jessen, 1972), al-though some derived forms may possess as many as12 radials (De Pinna, 1996). The propterygium ofmost actinopterygians is perforate (Jessen, 1972),allowing for passage of appendicular vasculatureand nerves to the anterior portions of the fin (Jan-vier, 1996). However, this character is absent in thestem actinopterygians Cheirolepis and Howqualepis(Long, 1988) and in the extant Polypterus and ismore accurately a synapomorphy of Actinopteri(sensu Patterson, 1982). Posterior to the proptery-gium are the mesopterygial radials. Like the prop-terygium, the mesopterygial radials articulate withthe scapulocoracoid proximally.

In actinopterygians, there are two phases of en-doskeletal patterning. In the first phase the initiallychondrogenic endoskeletal disc subdivides to formindividual radials via decomposition of interveningextracellular matrix. In teleosts, secretion of extra-cellular proteoglycans begins prior to formation ofthe radials. In the zebrafish Danio, proteoglycansecretion is correlated with the formation of theendoskeletal disc at early finbud stages (Grandeland Schulte-Merker, 1998). Other extracellular ma-trix components, namely Type II collagen, may alsobe secreted prior to disc subdivision in Danio (MCD,pers. obs). The second phase of development involvesthe condensation of the distal radials. Observationsfrom Polyodon development support previous inter-pretations that distal radials form as discrete con-densation foci after endoskeletal disc subdivision(Grandel and Schulte-Merker, 1998) in a mannernot unlike carpal/tarsal formation in tetrapods(Shubin and Alberch, 1986). However, signals medi-ating distal radial formation may involve ecto-dermal tissues owing to the appearance of the

lepidotrichia concomitant with distal radial conden-sation (see below).

Development of the pectoral endoskeleton in Poly-odon and Acipenser differs from the pattern seen inteleosts in a couple of important ways. At compara-ble stages, the pectoral fins of teleosts consist ofundifferentiated, flattened discs of chondrogenicmesenchyme, while the pectoral fins of Acipenseri-formes consist of a similar chondrogenic disc, butone that is attached along its posterior margin to achondrogenic condensation continuous with the en-doskeletal shoulder girdle. Thus, in Polyodon andAcipenser the metapterygium is already present atthe earliest stages of fin development (Figs. 5D,6B–D) prior to subdivision of the radials (Figs. 2A,5B). Another important difference between en-doskeletal patterning in teleosts and Acipenseri-formes is that in Polyodon and Acipenser some of theradials form after subdivision of the endoskeletaldisc. In Polyodon, only the first metapterygial radialforms by decomposition of surrounding chondro-genic mesenchyme (although see skeletal variationdiscussion below). The second, third, and fourthmetapterygial radials form as condensation fociwithin the nonchondrogenic mesenchyme betweenthe first metapterygial radial and the anterior mar-gin of the metapterygium. In this regard, themetapterygial radials form in a manner similar todistal radials or to the limb bones of tetrapods (Shu-bin and Alberch, 1986) and sarcopterygian fish (Se-mon, 1898; Joss and Longhurst, 2001). Indeed, theprimary morphological difference between themetapterygium of Acipenseriformes and that of sar-copterygians is in the presence of joints along theproximodistal axis (Davis, 2003).

The “composite” fin of Polyodon and Acipenser,possessing both a teleost-like endoskeletal disc anda sarcopterygian-like metapterygial condensation,has been generally overlooked in previous descrip-tions of Acipenseriform fin development (Sewertzoff,1926; Kryzanovsky, 1927; reviewed in Grandel andSchulte-Merker, 1998), although a figure in Kryza-novsky (1927, Abb.16) depicts the metapterygium asa continuous rod connected to the shoulder girdle inlarval Acipenser stellatus. Interestingly, a con-densed metapterygium attached to the flatten en-doskeletal disc has also been described for the chon-drichthyan Scyliorhinus canicula (Balfour, 1881;Scyllium canicula of the author). In describing thelarval fin endoskeleton in S. canicula Balfour writes(bracketed text added for clarity): “The plate [en-doskeletal disc] continuous with the basal bar[metapterygium] of the fin is at first, to a consider-able extent in the pectoral, and to some extent in thepelvic fin, a continuous lamina, which subsequentlysegments into rays.” A more detailed investigationinto fin skeletal patterning in other basal acti-nopterygians (Polypterus, Amia, and Lepisosteus)and in Chondrichthyes will be necessary to confirmwhether a common scapulocoracoid/metapterygial

