Skeletal Development in Xenopus Laevis (Anura: Pipidae)

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JOURNAL OF MORPHOLOGY 214:l-41 (1992)

Skeletal Development in Xenopus laevis (Anura: Pipidae)

LINDA TRUEB AND JAMES HANKEN Museum of Natural History, and Department of Systematics and Ecology, The University ofliansas, Lawrence, Kansds 66045-2454 (L.T.)and Department of Environmental, Population, and Organismic Biology, University of Colorado, Boulder, Colorado 80309-0334 (J.H.) ABSTRACT Postembryonic skeletal development of the pipid frog Xenopus laevis is described from cleared-and-stained whole-mount specimens and sectioned material representing Nieuwkoop and Faber developmental Stages 46-65, plus postmetamorphic individuals up to 6 months old. An assessment of variation of skeletogenesis within a single population of larvae and comparison with earlier studies revealed that the timing, but not the sequence, of skeletal development in X. laevis is more variable than previously reported and poorly correlated with the development of external morphology. Examination of chondrocranial development indicates that the rostral cartilages of X. laevis are homologous with the suprarostral cartilages of non-pipoid anurans, and sug- gests that the peculiar chondrocranium of this taxon is derived from a more generalized pattern typical of non-pipoid frogs. Derived features of skeletal development not previously reported for X. laevis include 1) bipartite formation of the palatoquadrate; 2) precocious formation of the adult mandible; 3) origin of the angulosplenial from two centers of ossification; 4) complete erosion of the orbital cartilage during the later stages of metamorphosis; 5) development of the sphenethmoid as a membrane, rather than an endochondral bone; and 6) a pattern of timing of ossification that more closely coincides with that of the pelobatid frog Spea than that recorded for neobatrachian species. o 1992 Wiley-Liss, Inc.

The literature describing skeletal develop- observations on laboratory-reared frogs. Os- ment in the pipid frogxenopus laevis is exten- sification sequence was tabularized for 32 sive and, in recent years, the species has cleared-and-single-stained specimens rang- become a "model system" for experimental ing in age from 20-365 days and arranged in studies of skull development (e.g., on laryn- four stages: premetamorphic Stages I (20-29 geal development: Sassoon and Kelley, '86; d) and I1 (31-39 d), metamorphic Stage I11 Sassoon et al., '86; Kelley et al., '89; on Meck- (40-49 d), and postmetamorphic Stage IV el's cartilage: Thomson, '86, '87, '89; on tooth (56-365 d). Bernasconi ('51) did not define development: Shaw, '79, '85, '86, '88). The his stages with morphological criteria; thus first accounts of the skull were provided more they are not easily compared to those of than a century ago by Parker (1876, 1881; = Nieuwkoop and Faber ('56). Moreover, no Dactylethra capensis, auctorum). The devel- data are provided on the development of the opment of the hyobranchial skeleton in X. cartilaginous precursors of the bony skele- laevis was described by Ridewood (1897) and ton, and Bernasconi's ('51) illustrations re- that of the larval chondrocranium by Kott- veal several errors and misinterpretations of haus ('33).Paterson ('39) reviewed the devel- the anatomy of the developing and adult skull. opment of the head of larval and adult X. A detailed account of the ontogenesis of laevis and corrected several errors of Kott- the vertebral column of Xenopus laevis was haus ('331, which unfortunately had been provided by Smit ('53), who examined sec- perpetuated by de Beer ('37) in his book on tioned specimens representing Stages 24-66 the development of the vertebrate skull. of Nieuwkoop and Faber ('56) of individuals The first comprehensive description of cra- collected from natural populations in South nial and postcranial ossification in Xenopus Africa and staged by J. Faber. As part of their laevis was by Bernasconi ('51),who based his study of the visceral arches, larynx, and vis- o 1992 WILEY-LISS,INC 2 L. TRUEB AND J. HANKEN ceral muscles of Xenopus laevis, Sedra and lessens the utility of the normal tables for Michael (’57) described changes in the skull staging wild populations.” during metamorphosis. Their specimens also Given these extensive and diverse research were collected from wild populations in South efforts, one would expect the osteology and Africa and comprise a total of 12 Nieuwkoop development of Xenopus laevis to be well and Faber (’56) stages: viz., Stage 55 (232 d; known. However, comparison of figures and “typical larval stage”), Stages 56-57 (k38-41 descriptions in Kotthaus (’33),Paterson (’39), d; “premetamorphic” stages before the expo- Weisz (’45a,b), Bernasconi (’51), Nieuwkoop sure of the forelimbs), Stages 58-65 (k44-54 and Faber (’56), Sedra and Michael (’571, d; “metamorphosing” stages), and Stage 66 Deuchar (’751, and Reumer (’85)reveals sig- (258 d; “newly metamorphosed”). The au- nificant discrepancies in the terminology of thors sectioned the heads of an unspecified both the larval chondrocranium and the adult number of specimens and based their descrip- skeleton. Moreover, none of these authors tions and illustrations on hand-generated described the striking differences in the devel- graphical reconstructions from these sec- oping and adult cranium of X. laevis as com- tions. Given the tedious nature of this method pared to non-pipidl anurans. and the absence of any statements about Sokol (’75) pointed out that the ethmoidal variation, it seems likely that Sedra and region of the skull of larval pipids is distinct Michael’s (’57) descriptions may be based on from that of all other anurans; he suggested only a single specimen of each stage. that on the basis of this and many other Cranial osteological data from Sedra and characters (e.g.,opercular structure, hyobran- Michael’s study were included among the chid and filter apparatus, prootic foramina criteria used to define the developmental and their nerves, palatoquadrate suspensori- stages in the widely used normal table of um), pipoid (i.e., Rhinophrynidae + Pipidae) development of Xenopus laeuis (Nieuwkoop larvae were derived with respect to all other and Faber, ’56). This work also contains gen- anurans exclusive of the Microhylidae. The eral, but abbreviated, narrative accounts of peculiarities of the ethmoidal region with the development of various parts of the skele- respect to the formation of the nasal capsule ton. These were written by a variety of con- in pipids were emphasized by RoEek (’89)and tributors and are based on examination of development of the ethmoidal area in Pipa sectioned material obtained from natural pop- pipa was described by RoEek and Veseljl(’89). ulations. The editors (Nieuwkoop and Faber, RoEek (’90) likened the ethmoidal endocra- ’56:7) carefully pointed out that intrastage nium of pipids to that of “labyrinthodont- variation was not studied, but that “It has grade” tetrapods, claiming that the two lar- . . . become evident . . . that a general varia- val ethmoidal configurations in anurans (i.e., tion of approximately half a stage to either the pipoid and non-pipoid types) could not side has to be taken into account.” have been derived from one another (contra Variation in osteological development in Sokol, ’75). Xenopus laevis was addressed briefly by An understanding of the structure of the Brown (’go), who examined small samples of mature skeleton (especially the cranium) re- wild-caught and laboratory-reared animals quires knowledge of its development. Fur- between Stages 49 and 60. Brown noted dis- ther, one can expect interspecific osteological crepancies in the timing and sequence of variation among adults to be correlated with ossification of certain elements when he com- variation in developmental patterns. In the pared his results with those of Bernasconi case of pipid frogs, the studies of Xenopus (’51). Brown (’80) reported that ossification laeuis clearly have historical precedence in is delayed in laboratory-reared X. laeuis as the literature as a baseline against which compared with wild-caught specimens and noted specific differences between his ob- ‘Throughout this paper, reference will be made to pipid and served timing and sequence of osteological pipoid frogs versus non-pipid and non-pipoid frogs. There are five Recent genera in the Family Pipidae-viz., Pipa in the New development in wild-caught X. laevis and World, Hymenochirus, Pseudhymenochirus, Xenopus, and Sih- those recorded by Nieuwkoop and Faber (’56). runu in the Old World (Cannatella and Trueb, ’88a,b).The super- family Pipoidea is composed of the Pipidae plus its sister taxon Brown (’80:28-29) correctly observed that Rhinophrynidae (containing only one Recent taxon, the New there is “considerably more variation in time World Rhinophrynus dorsalis).Currently, pipoid frogs are considered to be a highly derived group of primitive frogs that are of ossification than the one half stage stated thought to be most closely related to pelohatoid murans (e.g., in the normal table . . . [and that this] . . . Pelobatidae and Pelodytidae) (Cannatella,’85). SKELETAL DEVELOPMENT IN XENOPUS LAEVIS 3 other pipid data will be compared. However, TABLE 1. Comparison of three common normal tables several problems must be resolved before of development for anurans' such comparisons can be made. Disparities Taylor and Nieuwkoop and among published anatomical descriptions Kollros ('46) Faber ('56) Gosner ('60) must be resolved and intraspecific variation I 46 26 in the timing and sequence of osteogenic I1 47 27 events should be evaluated. - 48 - Herein, skeletal development of Xenopus I11 49 28 - 50 - laevis from Stage 46 (premetamorphic, feed- IV 51 29 ing larva) through Stage 66 (metamorphosed V 52 30 froglet) and for 6 additional months is de- VI 53 31 scribed and compared with results of previ- VII - 32 VIII - 33 ous studies on X. laevis and other anuran M 54 34 taxa. The account is organized into four ma- X 55 35 jor sections-chondrocranial development, XI - 36 metamorphic development of the cranium, XI1 - 37 XI11 56 38 postmetamorphic development of the cra- XIV 57 39 nium, and postcranial development. Within xv - 40 chondrocranial development, a description of XVI 58 - XVII 59 - the chondrocranium at Stage 53 immediately XVIII 60 41 prior to the appearance of ossification pre- XIX - - cedes an account of its development. Meta- xx 61 - morphic development of the cranium is orga- XXI 62 42 XXII 63 43 nized regionally (e.g., dermal investing bones, XXIII 64 44 nasal capsule),whereas remarks on postmeta- MV 65 45 morphic development are brief chronological XXV 66 46 summaries of the major changes that occur. 'An "-"indicates absence of equivalent stage. Adapted from Postcranial development is described region- Just et al. ('81) ally-the axial column, anterior appendicular skeleton, and posterior appendicular skeleton. Major developmental patterns observed ered formalin. Larvae were staged according are summarized following the detailed re- to the normal table of Nieuwkoop and Faber gional and temporal accounts. Chronologies ('56); to facilitate comparison of these data of developmental events and their variation with those of other taxa, developmental stages are presented in Tables 2-6 and Appendices of the three most commonly used normal A-D, respectively. tables of development (Taylor and Kollros, The Discussion includes comments on the '46; Nieuwkoop and Faber, '56; Gosner, '60) events of skeletogenesis in Xenopus laevis are compared in Table 1. Larval snout-vent that are unusual relative to what is known and tail lengths were measured; total length about the process in other anurans, and con- of larvae, as cited herein, is the sum of the sideration of the homologies of some cranial snout-vent and tail lengths, but total length elements of X. luevis. There are reasonably of postmetamorphic individuals is snout- extensive comparisons between the results of vent length. Specimens were stained differen- this study and those of previous studies on X. tially for bone and cartilage as whole mounts, laevis, along with comparisons of the skeletal following the techniques of Dingerkus and development between X. laevis and other an- Uhler ('77) and Wassersug ('761, as modified urans. The paper concludes with a brief com- by Hanken and Wassersug ('81). mentary on the reliability of ossification One hundred and fourteen individuals rep- events as staging criteria. resenting Stages 48-66 + 6 months of Xeno- pus laevis were measured; 102 specimens MATERIALS AND METHODS representing Stages 46-66 + 6 months were Adult Xenopus laevis were obtained from examined for osteological development and Carolina Biological Supply and maintained are deposited in the herpetological collection in the laboratory of James Hanken at the of the Museum of Natural History at The University of Colorado at Boulder. Breeding University of Kansas (KU 217886-987). For was induced by hormone injection and the Stages 46-54 and 66 + 4 months to 66 + 6 eggs obtained were reared at 18°C. Speci- months, only two specimens per stage were mens were preserved in 10% neutral-buff- available for osteological examination; each 4 L. TRUEB AND J. HANKEN of the other stages is represented by at least nium of pipids is the so-called “ethmoid” five individuals. region that lies anterior to the braincase be- Serial cross sections of the heads of three tween the palatoquadrate cartilages and ex- individuals were prepared (KU 219922, Stage tends forward above the lower jaw as the 63; KU 219923, Stage 64; KU 219921, Stage suprarostral plate. The anterior margin of 65). Specimens were fixed and preserved in this plate is slightly convex and supports the 10% formalin. The heads were removed and upper lip of the larvae. In dorsal aspect, the decalcified in a 5% formic acid-formalin solu- rostral portion is broad, thin, and flat; its tion for 3 d, after which they were dehy- lateral portions form distinct wings that are drated in an ascending ethyl alcohol series, &stally attenuate and united with an ante- cleared with Histoclear, and embedded in rior process of the palatoquadrate to form Paraplast Plus. Specimens were transversely the long, distinctive tentacular cartilage of sectioned at 5 and 10 p and stained following pipid larvae (Fig. 1). Posterior to the rostrum the modified Heidenhain’s Azan technique of and between the palatoquadrate cartilages, Baldauf (’58). Drawings were prepared with the aid of a the cartilage is thicker and bears a pair of dissecting microscope equipped with a cam- longitudinal channels that house the olfac- era lucida. tory nerves. The channels are confluent at their origin from the anterior end of the RESULTS brain, but they diverge anterolaterally from Chondrocranial development one another and are separated medially by a thickened area of cartilage termed the verti- The most complete descriptions of the cal septum. At this stage, the olfactory chan- chondrocranium of Xenopus laevis are those nels are unroofed anteriorly; a narrow strip of Kotthaus (’331, Paterson (’391, and Sedra of cartilage, the tectum anterius, forms the and Michael (’57). Kotthaus (’33) described anterior margin of the frontoparietal fonta- chondrocranial development from newly nelle and roofs the most posterior part of the hatched larvae (ca. Stage 40?)up to approximately Stage 53. Paterson’s (’39) description common olfactory channel. The cartilage is of chondrocranial structure was secondary to broadly united to the palatoquadrate carti- the main point of her paper-viz., an account lage on each side by a robust commissura of cranial nerves. Sedra and Michael’s (’57) quadratocranialis anterior (Fig. 1). account is the most complete and accurate, The broad, arcuate mandible projects ante- but the earliest stage that they describe is rior to the dorsal portion of the skull and is Stage 55 when cranial metamorphic changes composed of three elements that are synchon- are well underway. The descriptions in all of drotically united: paired Meckel’s cartilages these studies are based on examination of separated by a single infrarostral (fused infra- sectioned material and illustrations are hand- rostrals fide Sokol, ’75; inferior labial carti- drawn graphical reconstructions from sec- lage of Kotthaus, ’33, Paterson, ’39, Sedra tions, rather than illustrations of whole- and Michael, ’57). The posterior end of Meck- mount specimens. The material examined el’s cartilage is united with the anterior end herein has revealed some subtle anatomical of the palatoquadrate via the larval pars artic- differences in chondrocranial structure be- ularis. tween our specimens and those described in The palatoquadrate is short and consists of earlier accounts. two distinct parts; the anterior portion lies lateral to the “ethmoid” portion of the skull, Larval chondrocranium at Stage 53 (Fig. 1) whereas the posterior portion is associated The last larval stage prior to appearance of with the trabecula anterior to the otic cap- cranial ossification in whole-mount speci- sules. The anterior palatoquadrate articu- mens is Stage 53; hence, this stage is selected lates with the posterior end of Meckel’scarti- to represent the “mature” larval chondrocra- lage via the larval pars articularis. It bears an nium prior to the onset of metamorphic anteromedial process, the processus cornu changes in the skull. The chondrocranium is quadratus medialis, and an anterolateral pro- longer than wide, depressed, and wedge- cess, the processus cornu quadratus lateralis shaped in lateral aspect with the auditory (quadratoethmoidal cartilage of Paterson, region being the highest part of the skull. ’39);the latter unites with the lateral end of One of the most distinctive and controver- the rostral cartilage to form the tentacular sial (see Discussion) parts of the chondrocra- cartilage (Fig. 1). The commissura quadrato- Fig. 1. Xenopus laeuzs. Dorsal (left) and ventral pars articularis of the palatoquadrate; M c, Meckel’s (right) views of the chondrocranium of a Stage-53 larva. cartilage; musc p aud, muscular process of auditory Branchial baskets have been removed in both views and capsule; musc p pal, muscular process of palatoquadrate; only the right half of the ceratohyal is shown in the notoch, notochord; olf chan, olfactory channel; p cor quad ventral view. asc p, ascending process of the palatoquad- med, processus cornu quadratus medialis; quadeth c, rate; aud cap, auditory capsule; basal pl, basal plate;I quadratoethmoidal c; suboc bar, subocular bars of the basihyobr, basihyobranchiale; comm quadcran ant, com- palatoquadrate; suboc fen, subocular fenestra; supraros missura quadratocranialis anterior; epiotic em, epiotic I pl, suprarostral plate; t tect mar, taenia tecti marginalis; eminence of auditory capsule; fpar fon, frontoparietal tectant, tectum anterius; tect post, tectum posterius; ten fontanelle; hypoch comm, hypochordal commissure; infra- c, tentacular cartilage; thymus f, thymus foramen; venlat ros c, infrarostral cartilage; lar cr par, larval crista pa- p. ventrolateral process of the palatoquadrate. Scale = 2 rotica; lar otic p, larval otic process; lar pars art, larval mm. 6 L. TRUEB AND J. HANKEN cranialis anterior forms a broad bridge be- bears three foramina-two acoustic foram- tween the anterior palatoquadrate and the ina ventrally and an endolymphatic foramen medial part of the chondrocranium; posterior dorsal to and between the acoustic foramina. to the commissura, the palatoquadrate forms The jugular foramina exit the skull posteri- a flat subocular bar that encloses the anterior orly at the level of the occipital arch. The half of the subocular fenestra. The posterior pilae preoptica, metoptica, and antotica are part of the palatoquadrate consists of a ro- united dorsally by the orbital cartilage, which bust ventrolateral process that is expanded forms the lateral margin of the frontoparie- distally (basal process of Kotthaus, '33);prox- tal fontanelle anterior to the otic capsule. imally, the ventrolateral process is united to The posterolateral margin of the fontanelle is the braincase via the ascending process and formed by a narrow strip of cartilage medial to the otic capsule via the larval otic process. to the epiotic eminence-the taenia tecti mar- Previous workers (Paterson, '39; Sedra and ginalis; the posterior margin of the fonta- Michael, '57) restricted the term ventrolat- nelle is composed of a delicate, transverse eral process to the expanded ventral portion strip of cartilage between the otic capsules- of the process, and referred to the straplike, the tectum posterius (tectum synoticum of proximal cartilage as the ascending process. Kotthaus, '33). It is clear from the specimens examined that The auditory capsules are well developed the ascending process terminates at the level with prominent epiotic eminences (Fig. 1). of the otic process and subocular bar; thus, Anteromedially, a straplike bridge of carti- the ventrolateral process is the band of cartilage-the larval otic process-joins the otic lage with an expanded base that lies distolat- capsule to the ventrolateral process of the eral to the ascending process. Anterior to the palatoquadrate ventral and posterior to the ascending process, there is a flat anterior ascending process of the palatoquadrate. The projection that represents the posterior half larval crista parotica (larval otic process of of the subocular bar. The two parts of the Sedra and Michael, '57) is a platelike expan- subocular bar have a tenuous synchondrotic sion of cartilage at the anterolateral corner of connection; thus, there is not a single, robust the otic capsule. A small flange of cartilage borders the lateral and posterolateral margin subocular bar as depicted in the illustrations of the capsule and terminates in a slightly of Kotthaus ('331, Paterson ('391, and Sedra expanded process posterolaterally; together, and Michael ('57). these constitute the muscular process of the The braincase is broad and shallow, espe- otic capsule. The immense fenestra ovalis lies cially anteriorly. The floor is formed by the in the ventrolateral wall of the otic capsule. basal plate, which at the level of the subocular fenestra is pierced by the pair of craniopal- atine foramina anteriorly, and a pair of ca- Development of the chondrocranium, Stages rotid foramina posteriorly. Posteromedially, 46-52 between the otic capsules, the notochord per- Stage 46. In this, the earliest stage avail- sists in the floor of the braincase; the poste- able for examination, the parachordals flank rior border of the braincase is formed by a the notochord and unite anterior to it to form narrow cartilage bridge between the halves a narrow basal plate. The parachordals di- of the chondrocranium, the hypochordal com- verge anterolaterally from this point to form missure (Fig. 1).The lateral wall of the brain- the posterolateral margins of the broad basi- case is pierced by four foramina. The optic cranial fenestra (Fig. 2). (Parachordal termi- foramen separates the pila preoptica from nology follows Paterson "391.1 The lateral the pila metoptica posteriorly, which, in turn, margins are formed by the cranial trabecu- is delimited posteriorly by the oculomotor lae, which extend longitudinally forward from foramen. Slightly posterior and dorsal to the the parachordals to the large, flat ethmoidal oculomotor foramen is the small trochlear or trabecular plate. Lateral to the union of foramen. Both foramina exit the wall of the the anterior parachordal and the trabecula, braincase at the posterior level of the subocu- there is a diffuse condensation of cartilage lar fenestra just anterior to the ascending that represents the proximal part of the ven- process. The pila antotica forms the lateral trolateral process of the posterior palatoquad- wall of the braincase between the oculomotor rate. The anterior, posterior, and posterome- foramen and the large prootic foramen poste- dial parts of the auditory capsule are present rior to the ascending process. The wall sepa- lateral to the posterior parachordals (labelled rating the braincase and auditory capsule parachordal in Fig. 2). SKELETAL DEVELOPMENT IN XENOPUS LAEVIS 7 The anterior palatoquadrate is well devel- riorly, the processus cornu quadratus latera- oped and united with the ethmoid plate via a lis extends forward from the palatoquadrate broad commissura quadratocranialis ante- to the tentacular cartilage; a connection be- rior. Posteriorly, the anterior palatoquadrate tween the tentacular cartilage and the ala of bears a subocular bar; anteriorly, the muscu- the suprarostral cartilage is absent. There is lar process, processus cornu quadratus medi- chondrification around the entire auditory alis, and larval articular process are evident. capsule. The processus cornu quadratus lateralis is Stage 48. The basicranial fenestra is represented only by a diffuse condensation of small. The ventrolateral process of the poste- cartilage anterior to the palatoquadrate. rior palatoquadrate is lengthened, but not The suprarostral plate is well developed, expanded distally; proximally, the posterior but lacks a cartilaginous connection with the palatoquadrate bears a suborbital bar that rudimentary tentacular cartilage. The lower nearly meets the suborbital bar of the ante- jaw is represented by paired Meckel’s carti- rior palatoquadrate. The ascending process lages separated by the single infrarostral. of the palatoquadrate appears as a lateral Stage 47. The floor of the braincase is cartilaginous projection of the wall of the expanded (and the size of the basicranial braincase dorsal to the ventrolateral process. fenestra decreased) by proliferation of carti- The walls of the braincase are thicker and lage between the anterior parachordals. The longer, curving anteromedially around the trabeculae are thickened to form a trough in front end of the brain. Anteriorly, the tentac- which the anterior brain is lodged. The ven- ular cartilage is united with both the cornu trolateral process of the posterior palatoquad- quadratus medialis and the lateral flange of rate is united with the medial edge of the the suprarostral cartilage. The epiotic emi- trabecula. A proliferation of cartilage along nences are scarcely visible in the otic capsule. the dorsum of the trabecula represents the The hypochordal commissure constitutes a incipient lateral walls of the braincase. Ante- band of cartilage ventral to the notochord

Fig. 2. Rana temporaria (left; 7.5-mm larva) and basicranial fenestra; c, cartilage; comm quadcran ant, Xenopus lueuis (right;Stage 46), dorsal views of chondro- commissura quadratocranialis anterior; cor trabec, cornu crania of young larvae to illustrate differences between trabecula; infraros c, fused infrarostral cartilages; M c, non-pipoid and pipoid taxa. Arrows indicate palatoquad- Meckel’s cartilage; pl internas, planum internasale; suboc rate cartilages. Note in particular, differences in palato- bar, subocular bar of palatoquadrate, supraros pl, su- quadrate and rostra1 cartilages. Illustration of Ram prarostral plate; ten c, tentacular cartilage; venlat p, adapted from Gaupp (’06); lower jaw not shown. ant ventrolateral process of palatoquadrate. Scale = 1 mm parachordal c ,anterior parachordal cartilage; basicr fen, (Xenopus). 8 L. TRUEB AND J. HANKEN TABLE 2. Comparison of schedules of cranial ossification and mineralization in Xenopus laevis' Sedra and Staee This study Bernasconi ('51) Michael ('57) Brown ('80)

54 1. Frontoparietal 1. Frontoparietal -

55 2. Parasphenoid 2. Parasphenoid 1. Frontoparietal 1. Frontoparietal Exoccipital (55-56) 3. Exoccipital Parasphenoid Parasphenoid Prootic 65-57) Angulosplenial Exoccipital (medial) 4. Maxilla Prootic Teeth Angulosplenial (medial) Premaxilla

56 3. Angulosplenial 5. Prootic - (medial) 57 4. Maxilla 2. Maxilla Premaxilla Teeth

58 5. Premaxilla (58-59) 6. Nasal 2. Prootic Nasal (5&60) Exoccipital Maxilla Nasal 59 7. Dentary 3. Pars interna 3. Nasal plectri Operculum 60 6. Septomaxilla 8. Angulosplenial 4. Septomaxilla (lateral) Teeth Septomaxilla Premaxilla Dentary (60-62) Squamosal Teeth Angulosplenial Pterygoid Angulosplenial (lateral) (60-61) Dentary 61 7. Pterygoid (61-62) 9. Columella 5. Parsmedia plectri Tympanic annulus Pars externa (61-62) plectri Pars externa Tympanic an. plectri (6142) nulus 62 8. Pars media plectri 10. Vomer - (61-63) Squamosal(62-63) 63 9. Vomer (63-64) - 6. Pterygoid

64 10. Sphenethmoid - 7. Squamosal (64-66) 65 11. Parsarticularis

66 - - 8. Spenethmoid 66 + 1 month 12. Operculum 11. Sphenethmoid - Pars articularis 66 + 4 months - 12. Alarycarti- - lage

66 + 10 months - 13. Planmat- - orbitale

'Endochondral elements are in boldface. Developmental stages are those of Nieuwkoop and Faber ('56) and as applied to Bernasconi's ('51) data are only estimations. Groups of elements associated with a single number appeared simultaneously in the specimens examined. Ranges of stages in which elements were observed to appear in this study are indicated in parentheses, SKELETAL DEVELOPMENT iN XENOPUS LAEVIS 9 and unites the otic capsules. The occiput of ventrolateral process of the palatoquadrate the chondrocranium has begun to form and to produce the expanded base. the jugular foramina are evident. Stages 51-52. The tectum anterius, a narrow transverse band of cartilage roofing the Stage 49. The basicranial fenestra is olfactory channel just anterior to the brain, closed, and the paired craniopalatal and ca- forms the anterior margin of the frontoparie- rotid foramina are visible in the basal plate. tal fontanelle. The fontanelle is open posteri- Differential thickening of the intertrabecular orly, although a small spur of cartilage associ- plate begins to form the channels for the ated with the posteromedd margin of each olfactory nerves in the ethmoid area. The otic capsule signals the beginning of the tec- anterior and posterior palatoquadrates are tum posterius. The larval otic process, which united by fused subocular bars, which en- unites the otic capsule with the ascending close the subocular fenestra. The ascending process of the palatoquadrate, is complete. process of the palatoquadrate has fused with The base of the ventrolateral process of the the ventrolateral process, and the larval otic palatoquadrate is expanded posteriorly. Un- process appears as a posterior projection of til Stage 51, the anterior margin of the lower cartilage from the ventrolateral process near jaw lay approximately at the level of the its union with the ascending process. Elabo- anterior margin of the suprarostral cartilage; ration of the auditory capsule includes the however, the larger mandible now extends appearance of the larval crista parotica anter- beyond the rostral cartilages. olaterally, along with the lateral muscular process. Metamorphic development of the cranium, Stages 54-66 Stage 50. The primary change involves The sequence of cranial ossification is pre- elaboration of the anterodistal end of the sented in Table 2.