Fig. 6. Pectoral fin and girdle development in Acipensertransmontanus at Stage 42 in proximodistal section. Anterior isto the left. A: Stage 42 whole mount fin and girdle in ventral viewas reference for proximodistal sections B–F. Sections stainedwith H&E. B: Close-up of the common chondrogenic anlage of thescapulocoracoid and metapterygium. C: Common anlage of thescapulocoracoid and metapterygium 40 �m distal to B. D: Com-mon anlage of the glenoid ridge area and the metapterygium 40�m distal to C. E: Section through the proximal endoskeletal disc.F: Section through the endoskeletal disc showing the initial con-densations of the propterygium and mesopterygial radials. Scalebars � 100 �m. cop, coracoid process; dm, dorsal musculature; ed,endoskeletal disc; fco, coracoid foramen; gf, gill filament; gl, gle-noid ridge; lsc, lateral scapular process; me1-3, mesopterygialradials 1-3; mt, metapterygium; mtr1, metapterygial radial 1; pr,propterygium; pr/me1, common anlage of the propterygium andmesopterygial radial 1, prb, propterygial bridge; pn, pronephros;scp, scapular process; vm, ventral musculature


anlage and an anterior endoskeletal disc is an em-bryonic condition common to all gnathostomes pos-sessing pro-, meso-, and metapterygial radials. How-ever, the developing humerus in Ambystomamaculatum larvae forms a continuous condensationwith the scapulocoracoid (Burke, 1991), suggestingthat a common anlage of the pectoral girdle and themetapterygial axis may be a primitive gnathostomecondition.

Thus, the teleost developmental condition is dueto loss of the metapterygial axis and the early con-densation pattern of this element. As a result, theentire fin endoskeleton in teleosts forms via subdi-vision of the remaining endoskeletal disc. In con-trast, sarcopterygians would have lost the pro- andmesopterygial radials and the mechanism by whichthese elements form. The metapterygial appendageof sarcopterygian fish and tetrapods develops viacondensation of elements within a nonchondrogenicmesenchyme. This same mechanism forms the ho-mologous elements in Acipenseriformes, but is notobserved during the formation of teleost radials(Mabee and Trendler, 1996; Grandel and Schulte-Merker, 1998). However, the distal radials of te-leosts do arise as individual condensation foci (Bou-vet, 1968; Grandel and Schulte-Merker, 1998), andmay be the skeletal elements most developmentallysimilar to the sarcopterygian appendage skeleton.Together, these data support the hypothesis that thebasal osteichthyan, and possibly gnathostome, con-dition involved more than one mechanism of en-doskeletal patterning within the same fin.

Variation of the Endoskeletal Pattern

Several variant endoskeletal patterns are seen inPolyodon and Acipenser that differ from the “nor-mal” or most common arrangement (Fig. 7A,D). Thisvariation is confined to the metapterygium and themetapterygial radials, the only common exceptionbeing the appearance of an additional mesopterygialradial (Fig. 7B). However, there is a correlation be-tween variation in the number of mesopterygial ra-dials and variation in the number of metapterygialradials in Polyodon and Acipenser. There are nor-mally two mesopterygial radials in Polyodon (Fig.7D) and the usual complement for A. transmontanusis three radials (Fig. 7A). In Polyodon there arenormally four metapterygial radials articulatingwith the anterior margin of the metapterygium,while the normal complement in A. transmontanusis three. All specimens of Polyodon (n � 5; 17% oftotal) and Acipenser (n � 4; 13% of total) scored forthe presence of an additional mesopterygial radialpossessed one less metapterygial radial than thenormal complement. Furthermore, pectoral fins pos-sessing less than the normal complement of me-sopterygial radials were never observed. This pat-tern of variation suggests that supernumerarymesopterygials are due to a “slippage” or a slight