I- exoc exoc + proJ d Fig. 3. Xenopus laeuis. Dorsal (left) and ventral are depicted. angspl, angulosplenial; exoc, exoccipitd; (right) views of skull of metamorphosing larva (Stage exoc + pro, fused exoccipital and prootic; fpar, frontopar- 57; KU 217919). Cartilaginous chondrocranial elements etal; max, maxilla; pro, prootic; prsph, parasphenoid. shown in stipple pattern are identified in Fig. 1. Only Scale = 2 mm. right ceratohyale and right half of basihyobranchial keel 10 L. TRUEB AND J. HANKEN

Lpars ext plec pars med plec 1 Lfen ovalis

Fig. 4. Xenopus Zaeuis. Dorsal (left) and ventral parietal; inf pnas c, inferior prenasal cartilage; max, (right) views of skull of metamorphosing larva (Stage maxilla; pars artic, pars articularis of the palatoquadrate; 63; KU 217948). Position of dental ridge is shown by pars ext plex, pars externa plectri; pars med plec, pars broken line along venter of maxillae and premaxillae; media plectri; palatoq, palatoquadrate; pmax, premaxilla; individual teeth are not depicted. Cross-hatched pattern prsph, parasphenoid; pter, pterygoid; solum nas, solum indicates venter of frontoparietal visible owing to absence nasi; spmax, septomaxilla; sq, squamosal; tym ann, tym- of both cartilage and bone in the orbital region of the panic annulus. For identities of other bony elements braincase at this stage of development. Cartilage is shown refer to Fig. 9. Scale = 2 mm. in stipple pattern. fen ovalis, fenestra ovalis; fpar, fronto-

Dermal investing bones prootics in Stage 55. The parasphenoid forms Frontoparietal. This azygous bone is the from a single elongate, narrow center of ossi- first cranial element to appear. It develops in fication; it extends along the midventral floor Stages 54 and 55 from paired centers of ossi- of the braincase from between the otic cap- fication, each of which is long and slender sules (but does not reach the occiput) to the and located above the orbital cartilage along anterior edge of the developing frontoparie- the lateral margin of the frontoparietal fonta- tals. In the next four stages, the bone length- nelle. The bones grow in length and breadth, ens and develops its characteristic spear shape and by Stage 57 they are narrowly separated (Fig. 3), in which the posterior end (between medially and extend forward to the level of the otic capsules) is narrow and parallel-sided the tectum anterius (Fig. 3). They begin to and the midsection (just anterior to the otic fuse medially in Stage 58, and by Stage 60 capsules) is relatively broad with laterally the union is complete save for the small pari- convex margins. The sides of the parasphe- etal foramen. Anterior growth over the tec- noid converge to an acuminate anterior end tum anterius begins in Stage 60; by Stage 62 that lies beneath the trabecular plate ante- the frontoparietal lies posteriorly adjacent to rior to the braincase. Anterior growth is pro- the nasals in most specimens, and in Stage 63 nounced in Stages 60-62, and by Stage 63 it overlaps the posteromedial margins of these the tip of the parasphenoid dorsally overlaps bones (Fig. 4). Posterior growth is pro- the partes palatinae of the premaxillae (Fig. nounced from Stage 60, and by Stage 63 the 4). bone roofs the entire fontanelle and has Nasals. Narrowly separated, paired cen- achieved its postmetamorphic configuration. ters of ossification that give rise to the nasals Parasphenoid. This ventral bone appears appear in Stages 58-60. Initially, each nasal contemporaneously with the exoccipitals and forms from an arcuate ossification posterior SKELETAL DEVELOPMENT IN XENOPUS MVIS 11 infros c alsept c-, nasl/-

nasJ Lcomm quadcran ant tect nas sept prnax obl c7\ nasl [,-a1 c ant

pl an

patatoqJ .Lcomm quadcran ant Fig. 5. Xenopus laeuis. Development of rostral carti- cartilage; angspl (lat), lateral angulosplenial;angspl (med), lages and anterior part of the bony skull during Stage 59 medial angulosplenial; ant max proc, anterior maxillary (top; KU 217927) and Stage 60 (bottom; KU 217936). process; comm quadcran ant, commissura quadratocra- In Stage 59, note the origin of the alary cartilages from nialis anterior; fpar, frontoparietal; infros c, infrarostral the posteromedial margin of the suprarostral plate; the cartilage; M c, Meckel's cartilage; max, maxilla; musc premaxillae and maxillae lie on the dorsal surface of the pro, muscular process of palatoquadrate; nas, nasal; obl plate. Cross-hatched area between the alary cartilage and c, oblique cartilage; palatoq, palatoquadrate; pl antorb, the nasal represents the solum nasi. By Stage 60, the planum antorbitale; pmax, premaxilla; quadeth c, quadra- suprarostral plate is absent and the septomaxilla, tectum toethmoidalis cartilage; sept nas, septum nasi; spmax, nasi, and oblique cartilage have appeared. Posterior mi- septomaxilla; supraros pl, suprarostral plate; tect nas, gration of the palatoquadrate is well advanced at this tectum nasi; ten c, tentacular cartilage. Scale = 2 mm. stage. Cartilage is shown in stipple pattern. a1 c, alary to the external naris (Figs. 4,5).Growth seems the anteromedial tip of the nasal grows rap- to occur at an approximately equal rate at the idly forward between the nasal capsules to anterolateral and anteromedial ends of this produce the rostral process characteristic of crescentic bone so that the symmetry of the the adult. Fusion of the two nasals begins developing nasal is maintained until the com- with the anterior parts of the rostral pro- pletion of metamorphosis (Stage 65), when cesses and commences as early as Stage 66. 12 L. TRUEB AND J. HANKEN Vomer. The single adult vomer originates eroding, and the septomaxilla has appeared late in development (Stages 63-64) from a within the nasal capsule, Disappearance of pair of small ossification centers that flank the commissura quadratocranialis anterior the parasphenoid at the level of the planum in Stage 61 identifies the posterolateral edge antorbitale. Growth occurs medially and by of the trabecular plate (or the anterior mar- Stage 66, the two centers are fused to one gin of the subocular fenestra) as the planum another beneath the parasphenoid (Fig. 6). antorbitale (lamina orbitonasalis of some authors)-that is, the posterior wall of the na- Nasal capsule and septomaxilla sal capsule. All traces of the tentacular cartilage disappear by Stage 61, but a remnant of The development of the nasal capsule in- the cornu quadratus lateralis persists into volves complex changes in the trabecular and Stage 62. suprarostral plates. By Stage 56, approxi- By Stage 63, the posteroventral part of the mately the posterior two-thirds to three- septum nasi is fused to the medial planum fourths of the olfactory channels are roofed antorbitale. The floor of the nasal capsule is in cartilage; in effect, the tectum anterius smaller and consists of a band of cartilage extends anteriorly over the intertrabecular that extends from the medial part of the region to form a roof above the olfactory planum forward along the medial margin of tracts and the vertical septum of cartilage the choana and that is broadly separated that separates them (Fig. 3). Anterior growth from the septum nasi medially. The lateral of these elements continues into Stage 57, solum nasi consists of a strip of cartilage that accompanied by remodeling of the trabecular lies along the anterior margin of the choana. plate at the level of the external nares (Fig. It is fused with the lateral end of the oblique 5). This part of the plate, which lies at the cartilage to form the planum terminale. The lateral base of the suprarostral plate anterior anterior solum nasi is represented by a carti- to the olfactory foramina and anterior to the laginous process that grows forward toward level of the commissura quadratocranialis the premaxilla during Stage 64. By Stage 66, anterior, is depressed ventrally to form the the process extends dorsad behind the alary medial portion of the solum nasi. Concur- process of the premaxiIla to unite with the rently, the medial cartilage, which repre- alary cartilage. This looped connection be- sents the developing septum nasi, extends tween the alary cartilage and solum nasi has anteriorly between the external nares. been termed the superior prenasal cartilage In Stage 58, the nasals appear along the by Paterson ('39) and Sedra and Michael anterior margin of the tectum anterius; an ('57). It may represent both the superior and anterolateral proliferation of cartilage from inferior prenasal cartilages of non-pipid an- the septum nasi represents the beginning of urans. the tectum nasi posterolateral to the external In Stages 64 and 65, there is a proliferation naris. The alary cartilage arises from the of the alary cartilage posterolaterally and the posterior margin of the suprarostral carti- septum nasi anteriorly. By Stage 66, the ante- lage anterior to the external naris. The ob- rior end of the septum nasi is a robust, ex- lique cartilage is an independent chondrifica- panded rostral cartilage that lies between the tion in the dorsolateral area of the developing alary cartilages and the alary processes of the nasal capsule; it fuses with the tectum nasi premaxillae, and the alary cartilage forms medially in Stage 59. Anteroventral growth the anterior and lateral walls of the nasal of the portion of the trabecular plate medi- capsule. The dorsolateral roof of the nasal ally adjacent to the commissura quadratocra- capsule is composed of the expanded sep- nialis anterior produces the posterolateral tomaxilla, and the dorsomedial roof by the portion of the solum nasi. nasal, oblique cartilage, and small tectum Major changes occur in the rostral portion nasi. In contrast to the expansion of its dor- of the chondrocranium in Stage 60. Although sal roofing components, the cartilaginous the tentacular cartilage, along with the pro- floor of the nasal capsule becomes progres- cessus cornu quadratus lateralis and su- sively more restricted. prarostral process, is still present, most of the suprarostral plate has disappeared. At Posterior braincase and ear the same time the seDtum nasi extends ante- The exocciDital and DrOOtiC both form early riorly over the eroding suprarostral plate. in Stages 55-57 (Tabl;! 2). The appearance if Lateral to the developing nasal capsule, the the prootic may follow that of the exoccipital; commissura quadratocranialis anterior is the latter is present in all specimens by Stage SKELETAL DEVELOPMENT IN XENOPUS OZEVIS 13

Fig. 6. Xenopus lamis. Development of the orbital which also forms the anterior border of the prootic region of the braincase and palate between Stages 64 and foramen. Anterolateral margins of parasphenoid and 66 + 1 month (ventral views). A: Stage 64 (KU 2179551, posterior margin of united sphenethmoids beneath para- prior to appearance of vomer and sphenethmoid. B. Stage sphenoid are indicated by broken lines because these 65 (KU 217957). Vomer and sphenethmoid are present, bones are fused at this stage. Position of dentition on and parasphenoid has broadened between optic foramen maxilla and premaxilla is shown by light contour line; and auditory capsules. C: Stage 66 (KU 217964). Note individual teeth are not drawn. Cartilage shown in expansion of sphenethmoid and base of parasphenoid, stipple and ventral surface of frontoparietal by cross- and elaboration of lateral processes on parasphenoid in hatched pattern. aud cap, auditory capsule; fpar, fronto- region of prootic foramen. Broken lines inside parasphe- parietal; max, maxilla; optic f, optic foramen; pmax, noid indicate margins of auditory capsules visible through premaxilla, prsph, parasphenoid; pter, pterygoid; sph, the bone. D Stage 66 + 1 month (KU217971). The optic sphenethmoid. Scale = 2 mm. foramen is enclosed completely by the sphenethmoid, 14 L. TRUEB AND J. HANKEN 56, whereas the prootic is not present in all pears. Thus, in the sections and the whole- specimens until Stage 57. The exoccipital mounts examined, the preorbital portion of begins to ossify in the occipital arch in close the neurocranium is associated with the post- association with the prootic, which com- orbital part only by the frontoparietal dor- mences ossification in the medial wall of the sally and the parasphenoid ventrally, with auditory capsule (Fig. 3). Ossification from the side walls of the braincase lacking any the medial hemisphere of the otic capsule cartilage (Fig. 6A). In Stages 64-66, ossifica- progresses to the lateral hemisphere, and by tion appears in the ventrolateral area of the Stage 61 the capsule is well ossified with the braincase adjacent to the parasphenoid in exoccipital nearly completely fused with the front of the optic foramen in the ventrolat- prootic. The subspherical auditory capsule eral area of the braincase (Fig. 6B). From bears a marginal cartilage anteriorly and lat- this center, the thin, sheetlike sphenethmoid erally (Fig. 4).This cartilage gradually dimin- grows anteriorly and dorsally to form the ishes in size and becomes restricted to a lateral wall of the neurocranium between the narrow edge along the anterolateral margin optic foramen and the planum antorbitale. In of the auditory capsule by Stage 66, at which Stage 66, the ventral margins of the spheneth- point it represents the narrow crista parotica moids articulate with the lateral margins of of the adult. the parasphenoid and the dorsal margins lie External and middle ear elements appear adjacent to the frontoparietal; the bones do abruptly in Stage 62 (Table 2). The tympanic not enclose the orbitonasal foramina (Fig. annulus forms as a slender crescent of carti- 6C). In one specimen (KU 217966), the an- lage that is vertically oriented and lies anteri- teroventral corners of each sphenethmoid orly adjacent to the small, round pars ex- have proliferated medially to form a thin terna plectri. The latter is associated with ventral bridge uniting the two sides of the the distal end of the pars media plectri and, braincase above the parasphenoid just poste- at this stage, is ossified only distally. In suc- rior to the planum antorbitale. In the same ceeding stages, the pars externa plectri ex- specimen, there is posterodorsal prolifera- pands while the tympanic annulus grows tion of the bone to form the dorsal margin of around it and the pars media plectri ossifies the optic foramen; there is no evidence of in a distal-to-proximal direction. By Stage 66, cartilage or bone between the optic and the tympanic annulus forms a nearly com- prootic foramina. The posterior margin of plete ring around the pars interna plectri; it the prootic foramen is formed by the prootic remains incomplete posteriorly, at the union at the anteromedial corner of the otic cap- of the pars externa plectri and pars media sule. plectri. The pars media plectri at this stage is completely ossified and slightly expanded ba- Upper jaw sally in the area of the fenestra ovalis. The pars media plectri abuts a delicate disc of Maxilla. The first ossification of the adult cartilage, the pars interna plectri, that nearly upper jaw appears in Stage 57 as a slender spindle of bone located along the posterior fills the fenestra ovalis. Sedra and Michael margin of the suprarostral cartilage anterior (’57:46) identified the operculum as a “feebly to the naris in the region where the alary developed outgrowth from the posterior bor- cartilage will develop (Fig. 3). The bone der of the fenestra ovalis” in Stage 65. We lengthens laterally in Stages 58 and 59 (Fig. find no indication of an operculum in either 5). By Stage 60, when the suprarostral plate Stage 65 or 66 specimens in which the large has been replaced by the nasal cartilages, the fenestra ovalis seems to be bordered com- maxilla is displaced ventrally and bears dis- pletely in bone; however, the structure de- tinct partes facialis and palatina, and teeth. scribed by Sedra and Michael (’571, de Villi- Subsequently the bone grows posteriorly; at ers (’321, and Paterson (’39) is obviously Stage 60, the maxilla terminates at the ante- present in 1-2-month-old postmetamorphic juveniles and in adult specimens. rior end of the orbit, and by Stage 66 it subtends the eye. Premaxilla. The premaxilla forms in Orbital region of the braincase Stages 58 and 59. The paired bones are lo- Coincident with the restructuring of the cated at the tip of the snout and, initially, rostral chondrocranium between Stages 59 they are more closely associated with the and 60, the cartilage forming the lateral walls maxillae laterally than with one another me- of the braincase in the orbital region disap- dially (Fig. 5). The first part of the bone to SKELETAL DEVELOPMENT IN XENOPUS mvrs 15 appear is the alary process, which is under- A lain by a tiny, transverse bar of bone representing the pars dentalis. By Stage 60, teeth are associated with the premaxillae and the ma pars palatina is present. During Stages 61- 66, the premaxillae grow until they are narrowly separated from one another medially and from the maxillae laterally, and the pars palatina is as deep as the maxilla. The alary processes, which initially are uniform in width, slender, and only slightly laterally di- vergent from one another in frontal aspect, 6 become expanded dorsally; growth seems to be more rapid at the dorsolateral and dorsomedial corners of each alary process so that by Stage 66, the processes are dorsally bifur- cate. Mandible Angulosplenial. This dermal bone (angulare or goniale of some authors) invests Meck- el’s cartilage anteromedially and posteriorly; C unlike its counterpart in other anurans, it arises from two centers of ossification in Xe- nopus laeuis. The primary center appears in Stages 56 and 57 as a bony splint along the medial surface of Meckel’s cartilage (Fig. 3). By Stage 59, this ossification is approximately centered on the length of Meckel’s cartilage and covers about three-fourths its length; the bone is acuminate anteriorly, and blunt and wide posteriorly. In Stages 59 and 60, a secondary center of ossification (prearticular of Bernasconi, ’51) appears lateral to Fig. 7. Xenopus laeuis. Development of the mandible the posterior third of MeckeI’s cartilage (Fig. (ventral aspect) during metamorphosis and early postmetamorphosis. A Stage 63 (KU 217948). The angulo- 5). Proliferation of this center along with the splenial forms from lateral and medial centers of ossifica- posterior part of the medial center results in tion that are not united at this stage of development. The their fusion dorsally and ventrally (Fig. 7) so infrarostral is synchondroticallyunited to Meckel’s carti- that by Stage 66, the posterior third of Meck- lage (mandibular cartilage). B: Stage 64 (KU 217953). The two centers of ossification of the angulosplenial are el’s cartilage is encased in the cylindrical fused at this stage and the coronoid flange has begun to angulosplenial with only a knob of cartilage form. The dentary has increased in length anteriorly and protruding posteriorly. The coronoid process posteriorly, and the mandibular cartilage has expanded first appears in Stage 63 as a dorsomedial near the anterior tip of the angulosplenial. C Stage 66 + 1 month (KU 217969). Note the erosion of cartilage at elaboration of bone along the primary center the mandibular symphysis, the increased size of the of ossification adjacent to the developing coronoid flange, and recurvature of the posterior part of pterygoid. By Stage 66, the angulosplenial the mandible. The angulosplenial extends anteriorly, dor- invests nearly the entire medial surface of sal to a narrow shelf formed by the mandibular cartilage. Cartilage is shown in stipple pattern. angspl (lat), lateral Meckel’s cartilage. Its anterior tip remains angulosplenial; angspl (med), medial angulosplenial; cor acuminate and is underlain by an expansion flg, coronoid flange of the angulosplenial; den, dentary; of the anterior end of Meckel’s cartilage that mand c, mandibular cartilage. Scale = 1mm. appears in Stage 62. Dentary. The appearance of the dentary in Stages 6042 is associated with changes in rostral becomes straight and fuses dorsally the infrarostral cartilage. Prior to Stage 60, with Meckel’s cartilages; a partial separation this cartilage is distinct from Meckel’s carti- between these cartilages is visible in ventral lages and has a characteristic inverted chev- aspect through Stage 66. The dentary ap- ron shape (Figs. 1,3).At Stage 60, the infra- pears as a slender splint of bone on the outer 16 L. TRUEB AND J. HANKEN surface of the mandible at the union of Meck- dial corner of the otic capsule; this is the el's cartilage and the infrarostral. In subse- postpalatine commissure of Sedra and quent stages (Fig. 71, the dentaries retain a Michael ('57) and pseudobasal process of distinct medial separation and grow posteri- Paterson ('39). orly along the lateral aspect of the mandible. Pterygoid. This massive ventral compo- By Stage 66, the bone covers approximately nent of the suspensorium first appears in the anterior two-thirds of the external sur- Stages 61 and 62 as a diffuse center of ossifi- face of Meckel's cartilage, leaving only a small cation on the ventromedial surface of the area of the cartilage exposed laterally be- palatoquadrate (Figs. 4,6). It rapidly prolifer- tween the posterior end of the dentary and ates anteriorly and medially during Stages 64 the massive angulosplenial posteriorly. and 65. Anterior ossification lies along the Suspensorium ventromedial surface of the pterygoid process and represents the anterior ramus of the The massive changes in the suspensorium pterygoid. Medial ossification proceeds in a associated with metamorphosis commence in dorsomedial direction toward the ventral sur- Stage 60 with the initial erosion of the poste- face of the otic capsule. By Stage 66, the rior parts of the palatoquadrate (ventrolater- anterior ramus of the pterygoid extends along al process and its subocular bar), along with the pterygoid process adjacent to the maxilla, its connections to the neurocranium-the and the medial ramus is an extensive plate ascending and larval otic processes. As the underlying the lateral otic capsule and Eusta- posterior palatoquadrate diminishes and com- chian canal. pletely disappears by Stage 63, the anterior part undergoes extensive modification and Squamosal. This element appears as early migrates posteriorly beneath the eye and then as Stage 62, but is not present in all speci- posterodorsally to become associated with mens examined until Stage 63, when the the anterolateral corner of the otic capsule. ventral ramus appears as a sliver of bone This transformation involves the disappear- along the anterolateral edge of the palato- ance of the muscular process and the erosion quadrate medial to the developing pars ex- of the commissura quadratocranialis ante- terna plectri and tympanic annulus (Fig. 4). rior and elaboration of its remnants into a During Stage 64, the squamosal grows dor- robust suborbital bar (Fig. 4). The anterior sally and assumes an arcuate (curved anteri- portion of this bar is united to the planum orly) form in the lateral plane. Small zygo- antorbitale and laterally adjacent to the max- matic and otic rami are present in Stage 65 illa, and is termed the posterior maxillary and the base of the ventral arm is expanded. process. The continuation of this cartilage In Stage 66, the squamosal is still small. The diverges posteromedially from the maxilla, slender zygomatic process is directed antero- extends to the palatoquadrate cartilage, and laterally from the ventral shaft of the squamo- comes to be underlain by the pterygoid bone; sal and lies medially adjacent to the dorsal this posterior portion of the bar is identifi- edge of the tympanic annulus. The otic ra- able as the pterygoid process of the palato- mus is posteromedially oriented toward the quadrate cartilage in Stage 63 (Fig. 4). In zygomatic spur of the pterygoid process and Stage 66, a spur of cartilage develops from lies between the posterodorsal edge of the the dorsolateral aspect of the pterygoid pro- tympanum and the crista parotica. The ex- cess. The process is directed posterodorsally panded base of the ventral arm of the squamo- and later in development serves as a point of sal is configured into a broad, lateral flange articulation for the zygomatic ramus of the that embraces the posteroventral rim of the squamosal; hence, it is termed herein the tympanic annulus. zygomatic spur of the pterygoid process. Ow- Pars articuEaris of the palatoquadrate. ing to its small size, we did not observe the Ossification first invades the ventral, articu- otic process in the premetamorphic whole- lar end of the palatoquadrate cartilage in mount specimens; according to Sedra and Stage 66. Michael ('571, the adult otic process is pro- duced from the dorsal end of the palatoquad- Postmetamorphic development of the rate cartilage in Stage 64 and fuses with the cranium, Stages 66 -+ 1 month to 6 months crista parotica of the auditory capsule in Stage 65. Concomitantly, the medial region of the Stage 66 + 1month (Fig. 8) palatoquadrate grows toward a ventral carti- Within the first month of postmetamor- lage that extends ventrolaterally from be- phic development there are marked changes neath the prootic foramen at the anterome- in several cranial elements. The rostral pro- SKELETAL DEVELOPMENT IN XENOPUS LAEVIS 17

tym ann 7 rpars ext plec

Fig. 8. Xenopus Zuevis. Dorsal (left) and ventral of the prootic bones beneath the frontoparietal (dorsal (right) views of the skull of an early postmetamorphic view) and parasphenoid (ventral view) are indicated by individual, Stage 66 + 1 month (KU 217969). Note the dashed lines. Elements are identified in Fig. 9. Line along incomplete fusion of the nasals and the size of tympanic maxillae and premaxillae indicates position of teeth. annulus and pars externa plectri relative to those of adult Cartilage is indicated by stipple pattern. fpar, frontopari- (Fig. 9). The parasphenoid is fused with the spheneth- etal; nas, nasal; pars ext plec, pars externa plectri; pro, moid, the posterior extent of which is indicated by the prootic; prsph + sph, fused parasphenoid and spheneth- dashed transverse line in the ventral view. The margins moid tym ann, tympanic annulus. Scale = 2 mm. cesses of the nasals fuse with one another parasphenoid develops two pairs of acumi- medially. The frontoparietal expands such nate, lateral flanges-one pair on each side of that it widely overlaps the posteromedial sur- the bone. The anterior pair lies between the faces of the nasals anteriorly and covers the levels of the optic and prootic foramina, anterior half of the tectum posterius. Antero- whereas the posterior pair lies beneath the laterally, the frontoparietal develops a small, prootic foramina; each of these posterior flangelike process on each side in the region flanges is directed posterolaterally toward the of the planum antorbitale. In cross section, developing prootic. the roof of the braincase is distinctly vaulted, Elsewhere in the skull, the prootic ossifies rather than shallowly domed. In the lateral along the dorsolateral margin of the auditory braincase, the sphenethmoid grows posteri- capsule in the cartilage of the crista parotica. orly and posteroventrally to enclose com- The otic ramus of the squamosal develops a pletely the optic foramen and form a bony narrow otic plate that is associated with the anterior margin to the prootic foramen. The cartilaginous lateral margin of the crista par- sphenethmoids unite with one another ven- otica. The ventral ramus of the bone bears a tromedially and fuse to the parasphenoid be- broad lateral flange that is associated with low; thus, the orbital region of the braincase the funnel-shaped tympanic annulus later- now is completely enclosed by the spheneth- ally, and the zygomatic ramus now ap- moid laterally and ventrally, and the fronto- proaches the maxilla where it articulates with parietal dorsally. the zygomatic spur of the pterygoid process The skull in the area of the prootic fora- of the palatoquadrate. The tympanic annu- men is undergoing modification. At the an- lus and pars externa plectri are greatly en- teromelal corner of the otic capsule, the larged relative to the size of the skull. A prootic expands anteromedially (Figs. 6,8). cartilaginous pars interna plectri forms an Dorsally, the bone forms a distinct articula- expanded base to the pars media plectri and tion with the frontoparietal and ventrally it nearly fills the fenestra ovalis. There is a is growing toward the parasphenoid. The narrow margin of cartilage along the poste- 18 L. TRUEB AND J. HANKEN rrost “n a r i a Ie ’I7