shift in the position of the first metapterygial radial.As a result, metapterygial radial 1 lies in articula-tion with the glenoid ridge instead of the anteriormargin of the proximal metapterygium (Fig. 7B).Only one specimen was identified that had fewerthan the regular complement of metapterygial radi-als but had no additional mesopterygial radials, al-though this condition appeared to be the result of afusion of metapterygial radials 2 and 3 (arrow in Fig.7F). However, a specimen of P. spathula figured inGrande and Bemis (1991, fig. 21) does lack ametapterygial radial while retaining only two me-sopterygial radials. Some specimens possess super-numerary metapterygial radials (Fig. 7E). In thesecases, an additional condensation focus appears an-terior to the metapterygium and develops into anadditional radial.

Another form of variation commonly seen in bothPolyodon and Acipenser is variation of the morphol-ogy of the radials. In all but one case such variationwas limited to the metapterygium and themetapterygial radials. The one exception was thepresence of a novel condensation between a me-sopterygial radial and the glenoid ridge (arrow inFig. 6E). It is interesting to note that this novelcondensation was present in a specimen possessingan additional metapterygial radial as well. Othervariants observed include bifurcation of the distalend of a radial (Fig. 6C), fusion of adjacent conden-sation foci (arrow in Fig. 6F), and the presence ofpartial or complete joints along the metapterygium(arrowhead in Fig. 6F; arrow in Fig. 6G). Joints inthe metapterygium are generally located near eitherthe proximal or the distal ends, although joints canappear along mid-length (Grande and Bemis, 1991;see fig. 43 for the Chinese paddlefish Psephurusgladius). One specimen of Polyodon possessed anadditional metapterygial radial articulating withthe post-axial side of the metapterygium (arrow inFig. 6H). This particular condition is reminiscent ofthe post-axial radials seen in some sarcopterygians(e.g., Neoceratodus; Semon, 1898) and in somefossil elasmobranchs (e.g., xenacanthids; Hampeand Heidtke, 1997) and holocephalans (Stahl, 1999).

Variation in the Extracellular Matrix

As mesenchymal cells within the fin/limb bud dif-ferentiate into chondrocytes, they begin to synthe-size the proteins that will form the extracellularmatrix (ECM). Chief among these proteins arecartilage-specific proteoglycans and Type II colla-gen. The proteoglycans typically found in cartilagecan consist of hundreds of glycosaminoglycan chains(usually a mixture of chondrotin sulfate and keratinsulfate chains) covalently linked to a serine-rich coreprotein (Roden, 1980). These large proteins arehighly hydrophilic, forming hydrated gels that givethe ECM of cartilage a swelling pressure that resistscompressive forces (Alberts et al., 1983). The most


abundant proteins in gnathostome ECM are mem-bers of the collagen family. The three cartilage spe-cific collagens found in the gnathostome endoskele-ton are Types II, IX, and X, with Type II by far themost abundant (Castagnola et al., 1988).

In tetrapods, Type II collagen has been utilized asan earlier marker for chondrogenesis, generally ap-pearing prior to proteoglycan synthesis (Wake andShubin, 1998). Micromass cultures of mouse limbbud mesenchyme (Edwall-Arvidsson and Wrob-

lewski, 1996) and in vivo studies of chicken mandib-ular arch condensation (Mina et al., 1991) demon-strate that Type II collagen expression precedes thatof cartilage specific proteoglycans. In the endoskel-etal disc of zebrafish, proteoglycans (visualized byAlcian Blue) are present by 96 hpf (Grandel andSchulte-Merker, 1998), before detectable levels ofType II collagen are observed (MCD, pers. obs). TypeII collagen is first seen in the zebrafish scapulocora-coid (96 hpf) and then throughout the endoskeletal

Fig. 7. Variation of the endoskeletal pattern in Acipenser transmontanus (A–C) and Polyodon spathula (D–H). Pectoral fins areshown in ventral view for A. transmontanus and dorsal view for P. spathula. Anterior is to the left. A: Illustration of the “normal” finendoskeleton of A. transmontanus. B: Slippage of metapterygial radial 1 to articulate with the glenoid ridge instead of with theanterior surface of the metapterygium. C: Distal bifurcation of a radial. D: Illustration of the “normal” fin endoskeleton of P. spathula.E: Supernumerary radials. An additional metapterygial radial is present. Arrow denotes a novel condensation between mesopterygialradial 2 and the glenoid ridge. F: Distal bifurcation of a radial associated with one less metapterygial radial (arrow). This may involvefusion of the condensations for metapterygial radials 2 and 3. Arrowhead denotes novel joint formation along the metapterygium(compare to D). Space between mesopterygial radials 1 and 2 due to distortion of the specimen. G: Novel joint or incomplete joint nearthe base of the metapterygium. This condition is a common variant, scored in 30% of specimens older than 37 days poststaging (TL �32 mm, n � 30). H: Post-axial radial. Scale bars � 100 �m. dr, distal radial; gl, glenoid ridge; mt, metapterygium; mtr1, metapterygialradial 1; pr, propterygium.


disc (120 hpf), prior to disc subdivision (MCD, pers.obs.). In Acipenser the pattern of Type II expressionshows interesting similarities to both tetrapods andzebrafish. As in tetrapods, Type II collagen appearsrestricted to chondrogenic condensations and notthe surrounding mesenchyme. Unlike zebrafish,Type II collagen is not present in the undifferenti-ated endoskeletal disc (Fig. 4C,G). However, muchas in zebrafish, proteoglycans are present in theECM of the endoskeletal disc prior to accumulationof detectable levels of Type II collagen, the reverse ofthe condition in tetrapods.

Curiously, Type II collagen was never detected inthe glenoid ridge region of the scapulocoracoid inAcipenser (Fig. 4P,T,X). This may suggest the pres-ence of other collagens in this region, althoughmonoclonal antibodies for Types I, IX, and XI failedto stain the glenoid ridge (data not shown). Like-wise, an alternative monoclonal antibody to Type IIcollagen (CIIC1) produced similar results to the pri-mary Type II used in this study (II6B3), yet failed tostain the glenoid ridge. In larval Acipenseriformesthe glenoid ridge must act as a joint for the movingpectoral fin, even though the condensations of the finand girdle skeletons remain connected. The func-tional demands on the glenoid ridge may require adifferent ECM composition than the rest of thescapulocoracoid. Such variation in ECM compositionin cartilage may be the result of biomechanical stim-uli (Takahashi et al., 1998; Rabie et al., 2003). TheECM of the glenoid ridge may contain other collagentypes not assayed in this study or may possess col-lagens at concentrations below the sensitivity ofstandard immunostaining techniques. Alterna-tively, collagen may be absent from the glenoid ridgealtogether. Although collagen is the predominantECM protein in gnathostome cartilages, the carti-laginous endoskeletons of the extant agnathansPetromyzon (lampreys) and Myxine (hagfish) arenoncollagenous (Robson et al., 2000).