post rnax proc

-pro exoc Lprsph +

ptei pars ar

pro + ex0c-J Lprsph

Figure 9 SKELETAL DEVELOPMENT IN XENOPUS LAEVIS 19 rior margin of the fenestra ovalis that may capsules. Dorsally, the auditory capsules are represent an operculum. bridged by the frontoparietal, and ventrally In the lower jaw, the infrarostral cartilage by the posterior end of the parasphenoid, unites completely with Meckel’s cartilages which expands laterally across the medial laterally. Anteromedially, there is a partial edges of the prootic and forms the floor of the erosion of the infrarostral cartilage to form posterior braincase. an incomplete mandibular symphysis. The pars palatina of the premaxilla increases in Stage 66 3 months to adult (Fig. 9) depth and the lateral palatine process of this + element now underlies the anteromedial pro- Other than increase in overall size, changes cess of the pars palatine of the maxilla. The in the skull after 3 months postmetamorpho- pars articularis of the palatoquadrate is ro- sis are relatively minor. The most conspicu- bustly ossified. ous change is growth of the external plectral unit, the pars externa plectri and the tym- Stage 66 + 2 months panic annulus. At 3 months, the pars externa Changes in the cranium during the second plectri is clearly separate from the tympanic month postmetamorphosis primarily involve annulus. In dorsal aspect, the unit as a whole modification of the prootic foramen. The extends about half the length of the zygo- sphenethmoid grows posterodorsally over the matic ramus of the squamosal toward the eye foramen to form a bony dorsal margin. At from the posterior tip of the otic ramus (ca. this stage, the sphenethmoid remains inde- midlength of pars media plectri). In lateral pendent of the prootic and frontoparietal view, the dorsal margin lies at the level of the bones. The posterior flange of the parasphe- head of the squamosal and the ventral mar- noid nearly meets the anteromedial flange of gin is approximately even with the plane of the prootic to form an incomplete bridge be- the upper jaw. The structures increase tween the braincase and auditory capsule slightly in relative size during the next 3 beneath the prootic foramen. Continued an- months, but at 6 months are still much teromedial ossification of the margin of the smaller than they are in the adult. In mature prootic between its articulation with the specimens (Fig. 9), the pars externa plectri parasphenoid ventrally and the frontoparie- completely fills the area circumscribed by the tal dorsally forms the posterior border of the tympanic annulus. The plectral unit occupies prootic foramen. The width of the crista pa- the entire posterolateral aspect of the skull rotica is now about one-third ossified. De- behind the eye and extends nearly to the level spite the elaboration of the prootics anteri- of the occipital condyles. Its dorsal margin orly and laterally, there is no evidence of lies well above the squamosal and crista pa- medial elaboration of the prootics or exoccip- rotica, and the ventral margin below the level itals to complete the foramen magnum dor- of the jaw articulation. sally and ventrally or to unite the auditory Other, less striking modifications include lateral expansion of the ossified portion of the crista parotica, completion of the bony bridge between the parasphenoid and prootic Fig. 9. Xenopus laeuis. Dorsal (upper) and ventral ventral to the prootic foramen, and expan- (lower)views of the adult skull. KU 20957, male, 52.0 sion of the posterior end of the parasphenoid mm SVL, cleared and stained. Cartilage is shown in to form the ventral margin of the foramen regular stipple pattern;irregular stippling (e.g.,“nariale”) indcates mineralized cartilage. alary c, alary cartilage;cr magnum and the posteromedial floor of the par, crista parotica; fpar, frontoparietal; max, maxilla; braincase. Even in fully mature individuals, obl c, oblique cartilage; optic f, optic foramen; pars art, a gap exists between the anteromedial floor pars articularis of palatoquadrate; pars ext plec, pars externa plectri; pars int plec, pars interna plectri; pars of the otic capsule and the parasphenoid and med plec, pars media plectri; pl antorb, planum antorbit- sphenethmoid medially, and the spheneth- ale; pl term, planum terminale; pmax, premaxilla; post moid does not fuse to the overlying frontopa- max proc, posterior maxillary process; pro + exoc, fused rietal. In larger individuals, mineralization prootic and exoccipital;pro f, prootic foramen; prsph + sph, fused parasphenoid and sphenethmoid;prsph, paras- occurs in the cartilaginous planum antorbit- phenoid; pter, pterygoid; rost c, rostral cartilage; sept ale. The nasals are completely fused medially nas, septum nasi; sprnax, septornaxilla; sph, spheneth- and largely covered by the frontoparietal, moid; sq (zyg r), zygomatic ramus of squamosal; sq, squamosal; ted nas, tedum nasi; tym ann, tympanic which develops an irregular group of medial, annulus. Scale = 5 mm. longitudinal striations. 20 L. TRUEB AND J. HANKEN Postcranial development anterior presacrals. Ossification of the pro- Axial column cesses of Presacral VIII proceeds more rapidly than that of Presacrals V-VII, which are For a detailed account of the ontogenesis of not ossified in all specimens until Stage 66. the vertebral column in Xenopus laevis, refer In most specimens, the transverse processes to Smit ('53), who also provided a review of begin to lose their short, knobby appearance previous work on Xenopus (Ridewood, 1897) by Stage 64, and are elaborated into antero- and other anurans. The schedule of chondri- laterally oriented spinous processes by Stage fication and ossification of the axial column 66. The sacral diapophyses develop in carti- observed in this study is recorded in Table 3. lage in Stage 62, prior to the formation of the Vertebrae. The first parts of the verte- transverse processes of the posterior presa- brae to form are the neural arches, which in crals, but after those of the anterior presa- the youngest specimens available for exami- crals. The sacral diapophyses are the first nation (Stage 48) are present for Presacral transverse elements of the vertebral column Vertebrae I-VII. The neural arches of the to begin ossification in Stage 63, but do not last presacral, the sacrum, and three postsac- assume their expanded adult configuration ral vertebrae appear in an anterior-posterior until the first month postmetamorphosis. sequence, with that of the last vertebra (XII) Ribs. Ribs develop on Presacrals 11-IV occurring at Stage 62 (Table 3). Ossification before the transverse processes and at about commences in all the presacrals in Stages the same time that the centra and neural 55-57, and follows an anterior-to-posterior arches begin to ossify in Stage 57. By Stage sequence throughout the remainder of the 58, the rib on Presacral I11 is ossified in all column. The neural arches of the sacrum specimens. Development of the ribs of Presa- (1x1 ossify in Stage 57, and those of the first crals I1 and IV is delayed by comparison; postsacral (X) in Stage 58. Ossification be- ossification does not appear in all specimens gins as early as Stage 59 for XI, but is not until Stage 60 (Table 3). The ribs are synos- present in all specimens until Stage 64; XI1 totically united with the transverse processes does not begin to ossify until Stages 62-64. in Stages 64 and 65. At Stages 65 and 66, the Fusion ofvertebrae XI and XI1 first occurs in ribs of Presacrals I11 and IV are only about Stage 63, followed by fusion of XI, X, and IX 30% longer than those of Presacral I1 and in Stage 64. At Stage 66, all the neural arches scarcely more robust. In the first month post- are imbricate, although those of Presacrals I metamorphosis, the posterior two pairs of and I1 are narrowly separated. Within the ribs become expanded distally and increase first month after metamorphosis, neural greatly in length until they are approxi- spines that overlap the posteriorly adjacent mately twice the length of the anterior pair of vertebra appear on Presacrals IV-IX. They ribs. The distal end of Presacral Rib IV devel- form on Presacrals I11 and I1 during the ops a posterolateral curvature in Stage 64. In second and third months postmetamorpho- specimens 1 month postmetamorphosis, this sis, respectively. The neural process of Presa- rib is strong deflected posterolaterally at ap- cral I does not develop until the fifth month proximately its midlength, suggesting that posthatching and initially appears as a pair of growth in this rib is occurring between the small spines on either side of the midline; dstal curvature and the expanded distal end subsequent ossification occurs between these rather than at the distal tip. spines to produce the bluntly truncated, flat Urostyle. This terminal element of the neural spine of the adult. vertebral column is formed by fusion of Post- The first vertebral centrum (I) is identifi- sacral Vertebrae X-XI1 with the hypochord. able in cartilage in Stage 53; those of Presa- The hypochord forms in cartilage and bone crals 11-VI appear in Stage 54, and VII-IX in between Stages 60 and 62. By Stage 63, it is Stage 57. Ossification of Centra I-IV occurs ossified in all specimens, and in Stage 66 the between Stages 55 and 57, followed by Cen- hypochord fuses with the block of postsacral tra V-IX in Stage 57. vertebrae that lie dorsal and anterior to it. Cartilaginous transverse processes are present on Presacrals 11-IV in Stage 61 and ossification commences in Stage 64. Although Anterior appendicular skeleton the cartilaginous transverse processes of Pre- The anterior limb bud is present as early as sacrals V-VIII do not appear until Stage 63, Stage 48, the youngest specimens available they begin to ossify at the same time as the for examination. The long bones of the fore- SKELETAL DEVELOPMENT IN XENOPUS LAEVIS 21

TABLE 3. Comparison of schedules of axial chondrification and ossification in Xenopus Iaevis' This study Smit ('53) Bernasconi Stage ('51) Cartilage Bone Cartilage Bone

~ ~~ 48 - Neural Arches - Neural Arches I-VII V-VI

49 - Neural Arch VIII - Neural Arches VII-IX 50

51 - Neural Arch IX Neural Arches 1-111, x 52 - Neural Arch XI -

53 - Neural Arches X-XI Centrum I 54 1. Neural Arches Centra 11-v1 - - Neural Arches 1-111 I-IX Centrum I 2. Neural Arches - I!-VII 3. Neural Arches - VIII-IX 4. Neural Arch X - Centra 11-IV 55 Neural Arches Neural Arch XI1 - I-VIII Centra I-IV Transverse Pro- cesses 11-IV

56 5. CentraV-X - - Ribs, Presacral I11 - 6. Rib, Presacral Centra I-IX Centra I-IX I1 57 7. Ribs, Presacral Ribs, Presacrals Neural Arches Ribs, Presacrals - Ill SI-IV IX-x 11, IV Neural Arch XI Centra V-IX Hypochord Centrum XI

58 8. Ribs, Presacral - Ribs, Presacrals - Ribs, Presacrals Iv 11-IV 11-111 Neural Arch XI1 Centrum XI1 59 Neural Arches - v-XI

60 - Hypochord Hypochord Neural Arch XI1 Hypochord

61 - Transverse Pro- - - Neural Arch XI cesses 11-IV

62 - Neural Arch XI1 - Sacral diapophysis - Sacral diapophysis 63 9. Sacral diapoph- Transverse Pro- Sacral diapophysis - - ysis cesses V-VIII

64 Transverse Pro- - Sacral diapophysis cesses 11-VIII Transverse Pro- cesses V-VIII

~ ~~ ~~ ~ ~ 'Developmental stages are those of Nieuwkoop and Faber ('56) and as applied to Bernaseoni's('51) data are only estimations Groups of elements associated wth a single number or stage appeared simultaneouslyIn the speclmens examined 22 L. TRUEB AND J. HANKEN limb and the ulnare and pre- and postaxial and clavicle become synostotically united (Fig. centralia are the first elements of the ante- 10). The final component to differentiate is rior appendicular skeleton to differentiate, the sternum which arises from a pair of chon- and are followed by the distal carpals and drifications associated with the posterior epi- metacarpals primarily during Stages 54-57. coracoid cartilages between Stages 62 and 65. Formation of the pectoral girdle is approxi- At Stage 66, the clavicle is a slender, curved mately contemporaneous with that of the element; the medial end of the bone is acumi- phalanges and takes place primarily after nate and curves anteriorly along the margin Stage 56 (Table 4). Sesamoid cartilages are of the procoracoid cartilage. The epicoracoid present beginning in Stage 62. cartilages are separate throughout most of their lengths but overlap posteriorly between Humerus, radioulna, ulnare, pre- andpost- the coracoid bones. The sternum is a single, axial centralia and metacarpals. Many limb shallow, arcuate cartilage. The glenoid end of buds in specimens of Stages 52-54 were dam- the slender coracoid is about equal in width aged during preparation and lack the fore- to the sternal end, but ossified more com- limb; hence, we cannot determine exactly at pletely. In the first month postmetamorpho- which of these stages the elements appear. sis, the medial configuration of the clavicle The humerus, radioulna, ulnare, and Meta- changes markedly; the medial end of the carpals I-IV are the earliest elements to dif- bone grows posteriorly over the procoracoid- ferentiate and in our sample are present in epicoracoidcartilage, thereby becoming broad Stages 55 and 56. These are followed slightly and blunt. At the same time, the sternal end later by the pre- and postaxial centralia and of the coracoid has ossified and now is slightly Distal Carpals 1-111 in Stages 55-57. The larger than the glenoid terminus. The ster- first elements of the forelimb to ossify are the num has increased in depth by growth along humerus and radioulna in Stages 57-58, fol- the posterior margin, and the epicoracoid lowed by the metacarpals. Metacarpals II-IV cartilages are synchondrotically united with ossify in Stages 58-59, whereas the ossifica- one another between the coracoids. By the tion of Metacarpal I extends through Stages fifth month postmetamorphosis, the procora- 58-62. coid cartilages are united with one another Phalanges. The earliest phalangeal ele- anteromedially. ments to differentiate are the proximal pha- Posterior appendicular skeleton langes of Digits I11 and IV in Stage 56; by Stage 57, all phalanges are present in carti- Like the forelimb, the limb bud of the hind lage. Ossification proceeds more gradually limb is present in Stage 48, but the elements and begins in Stage 60 when bone appears in are preformed in cartilage about one stage the proximal phalanges of Digits 1-W, how- earlier than their counterparts in the fore- ever, ossification of these elements is not limb. The long bones of the hind limb differ- present in all specimens until Stage 63. The entiate first along with the metatarsals and second proximal phalangeal elements of Dig- naviculare, followed by the other tarsals and its 111 and IV ossify between Stages 61 and finally the phalangeal elements and the pel- 65; bone first appears in the second proximal vic girdle (Table 5). phalangeal element of Digit I1 in Stages 63- Femur, tibiafibula, fibulare, tibiale, tarsal 65-approximately the same time frame dur- elements, and metatarsals. The long bones ing which the terminal phalangeal elements (femur, tibiafibula, tibiale, and fibulare), na- ossify in Digits I11 and IV. The final phalan- viculare, and Metatarsals II-V are differenti- geal element to ossify is the terminal member ated in all specimens by Stage 55 and in one of Digit I in Stages 64-66. specimen by Stage 54; the posterior limbs of Pectoral girdle. The first evidence of the Stage-53 specimens were damaged, thus pre- pectoral girdle is the scapular cartilage in venting observations. Metatarsal I differenti- Stage 56, and by Stage 57, the coracoid, epi- ates along with the tarsal elements in Stages coracoid, procoracoid, and suprascapular car- 55-56, and the prehallux is apparent in all tilages are present. Ossification proceeds rap- specimens by Stage 57. By Stage 58, all of the idly in Stages 58-62. The coracoid begins to long bones and metatarsals are ossified; all ossify in Stages 58-60 at the same time the tarsal elements remain cartilaginous through clavicle and cleithrum appear. The last part Stage 66. of the girdle to ossify is the scapula in Stages Phalanges. All phalangeal elements dif- 62-65; during Stages 64 and 65, the scapula ferentiate between Stages 55 and 57, contem- SKELETAL DEVELOPMENT IN XENOPUS LAEVIS 23