Type II collagen was not observed in the fin en-doskeleton of Polyodon until late larval stages, andthen only in the scapular and coracoid processes andthe distal elements of the fin (data not shown). Allspecimens of Polyodon immunostained for Type IIcollagen stained positively in other parts of the body,primarily the jaws and branchial apparatus. Mono-clonal antibodies against Types I, IX, XI, and analternative Type II collagen failed to stain the finendoskeleton in Polyodon. As in the case of the gle-noid ridge of Acipenser, either collagens are presentthat are not recognized by the antibodies available,the concentration of collagen protein is too low to bedetected, or portions of the fin endoskeleton in Poly-odon are composed of noncollagenous cartilage.Cloning and in situ expression of the Type II colla-gen gene in Acipenser and Polyodon, as well as up-stream genes implicated in the expression of Type IIcollagen, such as the transcription factor Sox 9 (e.g.,

Yan et al., 2002), would shed light on these obser-vations.

Development of the Dermal Fin Rays

Actinotrichia comprise the primary structuralsupport of the larval finfolds of Polyodon and Aci-penser. Actinotrichia first appear along the distalmargin of the finbud mesenchyme and elongate dis-tally within the developing ectodermal finfold (Fig.3A). In both taxa the proximal margins of the acti-notrichia overlap the distal margin of the endoskel-etal disc (arrows in Fig. 3A,B). The actinotrichiaalong the anterior margin of the finfold overlap thedeveloping endoskeleton more completely than doactinotrichia along the posterior margin, althoughthis is not clearly discernable until later stages (Fig.3D). A similar pattern of actinotrichial developmenthas been described for the neopterygians Salmo(Geraudie and Landis, 1982) and for Danio (Grandeland Schulte-Merker, 1998), although in both casesactinotrichia do not appear to overlap the distalmargin of the endoskeletal disc, overlapping onlythe chondrogenic mesenchyme of the presumptivedistal radials.

Actinotrichia are composed of elastoidin (Ger-audie, 1977), a protein composed of �1 type collagenhomotrimers (Kimura et al., 1986). Actinotrichia inboth Acipenser and Danio stain positively for Type Icollagen (data not shown), suggesting that elastoi-din may crossreact with the antibody for Type Icollagen or that the actinotrichia in these taxa maycontain Type I collagen (a heterotrimer of �1 and �2type collagen chains). Previous studies of acti-nopterygian fin development have observed migra-tion of distal finbud mesenchyme into the finfoldconcomitant with actinotrichial formation (Geraudieand Landis, 1982; Wood, 1982). Thus, the acti-notrichia appear to play a role in organizing mesen-chymal invasion of the finfold, perhaps through amechanism of contact guidance (Wood and Thoro-good, 1984). In Polyodon and Acipenser, mesenchy-mal invasion of the finfold follows a similar pattern,as evidenced by the appearance of diffuse AlcianBlue within the proximal portions of the fin-fold shortly after actinotrichia first appear (Figs.2B, 4A).

The stages of Acipenser transmontanus availablein this study did not allow for observation of lepi-dotrichia formation. However, in Polyodon lepi-dotrichia are first visible around 37 days poststagingbut do not stain with Alizarin Red until a few dayslater, suggesting a delay between formation of thecollagenous prototype of each ray and subsequentcalcification. Jointing of the lepidotrichia first beginsin the proximal portions of the each ray, with moreanterior lepidotrichia initiating joint formation ear-liest. Thus, joint formation follows the same generalorder as lepidotrichial condensation described forSalmo (Geraudie and Landis, 1982) and for Danio


(Grandel and Schulte-Merker, 1998), starting at theanteroproximal corner of the finfold and proceedingposteriorly and distally. This order of lepidotrichialformation and subsequent jointing appears to be aprimitive condition for Osteichthyes. Ontogeneticseries of the Devonian fossil sarcopterygian Eusthe-nopteron show that larval specimens possess un-jointed lepidotrichia, but that as body length in-creases jointed lepidotrichia appear (MCD, pers.obs.). Certain osteichthyan groups have conver-gently lost jointing as well, resulting in elongaterods of bone. The Devonian sarcopterygian Sau-ripterus possesses unjointed lepidotrichia that areunique in their extensive ventral and dorsal overlapof the fin endoskeleton (Davis et al., 2001, 2004). InSyngnathidae (seahorses and pipe fishes), long un-jointed demi-rays are present in pectoral and dorsalfins. The loss of ray jointing may reflect specializedlocomotor strategies. In Sauripterus the unjointedlepidotrichia and their overlap of the fin endoskele-ton result in a stiffened appendage that may havebeen used to push against the substrate or watercolumn (Davis et al., 2004). Likewise, the stiffeneddemi-rays of syngnathids may reflect the uniquelocomotor requirements of these fishes (Consi et al.,2001).

Apical Ectodermal Fold and RelativeSkeletal Contributions

The relative contribution of the endoskeletal radi-als and dermal rays to the fin skeleton is one of thekey morphological differences between ray-finnedand lobe-finned designs (Shubin and Davis, 2004).The general trend is such that actinopterygians pos-sess elaborate dermal fin skeletons with a relativelysmall endoskeleton, while sarcopterygians possesselongate fin endoskeletons with a lesser contributionto fin surface area from the dermal rays. Teleostsand tetrapods represent the extremes of these con-ditions, possessing primarily dermal or exclusivelyendoskeletal appendages, respectively. Phylogeneti-cally basal actinopterygians and sarcopterygians of-ten possess intermediate patterns of dermal andendoskeletal contribution. For example, the pectoralfin skeleton of Sauripterus consists of an elaborateendoskeleton sandwiched between massive un-jointed dermal rays (Davis et al., 2001, 2004). Sucha morphology would not have been predicted bymodels of paired fin development based on modelorganisms (Thorogood, 1991; Sordino and Duboule,1996). In particular, our understanding of the cellu-lar and molecular events behind fin skeletogenesis isbased on observations in a few teleosts, particularlyDanio. In Danio, proliferation of finbud mesen-chyme ceases when the distally flanking structureknown as the apical ectodermal ridge begins to elon-gate to form the apical ectodermal fold or finfold(Sordino and Duboule, 1996). This correlation be-tween cessation of proliferation in the cellular pop-

ulations fated to form the endoskeleton, formation ofthe finfold, and initiation of dermal fin skeleton de-velopment led to the hypothesis that a simple het-erochronic shift in when ridge-fold transition takesplace could explain the different skeletal morpholo-gies seen in actinopterygians and sarcopterygians(Thorogood, 1991). There are two primary predic-tions based on this hypothesis. First, there will be arelative trade-off in the contribution of each skeletaltype due to the necessary temporal segregation be-tween endoskeletal proliferation and dermal ray for-mation. As a result, one should not see fin morphol-ogies that consist of large endoskeletons and largedermal ray skeletons. Second, there should be littleor no proximodistal overlap between the endoskele-ton and the dermal rays unless the dermal rays wereto extend or elongate proximally during develop-ment. The fins of Sauripterus demonstrate thatthese predictions do not hold true for all gnathos-tomes.

Polyodon and Acipenser are comparable to Sau-ripterus in possessing relatively elaborate, distal-ized endoskeletons and large dermal fin rays. Like-wise, these Acipenseriformes feature a broadproximodistal zone of overlap between their dermalrays and the fin endoskeleton. At the earliest stagesof finfold elongation (after ridge-fold transition) ac-tinotrichia can be seen overlapping the distal mar-gin of the endoskeletal disc (Fig. 3A,B). When thecollagenous framework of the lepidotrichia first ap-pears in Polyodon (37 days poststaging), this samerelative degree of overlap with the radials is main-tained (Fig. 3D). Distal radials begin to condensewithin the proximal finfold concomitant with lepi-dotrichial formation. Thus, the degree of overlapbetween the dermal fin skeleton and the proximalradials is determined at very early stages, with noevidence of subsequent migration or elongation ofthe dermal skeleton to overlap the radials. Further-more, initiation of actinotrichial formation begins atStage 42 in Polyodon and Stage 40 in Acipenser,shortly after initial elongation of the finfold (Stage39 in Polyodon, Stage 38 in Acipenser) and prior tomost of fin endoskeletal development. Thus, in con-trast to the condition seen in teleosts, cessation ofendoskeletal proliferation is not correlated with for-mation of the apical ectodermal fold in Acipenseri-formes. These observations suggest that correlationof these two events in teleosts may represent a de-rived condition and that formation of the dermalrays and formation of the fin endoskeleton are nei-ther temporally nor spatially segregated in all gna-thostomes.