TABLE 4. Comparison of schedules of forelimb chondrification and ossification in Xenopus laevis' This study Nieuwkoop and Faber ('56) Bernasconi Stage ('51) Cartilage Bone Cartilage Bone Brown ('80) 55 - Humerus Radioulna Ulnare Postaxial centrale Distal Carpals 11-111 Metacarpals I-IV 56 - Distal Carpal I - Humerus Humerus Phalanx 111-1 Radioulna Radioulna Phalanx IV-1 Carpalia Corocoid Scapula Metacarpals I-IV Scapula Procoracoid Coracoid 57 1. Humerus Phalanges 1-1-2 Humerus (57-58) Suprascapula - Humerus Radioulna Phalanges 11-1-2 Radioulna (57-58) Radioulna Phalanges 111-2-3 Metacarpals Phalanges IV-2-3 Coracoid Epicoracoid and procoracoid Suprascapula 58 2. Metacarpals - Metacarpal I Clavicle Metacarpals I-IV Clavicle Phalanges (58-60) - Clavicle Metacarpals 11-IV Cleithrum 3. Coracoid (58-59) Scapula 4. Cleithrum Clavicle (58-59) Phalanges Cleithrum (58-60) Coracoid (58-60) 59 - Cleithrum Coracoid 60 5. Scapula - Scapula (60-62) - - - Phalanx 1-1 (60-63) Phalanx 11-1 (60-62) Phalanx 111-1 (60-63) Phalanx IV-1 (60-63) 61 Phalanx 111-2 Sternum (6 1-64) Phalanx IV-2 (61-64) 62 - Sternum Sesamoids 63 - - Phalanx 11-2 (63-65) Phalanx 111-3 (63-66 + 1 month) Phalanx N-3 (63-66 + 1 month) 64 Phalanx 1-2 f 64-66 + 1 month)

LDevelopmental stages are those of Nieuwkoop and Faber ('56) and as applied to Bernasconi's data are only estimations. Groups of elements associated with a single number or stage appeared simultaneously in the specimens examined. Ranges of stages in which elements were observed to appear in this study are indicated in parentheses. Bernasconi ('51) and Brown ('80) recorded only ossification events. 24 L. TRUEB AND1 J. HANKEN poraneous with those of the forelimb. The first proximal phalanges of Digits 11-V are A lavicle + scapula the first to form (Stage 55) and are followed by the first phalanx of Digit I, second phalanges of Digits I1 and V, and second and third phalanges of Digits 111 and IV. The last phalangeal elements to appear in cartilage are the terminal phalanges of Digits I, IV, and V in Stage 57. Phalangeal ossification begins in Stage 58 when it is present in all the phalanges of Digits I1 and I11 and the proximal two phalangeal elements of Digit IV, and the first proximal phalanx of Digit V. Ossification appears in the third phalangeal element of Digit IV and second phalangeal element of Digit V in Stage 59. The terminal phalanges of Digits IV and V are the last to commence ossification in Stage 60. By Stage 65, all phalangeal elements of the hind limb have begun ossification in all specimens examined, whereas those of the forelimb have not. Pelvic girdle. The ilium forms relatively early (Stage 55) in cartilage and commences ossification in the posterior part of the ilial shaft by Stage 57. The ischium and pubis are not apparent before Stage 60. Although is- chid ossification can appear posterodorsal to the acetabulum as early as Stage 60, it is not developed in all specimens until Stage 63. Bone does not appear in the pubis until postmetamorphosis (Stage 661, at which time a disclike center of ossification develops in the pubic cartilage. The disc is oriented vertically with its flat surfaces facing anteriorly and posteriorly. The epipubis appears in Stages 60-63 as a small, triangular plate of cartilage. The truncate apex is attached to the midventral surface of the cartilaginous pubes. The epipubis lengthens, and by Stage 66 it has a moderately narrow proximal shaft and expanded terminus that is pointed ante- Fig. 10. Xenopus lueuis. Development of the pectoral riorly; the anterior margin becomes more girdle duringlate metamorphosis and earlypostmetamor- phosis. Girdles are drawn in ventral view with left supra- blunt in subsequent development. scapulae removed, and scapulae and right suprascapulae Within the first month postmetamophosis, deflected into the ventral plane. A: Stage 64 (KU 217955), the paired ischia are united synostotically, prior to appearance of sternal elements. B: Stage 65 (KU whereas the ilia and pubes remain separate 217958). Sternum appears as two posteromedial chondrifications adjacent to expanded ends of epicoracoid carti- from one another and the ischia. The ilial lages. C: Stage 66 (KU 217963). There is a marked shaft is expanded anteriorly and develops a expansion in the suprascapular cartilage and the thick- small crest along its dorsolateral margin. By ness of the clavicular portion of the fused clavicle and scapula; the sternal chondrifications have united to form Stage 66 + 2 months, the posteromedial area a single, shallow sternal plate. D Stage 66 + 1 month of the pubes behind the epipubis and anterior (KU 217969). Note development of posterior arm of pubic ossifications has begun to mineralize. cleithrum and medial end of clavicle. Cartilage is shown By 6 months postmetamorphosis, the ilial in regular stipple pattern. c, cartilage; fen, fenestra. Scale = 1mm. crest is well developed, but the pubic ossifications remain discrete from one another as they do in the adult. SKELETAL DEVELOPMENT IN XENOPUS LAEVIS 25 SUMMARY OF OSTEOLOGICAL DEVELOPMENT earliest and most protracted sites of bone Chondrification and ossification sequences formation are the cranium (Stage 54) and the are presented in Tables 2-5; variation in axial column (Stage 55). The appendicular these features is assessed in Appendices A-D. skeletons commence ossification two or three Figure 11 depicts a generalized scheme of stages later (Stage 571, with the posterior ossification timing on a regional basis. The appendicular skeleton ossifying more rapidly

TABLE 5. Comparison of schedules of hind-limb chondrification and ossification in Xenopus laevis' Nieuwkoop and This study Faber ('56) Bernasconi Brown Stage ('51) Cartilage Bone Cartilage Bone ('80) 54 - Femur - Femur - - Tibiafibula Tibiafibula Tibiale Tibiale Fibulare Fibulare Naviculare Ilium Metatarsals 11-V Ischium 55 - Metatarsal I Tarsals 1-11 Phalanx 11-1 Phalanx 111-1 Phalanx IV-1 Phalanx V-1 Ilium 56 - Prehallux - Femur - Phalanx 1-1 Tibiafibula Phalanx 11-2 Tibiale Phalanx 111-1 Fibulare Phalanges IV-2-3 Metatarsals Phalanx V-2 57 1. Femur Phalanx 1-2 Femur (57-58) - Phalanges Femur Tibiafibula Phalanx IV-4 Tibiafibula Tibiafibula Tibiale Phalanx V-3 Tibiale (57-58) Ilium Fibulare Fibulare (57-58) 2. Ilium Metatarsals I-V (57-58) Ilium (57-58) 58 3. Metatarsals - Phalanges 11-1-2 (58-60) - Phalanges Phalanx 111-1 (58-59) Phalanx 111-2 (58-60) Phalanx IV-I (58-59) Phalanx IV-2 (58-60) Phalanx V-l(58-60) 59 - - Phalanges 1-1-2 (59-60) - - - Phalanx IV-3 (59-62) Phalanx V-2 (59-62) 60 4. Ischium Ischium Phalanx IV-4 (60-65) Epipubis Ilium Tibiale 5. Pubis Pubis PhalanxV-3 (6045) Ischium Fibulare Epipubis Ischium (60-63) Pubis Metatarsals 61-65 66 66 + 1month 66 + 2 months 6. Prehallux - - - - - 66 + 2-5 months - - 66 + 6 months 7. Tarsals -

'Developmental stages are those of Nieuwkoop and Faber ('56)and as applied to Bernasconi's ('51)data are only estimations. Groups of elements associated with a single number or stage appeared simultaneously in the specimens examined. Ranges of stages in which elements were observed to appear in this study are indicated in parentheses. Bernasconi ('51) and Brown ('80) recorded only ossification events. 26 L. TRUEB AND J. HANKEN suspensorium is the next unit to develop; whereas the formation of the pterygoid is complete at Stage 65, that of the squamosal and the plectra1 apparatus occupies several months postmetamorphosis. Axial patterns As expected, the core of the axial column- the centra and neural arches-includes the first elements to ossify; however, the development of the postsacral components Werte- brae X-XII) is delayed and occupies considerably more developmental time than the anterior part of the vertebral column. The separate designation of the urostyle in Fig- ure 11 is somewhat misleading, given that this element is formed by a fusion of the hypochord with the fused postsacral vertebrae. The onset of urostyle ossification is defined by the appearance of ossification in the hypochord, and the terminus as the fusion of the postsacral vertebrae with the hypochord. If one considers the number of stages re- quired for at least partial ossification of an element in all specimens examined as an Fig. 11. Xenopus laeuis. Timing of ossification events. Data plotted represent only the initiation of ossification index to the speed of formation, then ossifica- within each skeletal unit; thus, all elements of the brain- tion of the ribs and transverse processes does case, for example, have begun to ossify by Stage 66, but not follow the anterior-to-posterior sequence ossification of this structure is not complete by Stage 66. that one might expect. Thus, the second pair Feeding begins at Stage 45. of ribs ossifies more rapidly than either the first or third pairs. All ribs develop prior to than the anterior, some components of which the appearance of transverse processes. The do not begin to ossify until the first month transverse processes of all presacral verte- after metamorphosis. brae commence ossification simultaneously, but the process is most rapid in Presacrals Cranial patterns 11-IV and VIII (1-2 stages), whereas ossifica- Of all parts of the skeleton, the bony hous- tion of the processes of Presacrals V-VII ing for the brain undergoes the earliest and requires three stages. most protracted period of development (Stag- es 54-66; Fig. 11).The first parts to form are Appendicular patterns the roof and the floor represented by the The last parts of the skeleton to ossify are frontoparietal and parasphenoid, respectively. the girdles and appendages beginning in Stage Thereafter, the auditory capsules and poste- 57. The posterior appendicular skeleton ossi- rior skull begin to ossify as the prootics and fies more rapidly than the anterior skeleton exoccipitals. Ossification of the lateral walls (Fig. 11). In the posterior appendicular skele- of the braincase does not commence until ton, the long bones, metatarsals, and girdle Stages 64-66 when the sphenethmoid ap- commence ossification simultaneously, pears. The braincase is not fully formed until whereas in the anterior skeleton, the begin- 2 months postmetamorphosis and undergoes ning of metacarpal and pectoral-girdle ossifi- significant alteration in the otic-prootic re- cation is delayed a stage relative to the poste- gion during the 6 months following metamor- rior skeleton. Also, phalangeal ossification of phosis. the manus is delayed by a stage relative to The mandible and upper jaw are the sec- that of the pes and requires one stage more ond and third parts of the osseous skull to for completion. In the manus, the proximal form, followed by the bones of the nasal phalangeal elements form first and contempo- capsule, the nasals and septomaxillae. The raneously with one another, followed by the SKELETAL DEVELOPMENT IN XENOPUS LAEVIS 27 second phalangeal elements of Digits I11 and configuration, which primitively occurs in IV. The second (terminal) phalangeal ele- late metamorphosis, in the embryonic period ment of Digit I1 then ossifies at the same of X. laevis represents an example of preco- time as the third (terminal) elements of Dig- cious metamorphosis similar to that which its I11 and IV.The terminal phalanx of Digit I occurs in the leptodactylid frog Lepidobatra- is the last to ossify. Thus, the general pattern chus. This derived developmental pattern was is proximal to distal with ossification proceed- defined by Hanken ('92) as one in which ing more rapidly in longer digits and in a metamorphorphic events in typical anurans postaxial-to-preaxialsequence. The same pat- are advanced into the embryonic period, and tern is evident in the pes. the development of larval components is cor- DISCUSSION respondingly eliminated. Novel features of cranial development in The timing of palatoquadrate development Xenopus laevis in Xenopus laevis also is peculiar. In most anurans, the palatoquadrate develops early There are several noteworthy and some as a massive bar of cartilage that is firmly unique features of chondrocranial develop- attached posteriorly to the postorbital neuro- ment in Xenopus laevis relative to non-pipid cranium by the ascending process and anteri- anurans. First, the area anterior to the tra- orly to the trabecular or ethmoidal part of becular plate is developed as a single plate of the neurocranium by the commissura qua- cartilage, the suprarostral plate, instead of dratocranialis anterior. In X. laevis, the ante- one or more suprarostral cartilages derived rior palatoquadrate develops early (ca. by from paired cornu trabeculae (Fig. 2). Sec- Stage 39). However, the posterior part of the ond, the trabecular plate lying between the palatoquadrate is transient with respect to commissurae quadratocranialis anterior is the anterior part; it develops several stages much longer (and hence, also the commissu- later (Stages 45-46], begins to erode at Stage rae) than its counterpart in non-pipids. 61, and has disappeared at Stage 63, three Whereas the anterior margin of the basicra- stages before the completion of metamorpho- nial fenestra lies between the commissurae sis. In non-pipid anurans such as the pelo- in most anurans, in X. laevis it lies well batid Spea, the configuration of the posterior posterior to these structures (Fig. 2). Third, part of the palatoquadrate and the nature of and perhaps most unusual, the palatoquad- its attachment to the neurocranium change rate forms in two parts rather than as a during the late stages of metamorphosis, but single unit as in non-pipids. Each part forms the structure does not disappear (Wiens, '89). in the same position relative to the para- Thus, there seems to have been a structural chordals and trabeculae (and later, the brain- and temporal repatterning of palatoquadrate case), but development of the posterior por- development in X. laevis relative to most tion is delayed relative to the anterior portion other anurans. and to its formation in other anurans. Probably the most bizarre feature of cra- Even before feeding, which commences at nial development in Xenopus laevis is the Stage 45 in Xenopus laevis, the mandible orbital region of the braincase. As in other (Paterson, '39:Fig. 22b) resembles in many anurans, the orbital region initially forms in respects that of the mature premetamorphic cartilage. Whereas in non-pipid anurans, the tadpole of Stage 54 and the adult. Meckel's cartilage usually is replaced partially or cartilages are long, curved structures that wholly by endochondral bone (sphenethmoid are separated anteromedially by the single and anterior part of the prootics), in X. laevis infrarostral that later fuses with them to the cartilage is resorbed between Stages 59 form a continuous mandibular arcade to and 60 and the orbital walls of the braincase which dermal investing bones are applied then are formed by membrane bones that medially and laterally to form the adult lower develop at metamorphosis. jaw (Fig. 7). In effect, these cartilages assume an initial configuration similar to that of Questions of homology non-pipid anurans at the end of metamorpho- The rostral chondrocranium sis. This was recognized by Sokol ('75:19), InXenopus, the rostral portion of the chon- who observed that "a cenogenetic structural drocranium anterior to the braincase has complex is no longer being maintained by been variously termed the ethmoid cartilage selection pressure and is being eliminated in or plate (Paterson, '39; Sedra and Michael, favor of a more direct developmental '57; RoEek and Veself, '89), planum interna- pattern." Early formation of the adult jaw sale (RoEek, '89, 'go), and Gaumendach (pal- 28 L. TRUEB AND J. HANKEN atal roof; Kotthaus, ’33). Sokol(’75) referred (Cannatella and Trueb, ’88a,b; Cannatella, to the anterior plate as a suprarostral, reason- ’85) argue for the derivation of the pipid ing that it is homologous with the supraros- condition from the plesiomorphic larval con- tral cartilages of non-pipid anurans (contra dition of their archaeobatrachian relatives Starrett ”731, RoEek ”891, and RoEek and (eg., pelobatoids, discoglossids, bombina- Vesely “891). He maintained that in pipids, torids, and ascaphids). To argue the alter- as well as microhylids, this cartilage arises as nate hypothesis-that is, that the chondro- an anterior extension of the trabeculae and crania of pipid and nonpipids could not have that it fails to differentiate into the complex been derived from one another-is tanta- and variable multipartite suprarostral sys- mount to suggesting a diphyletic origin of the tem characteristic of beaked larvae. For rea- Anura; we know of no evidence in support of sons discussed below, we adopt the terminol- this hypothesis, but there is a substantial ogy of Sokol(’75). body of evidence supporting the monophyly RoEek and Vesely (’89) argued that topo- of the Anura (Trueb and Cloutier, ’91a,b). graphical and developmental differences be- Thus, it seems most reasonable to assume tween the cornua trabeculae of non-pipid an- that the rostral cartilage ofXenopus is homol- urans and the planum internasale or ethmoid ogous with the suprarostral of non-pipoids, plate of pipids precluded their derivation from pending the discovery of evidence to the con- one another, despite the fact that both seem trary such as a tadpole possessing both a flat to be derived from the same region of cranial platelike anterior extension of the planum neural crest (Sadaghiani and Thiebaud, ’87). internasale and suprarostral cartilage. In the RoEek (’89) pointed out that in non-pipid unlikely event that such a larva were found, anuran larvae, all adult ethmoidal structures the hypothesized homology would fail the except the solum nasi are derived from new “conjunction test” of Patterson (’82) be- cartilaginous tissue that appears between the cause both structures would have been found cornua trabeculae and ultimately fuses with to occur in the same organism. them; the solum nasi arises from the cornua trabeculae. In pipids, the adult ethmoidal OrbitaI region of the braincase structures also arise from new cartilaginous tissue (RoCek, ’89);the cartilage appears dor- The peculiar development of the orbital sal to the ethmoid plate in the same relative neurocranium in Xenopus laevis has been position as it does in non-pipids. The major noted in passing by other workers. Paterson developmental difference between pipids and (’39:184) described the sphenethmoid region non-pipids is that the larval ethmoid carti- of young Xenopus as being occupied by “a lage, unlike that of non-pipids, eventually very thin bone which forms the lateral wall of disappears and does not contribute to the the cranium anterior to the optic foramen” nasal capsule of the adult (Kotthaus, ’33; and which “joins the fronto-parietalia with Sedra and Michael, ’57; this paper). Thus, it the cartilaginous floor of the cranium in front, seems highly likely that one of two situations and rather more posteriorly it is confluent prevails; the larval rostral cartilage either with the parasphenoid.” She commented that represents a fusion and simplification of the the bone “is obviously homologous with the cornua trabeculae of beaked tadpoles, or in 0s en ceinture, but it does not possess the pipids, the cornua trabeculae fail to develop large marrow cavities that characterize it in and a flat, platelike, anterior extension of the other Anura.” Sedra and Michael (’57:49) planum internasale develops in their place. similarly observed that “Each side wall [of It is clear that the configuration of the the braincase] is mainly ossified in front of rostral chondrocranium in Xenopus laeuis the optic foramen” by a bone they called the differs from that typical of non-pipoid orbitosphenoid. They reported that the re- anurans; however, we argue that the develop- mainder of the side wall is membranous, but mental differences between X. laevis and non- depicted the unossified portions of the orbital pipoid anurans are less trenchant than RoEek neurocranium as cartilage (Sedra and and Vesely (’89)would have us believe. More- Michael, ’57:Figs. 32,35-37,391. Our sections over, deviations from the ancestral develop- of heads in Stages 63-65 similarly reveal that mental pattern do not intrinsically constitute cartilage is absent and that in Stages 64 and an argument against homology of the result- 65 there is thin bone lacking marrow cavities ing structures (Patterson, ’77). Parsimony and surrounded by connective tissue in the and the phylogenetic logic of other evidence orbital region of the braincase. SKELETAL DEVELOPMENT IN XENOPUS LAEVIS 29 One can argue that the orbitosphenoid of gous with the sphenethmoid of other anurans. salamanders and the sphenethmoid of an- Thus, following Patterson ('77),the sphen- urans are homologous because both are endo- ethmoid of X. laevis is an endoskeletal bone chondral elements that form from a pair of that is not preformed in cartilage owing to a ossifications-one on either side of the brain- regression of cartilage or change in growth case medial to the orbit. In the case of pattern; as such, it is similar to the mem- salamanders and most anurans, ossification brane basisphenoid of teleosts, which is con- appears anterodorsally and spreads posteri- sidered a phylogenetic homologue of the endo- orly and ventrally toward the optic foramen chondral basisphenoid of Amia, paleoniscoids, and parasphenoid, respectively. Because the and primitive pholidophorids (Patterson, '77). cartilage in which the sphenethmoid or orbitosphenoid forms is continuous with the Comparison to previous studies of skeletal lamina orbitonasalis or planum antorbitale development in Xenopus laevis and the septum nasi of the olfactory capsule, Comparisons among the various studies of anterior ossification variably encloses the or- chondrification and ossification of Xenopus bitonasal foramen in bone and may invade laevis are problematic. First, the sources of the septum nasi. In most taxa, the pair of the specimens examined varies; thus, Sedra ossifications unite ventrally above the para- and Michael ('571, Nieuwkoop and Faber sphenoid and dorsally anterior to the level of ('561, Smit ('531, and part of Brown's ('80) the frontoparietal fontanelle to form the gir- material were wild-caught, whereas Bernas- dle bone that houses the anterior end of the coni's ('51) material and the specimens re- brain. Xenopus laevis, however, differs from ported on herein were reared in the labora- salamanders and non-pipid anurans by a com- tory. Brown's ('80) results suggest that bination of significant features: 1) ossifica- ossification in wild-caught larvae begins ear- tion of the sphenethmoid seems to be mem- lier and proceeds more rapidly than it does in branous rather than endochondral; 2) the laboratory-reared populations; however, these sphenethmoid forms only the posterolateral developmental differences (as well as differ- border of the orbitonasal foramen, and is ences among laboratory-reared populations) distinct from rostra1 cartilages and any ossifi- simply may have resulted from one or more cation that might occur in them; 3) it com- environmental variables such as tempera- mences ossification posteroventrally rather ture, density of larvae, and photoperiod. Sec- than anterodorsally; 4)after metamorphosis, ond, specimens have been prepared in a vari- the sphenethmoid spreads posteriorly to- ety of ways that may not be directly ward the prootic such that it surrounds the comparable. Bernasconi ( '51) used alizarin- optic foramen and forms the anterior border stained whole mounts, whereas the speci- of the prootic foramen (discussed above); and mens examined by Brown ('80)and most of 5) the dorsal margin of each sphenethmoid those examined herein are whole mounts dou- fuses with the frontoparietal, and midven- ble-stained for cartilage and bone; Sedra and trally, the bones unite with one another and Michael ('57), Paterson ('39),Nieuwkoop and fuse with the parasphenoid below. If one Faber ('561, and Smith ('53) examined sec- were to use these developmental data to spec- tioned material. Third, Bernasconi's ('51)and ulate that the sphenethmoid of X. laevis (and Paterson's ('39)results are calibrated by lar- possibly other pipids as well; Trueb, personal val size and age and, thus, cannot be com- observation) is not homologous to the pared readily with one another or with stud- sphenethmoid of other anurans, this would ies based on the normal table of Xenopus imply that the bone was missing in the ances- laevis development (Nieuwkoop and Faber, tor of pipids. Were this the case, the anterior '56).As shown in Figure 12, the total lengths braincase could be considered a neomorphic of X. laevis larvae are highly variable, espe- structure that is topologically homologous cially during the stages of cranial ossification and functionally analogous, but not phyloge- (Stages 54-64). Fourth, the range of ossifica- netically homologous, to the sphenethmoid tion events in Brown ('80) is limited owing to of nonpipid anurans. In the absence of evi- the author having only Stages 55-60 avail- dence of a pipid ancestor that lacks a able for examination. Fifth, none of the previ- sphenethmoid, however, one must assume ous studies addressed individual variation in instead that development of the spheneth- developmental timing. We found that the moid has been altered and represents a de- sequence of events does not vary, but that rived feature. but that the bone is homolo- the onset of chondrification and ossification 30 L. TRUEB AND J. HANKEN 90

vE C 50 5c 3 40 -0 c 30

Developmental Stage

Fig. 12. Xenopus laeuis. Variation in total length of larvae plotted against developmental stage. Means are indicated by closed circles; ranges are represented by vertical lines. The sample size measured in each stage is indicated by the terminal digit of the categories on the X-axis; thus, “St.48.3” indicates that three specimens of Stage 48 were measured. varies depending on the skeletal element (Ap- Figure 13. The general chronology of ossifica- pendices A-D). Finally, the terminology of tion of Sedra and Michael seems to be de- cranial elements is inconsistent and/or incor- layed by two or three stages relative to the rect (Table 6). Bernasconi (’51) apparently data from this study and that of Brown (’80). noted two centers of ossification for the angu- This result is somewhat surprising given that losplenial-viz., his goniale and articular. Se- in a comparative assay of osteogenic events dra and Michael (’57) did not distinguish in Bornbina, Hanken and Hall (’88) found between the exoccipital and prootic, referring that bones were fully differentiated in sec- to both as “occipito-prootic ossification.” tioned specimens some six stages before they Paterson (’39)distinguished parts of the fron- were detectable in cleared-and-stained whole- toparietal and sphenethmoid regionally, but mounts. Exceptions to this generalization for not developmentally. Bernasconi (’51) appar- Xenopus laevis are the parasphenoid, part of ently misidentified several elements. Thus, the angulosplenial, septomaxilla, and den- his supraethmoid is the nasal, his nasale is tary, which appear at the same stages, and the septomaxilla, and his palatinum and nar- the plectral apparatus and premaxilla, which iale are mineralized cartilage of the planum appear earlier and later, respectively, than in antorbitale and alary cartilage, respectively. this study. Except for the plectral apparatus, Notwithstanding these limitations, it is pos- the sequence of appearance of cranial ele- sible to make some general comparisons. The ments is the same in this study and Sedra cranial ossification data of this study, Sedra and Michael’s. Curiously, Sedra and Michael and Michael (’5’71, Bernasconi (’51), and do not mention the vomer. Relative to this Brown (’80) are compared in Table 2 and study and those of Sedra and Michael (’57) SKELETAL DEVELOPMENT IN XENOPUS LAEVIS 31

TABLE 6. Comparison of nomenclature used in this and other descriptions of osteological development in Xenopus laevis' Sedra and Michael This study Bernasconi ('51) Brown ('80) Paterson ('39) ('57) Angulosplenial, Goniale Prearticular Angulare Goniale medial Angulosplenial, Articular Angulare Goniale lateral Exoccipital Pleuroccipitale Occipito-prootic ossification Frontoparietal - Frontoparietal (part) - Supraethmoid (part) Nasal Supraethmoid - Pars articularis of Quadrate - the palatoquadrate Prootie - - Occipito-prootic ossification Sphenethmoid Sphenethmoid Sphenethrnoid 0sen ceinture (part) Orbitosphenoid Pleurosphenoid (part) Septomaxilla Nasale - - - Mineralization of Nariale - - - alary cartilage

Mineralization of Palatinurn ~ - - planum antorbitale

'Bones not listed are named in the same way in all five studies and Brown ('80)' Bernasconi's chronology is ('56) recorded the scapula and phalangeal peculiar in that the prootic ossifies late and elements to ossify earlier and the sternum to the squamosal and pterygoid bones ossify chondrify earlier. The primary difference be- early. tween our results and Bernasconi's ('51) is In general, we think that Smit ('53) the earlier ossification of the phalangeal ele- achieved a finer resolution of skeletal develop- ments in his schedule. ment of the axial column based on his study With respect to the posterior appendicular of sectioned material than we did in our skeleton (Table 5), there are some striking examination of whole-mount specimens. The differences between the results reported overall pattern of events is similar in this herein and those of Nieuwkoop and Faber study and Smit's ('53) except for the timing ('56). They reported the ilium and the isch- of the chondrification of the transverse pro- ium to chondrify one and five stages earlier, cesses of Presacrals 11-IV; in his schedule respectively, than we found them. According these structures appear in Stage 55, whereas to Nieuwkoop and Faber ('33,the long bones, we did not find them until Stage 61 (Table 3). metatarsals, and phalangeal elements ossify Furthermore, we were unable to identify the one stage earlier than reported here, and cartilage precursors of Centra VII-IX, which ossification of the ilium occurs three stages were first evident in bone at Stage 57. later in their developmental scheme, but the The development of the anterior appendic- ischium ossifies at the same stage. They also ular skeleton reported in Nieuwkoop and reported the pubis to be ossified at Stage 60, Faber ('56) deviates somewhat from the re- whereas we found it to chondrify at this stage sults of this study, Bernasconi ('511, and but not ossify until Stage 66. The sequences Brown ('80) (Table 4). For example, Nieuw- of ossification observed by Bernasconi ('51) koop and Faber ('56) reported the proximal and Brown ('80) correlate exactly with that limb bones to chondrify and ossify in Stage of this study; however, Brown did not ob- 56, whereas we noted chondrification in Stage serve ossification of the tibiale, fibulare, and 55 and ossification in Stage 57. Similarly, metatarsals until three stages later than in Nieuwkoop and Faber ('56) reported the cor- this study. This developmental hiatus in acoid present in bone and cartilage in Stage Brown's ('80) chronology is peculiar, given 56, whereas we did not find it chondrified that each of the other studies reported the until Stage 57 and ossified until Stage 58. long bones and metatarsals to ossify early Relative to this study, Nieuwkoop and Faber and nearly simultaneously. 32 L. TRUEB AND J. HANKEN