CONCLUSIONS

In both Polyodon spathula and Acipensertransmontanus different portions of the pectoral finendoskeleton develop via two different mechanisms.In particular, there is a distinct difference between


the pattern of chondrogenesis of the metapterygiumand the anterior radials. The anterior pectoral radi-als of Acipenseriformes, the propterygium and me-sopterygial radials, are considered homologous tothe pectoral radials of teleosts. As in teleosts, theseelements develop by subdivision of an initially chon-drogenic disc of cells. On the other hand, the Aci-penseriform metapterygium is considered homolo-gous to the monobasal fin skeleton of sarcopterygianfish and tetrapods. The metapterygium is initiallycontinuous with the pectoral girdle in Polyodon andAcipenser; a condition similar to that described forthe humerus and scapulocoracoid of the tetrapodAmbystoma (Burke, 1991). The metapterygium elon-gates in a proximal to distal direction through sub-sequent stages as metapterygial radials condensewithin the adjacent nonchondrogenic mesenchyme.Thus, the patterning mechanisms observed in theAcipenseriform fin reflects the putative homologiesof respective elements based on fossil and morpho-logical evidence. As such, the majority of extantosteichthyan diversity (i.e., teleosts and tetrapods)can be characterized by loss of different portions ofthe primitive developmental condition. In teleosts,the metapterygial axis is lost, resulting in a finendoskeleton formed exclusively by the endoskeletaldisc and the distal radials. Sarcopterygian fins havelost the endoskeletal disc and the distal radials,retaining only the metapterygial axis. These pat-terns of loss, in turn, may have allowed remainingskeletal structures to evolve in new ways. In tetra-pods, evolutionary patterns of skeletal loss also in-clude components of the dermal fin skeleton. Indeed,the loss of the paired fin lepidotrichia is a key nov-elty correlated with the origin of tetrapod limbs.

The patterns of variation seen in this study sug-gest a level of developmental independence betweenthe formation of the anterior radials and themetapterygial axis. Such independence is supportedby the convergent loss of anterior radials in fossilchondrichthyans and sarcopterygians and the loss ofthe metapterygium in teleosts. Likewise, basal gna-thostome taxa vary in the number of endoskeletalradials anterior to the metapterygium, suggestingthat variation in the pattern of endoskeletal discsubdivision may evolve without constraint on themetapterygium.

ACKNOWLEDGMENTS

Embryos of Polyodon spathula and invaluabletechnical support were provided by Steve Kahrs andthe employees of Osage Catfisheries Inc., OsageBeach, Missouri. We thank Michael Coates, VictoriaPrince, Melina Hale, Robert Ho, and members oftheir respective labs at the University of Chicago forassistance with ideas, techniques, and equipment.M.C.D. thanks William Bemis for sharing insightson, and his enthusiasm for, sturgeons and paddle-

fish. A.F. thanks John Postlethwait for support dur-ing sturgeon collection. The monoclonal antibody fortype II collagen (II-II6B3) was originally developedby Thomas F. Linsenmayer of Tufts Medical Schooland was obtained from the Developmental StudiesHybridoma Bank under the auspices of the NICHDand maintained by the University of Iowa, Depart-ment of Biological Sciences.

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Pectoral Fin and Girdle Development in the Basal...

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Transcript of Pectoral Fin and Girdle Development in the Basal...