, -3 , -2 , -1 , 0 , +1 , +2 , +3 , tropicalis between Stages 55 and 60, and I I I I I I I Trueb provided a partial summary of Fnlr,&A ! ('85) cranial development of Rhinophrynus dorsalis (Rhinophrynidae). Early ossification (i.e., ; Prsph:55 : the first 5 cranial bones) is the same in the three pipid (Xenopus and Silurana) taxa; : Exoc:55-56 ' however, the maxilla, premaxilla, and septomaxilla develop one or two stages later in I Pro: 55-57 I X. borealis and S. tropicalis than in X. laevis (Brown, '80). Timing of ossification of the long bones of the fore- and hind limbs, metacarpals, clavicle, cleithrum, and the ilium I, I Max:57 : varies slightly, but all these bones develop within one or two stages of one another. The most striking difference noted by Brown ('80) 1 Nas:58SO was in the timing of ossification of the tibiale, i-j fibulare, and metatarsals, which ossify be- : Spmax:60 i tween Stages 56 and 57 in X. borealis and S. ' Den:6&62 ' tropicalis, but not until Stage 60 in X. laevis. (See Trueb, '85 for a summary.) However, I ' Ang(lat): 6-1 ~ the results of this study which show these i-; elements to ossify in Stage 57 in X. laevis do not substantiate this claim. I Tyrn ann B Pars ext plec: 61-62 Published data on the development of the j-j living sister taxon of the pipids, Rhinophry- nus dorsalis, are incomplete. Nonetheless, the information provided by Trueb ('85) indicates some interesting similarities and differences. The prootic of Rhinophrynus develops later than the exoccipital rather than at the same time, as it does in X. laevis. Ossification of the sphenethmoid is evident in Rhinophry- nus at the onset of metamorphosis (ca. Gos- Fig. 13. Xenopus laevis. Timing of ossification of ma- ner Stage 41 = Nieuwkoop and Faber Stage jor cranial elements in this study (black bars) compared 601, whereas the sphenethmoid of X. laevis with data from Sedra and Michael ('57; stippled bars), does not develop until metamorphic climax Brown ('SO; vertically hatched bars), and Bernasconi ('51; diagonally hatched bars). Zero refers to the stage at (Nieuwkoop and Faber Stages 64-66). The which we observed first ossification; the timing of earlier septomaxilla and pterygoid of Rhinophrynus observations by other authors is indicated by negative form later than in X. laevis. values (e.g., - 1 = 1 stage earlier), whereas the timing of later observations is indicated by positive values (e.g., + 1 = 1stage later). For detailed comparisons, see Table Non-pipoid taxa 2. Ang(lat), lateral angulosplenial; Ang(med), medial angulosplenial; Den, dentary; Exoc, exoccipital; Fpar, fron- Pelobatid frogs are a member of the super- toparietal; Max, maxilla; Nas, nasal; Pars ext plec, pars family Pelobatoidea, which is the sister group externa plectri; Pars med plec, pars media plectri; Pmax, of the Pipoidea (i.e., Rhinophrynidae + Pipi- premaxilla; Pro, prootic; Prsph, parasphenoid; Pter, pterygoid; Sphen, sphenethmoid; Spmax, septomaxilla; Sq, dae fide Cannatella "851). Using the ontoge- squamosal; Tym ann, tympanic annulus; Vom, vomer. netic study of the pelobatid frog, Spea bombi- frons, by Wiens ('891, it is possible to compare skeletogenesis in these archaeobatrachian taxa. Development of Xenopus laevis compared to The same bones form first in the cranium other anurans of both Spea and Xenopus laevis (viz., the Pipoid taxa frontoparietal, parasphenoid, exoccipital, and There are limited data published for three the prootic); however, the prootic forms ear- other pipoid taxa. Brown ('80) provided infor- lier in X. laevis than it does in Spea. The mation on ossification sequences of Xenopus upper jaw, nasal, and septomaxilla form in borealis and Silurana (Xenopus, auctorum) the mid-sequence of cranial development in SKELETAL DEVELOPMENT IN XENOPUS LAEVIS 33 both ha.In X. laeuis, the angulosplenial of raneous with the first three skull bones and the mandible ossifies from two centers (unre- in the stage preceding ossification of the ported in any other anuran) and the larger, prootic (ca. Gosner Stage 36 = Nieuwkoop medial part ossifies before the upper jaw, and Faber Stage 55). In the hylid, Hyla lanci- nasal, or septomaxilla. In Spea, the angu- formis, and the ranid, Rana pipiens, losplenial ossifies after them, at approxi- ossification of the skull and vertebral column mately the same time the lateral part of the is present in Gosner Stages 29 and 30 (= angulosplenial appears in X. laevis. The Nieuwkoop and Faber Stages 51 and 521, vomer develops late in X. laevis in compari- respectively, and the appendicular skeleton son with Spea-that is, after the pterygoid develops in Gosner Stages 35-38 ( E Nieuw- rather than before. The plectral apparatus of koop and Faber Stages 55-56) (de Sa, '88; X. laevis appears well before metamorphosis Kemp and Hoyt, '69). On the basis of these (Nieuwkoop and Faber Stages 59-61), data, Wiens ('89) hypothesized that the ossi- whereas that of Spea develops postmetamor- fication of the skull and vertebral column is phically (i.e., after Gosner Stage 461, along delayed in Spea relative to Hyla and Rana. with the sphenethmoid. With regard to tim- Insofar as we can equate the developmental ing (but not composition) of ossification of stages of the various staging tables (Table 11, the braincase, Spea and X. laevis are more it is apparent in Figure 11 that in Xenopus similar to one another than the latter is to laevis, the skull and vertebral column form Rhinophrynus. slightly earlier (ca. Gosner Stages 34 and 35) All bony elements of the postcranial skele- than in Spea, but significantly later than in ton of Spea, except the prehallux, ischium, Rana or Hyla. However, X. laevis differs from carpals, and tarsals, begin to ossify contempo- each of these taxa in the late development of

[21 Bombina orientalis Xenopus laevis Bufo boreas [II Rana pipiens 6-

5- m a, 0) m G 4- u- 0 L a, E 3- z3

Fpar Exoc Prsph Spmax Pmax Vomer Nasal Max

Fig. 14. Temporal variation in the ossification of cranial bones among four taxa of anurans. Numbers on the ordinate refer to the number of developmental stages in which an element has been observed to ossify. Data for Bornbina orientalis are from Hanken and Hall ('841, for Bufo boreas from Gaudin ('781, and for Rana pipiens from Kemp and Hoyt ('69). Exoc, exoccipital; Fpar, frontoparietal, Max, maxilla; Pmax, premaxilla; Prsph, parasphenoid; Spmax, septomaxilla. 34 L. TRUEB AND J. HANKEN

Bombinu orientalis Xenopus laevis Bufo boreas Rana pipiens

Angulo Angulo Squa Dent Pter Prootic Sph (med) (lat)

Fig. 15. Temporal variation in the ossification of cranial bones among four taxa of anurans. Numbers on the ordinate refer to the number of developmental stages in which an element has been observed to ossify. Data for Bornbina orientalis are from Hanken and Hall ('841, for Bufo boreas from Gaudin ('78), and for Rana pipiens from Kemp and Hoyt ('69). In Xenopus laeuis, the angulosplenial forms from two centers of ossification (medial and lateral), which are represented separately; the data for the two centers are combined for comparison with the formation of the bone in the other taxa. Angulo, angulosplenial; Dent, dentary; (lat), lateral portion; (med),medial portion; Pter, pterygoid Sph, sphenethmoid; Squa, squamosal. the appendicular skeleton which does not the question arises as to the reliability of begin to ossify until Gosner Stages 39-40. these features as staging criteria. The data Timing of ossification relative to for cranial bones inX. laevis are arrayed with developmental stages those for laboratory-reared developmental series of Bombina, Bufo, and Rana in Figures The developmentaltable of Nieuwkoop and 14 and 15. Examination of these figures Faber ('56)uses external morphology for stag- shows that cranial development in X. Zaevis ing living and whole preserved specimens of is, in many instances, less variable than that Xenopus taevis, as do Gosner's ('60) and other observed in the other taxa, although based on schemes for non-pipid anurans. However, Nieuwkoop and Faber's table also includes the sample examined here, only three cranial internal criteria, of which ossification and. bones show fidelity to single stages: the para- chondrification events are an important part sphenoid, septomaxilla, and maxilla. Even in their diagnoses of various stages. Hanken greater variation is apparent in the develop- and Hall ('84) demonstrated that cranial ossi- ment of the postcranial skeleton (Appendices fication in Bombina orientalis is poorly corre- B-D). These results suggest that ossification lated with the development of external mor- is an imprecise staging criterion and that phology. Given the importance of skeletal features of soft anatomy may be more reli- developmental features in Nieuwkoop and able diagnostic tools of stages of larval de- Faber's ('56) table of normal development, velopment than ossification events in SKELETAL DEVELOPMENT IN XENOPUS LAEVIS 35 Nieuwkoop and Faber's ('56)developmental Hanken, J., and R.J. Wassersug (1981) The visible skele- table for laevis. ton. Funct. Photog. 16:22-26,44. X. Just, J.J., J. Kraus-Just, and D.A. Check (1981) Survey ACKNOWLEDGMENTS of chordate metamorphosis. In L.I. Gilbert and E. Frieden (eds): Metamorphosis: A Problem in Develop- We are indebted to Cliff Summers who mental Biology. New York and London: Plenum Press, aided in the rearing, measuring, and prepara- pp. 265-326. Kelley, D., D. Sassoon, N. Segil, and M. Scudder (1989) tion of specimens during his postdoctoral Development and hormone regulation of androgen re- tenure at the University of Colorado at Boul- ceptor levels in the sexually dimorphic larynx of Xeno- der. Gary Ten Eyck of The University of pus laeuis. Dev. Biol. 131:lll-118. Kansas at Lawrence prepared the serial cross Kemp, N.E., and J.A. Hoyt (1969) Sequence of ossification in the skeleton of growing and metamorphosing sections of the heads of larvalxenopus laevis. Rana pipiens. J. Morphol. 129:415-444. This research was supported by NSF grants Kotthaus, A. (1933) Die Entwicklung des Primordial- BSR 85-08470 and DCB 90-19624 and NIH Craniums von Xenopus laevis bis zur Metamorphose. grant DE 05610. Z. Wissensch. Zool.144:510-572. Nieuwkoop, P.D., and J. Faber (1956) Normal Table of LITERATURE CITED Xenopus laevis (Daudin). A Systematical and Chrono- logical Survey of the Development from the Fertilized Baldauf, R.J. (1958) A procedure for the staining and Egg till the End of Metamorphosis.Amsterdam: North- sectioning of the heads of adult anurans. Texas J. Sci. Holland Publ. Co. 10t448-451. Parker, W.K. (1876) On the structure and development Bernasconi, A.F. (1951) Uber den Ossifikationsmodus of the skull in the Batrachia. Part 11. Proc. Zool. Soc. bei Xenopus laevis Daud. Mem. SOC.Helvet. Sci. Nat. London 24:601469. 79r191-252 + 2 pls. Parker, W.K. (1881) On the structure and development Brown, S.M. (1980) Comparative Ossification in Tad- of the skull in the Batrachia. Part 111. Philos. Trans. poles of the Genus Xenopus (Anura, Pipidae). Master's Roy. SOC.Lond. [Biol.] 1:l-266 + 44 pls. thesis. San Diego State University, San Diego, CA. Paterson, N. (1939) The head of Xenopus laevis. Q. J. Cannatella, D.C. (1985) A Phylogeny of Primitive Frogs Microsc. Sci. 81:161-234. (Archaeobatrachians). 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APPENDIX A. Schedule of ossification of cranial elements in Xenopus laevis' Developmental stapes (Nieuwkoop and Faber, '56) Bone 54 55 56 57 58 59 60 61 62 63 64 65 66

~ ~~ ~ ~ Frontoparietal ll21/7+ + + + + + + + + + + Parasphenoid 717 + + + + + + + + i i + Exoccipital 4/76/6+ + + + i + + + + + Prootic 417 516 515 + + + i + i + + + Angulosplenial (medial) 516515+ + + + + + i + i Maxilla 515 + + + + + + + + + Premaxilla 415 515 + + + + + + + Nasal 315 415 515 + + + i i + Septomaxilla 5/5+ + + i + + Teeth 515+++++i Dentary 215 315 515 + i i + Anguiosplenial (lateral) 215 515 + + + i + Pterygoid 215 515 + + i + Tympanic annulus 315 515 + + + + Pars externa plectri 315 515 + + + + Pars media plectri 415 515 i + i Squamosal 115 515 i + i Vomer 415 515 + i Sphenethmoid 415 215 515 Pars articularis 515

'The number to the left of solidus indicates the number of individuals in which the element is ossified; the number to the right is the total number of specimens examined in that stage. A "+" indicates that the bone was present in all indwiduals examined.

APPENDIX B. Schedule ofaxial skeletogenesis in Xenopus laevis'

~~ ____ Developmental stages (Nieuwkoop and Faber, '56) Element 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 Neural Arch I Cartilage 22 + + + + + i + + Bone 411 516 515 i + + i i i i + i Neural Arch I1 Cartilage 212+ i i + + i + + Bone 417 516 515 + i + i + + + + i Neural Arch I11 Cartilage 212+ + i + i + + + Bone 411 516 515 i i i i + + + + + Neural Arch IV Cartilage 1/2+ + + + + + + + Bone 317 516 515 + i i i + i i + + Neural Arch V Cartilage 112 112 212 i + + + + + Bone 311 516 515 + + + + i i + + i Neural Arch VI Cartilage 112 112 212 i + + i + i Bone 217 416 515 + + i i i i i + i Neural Arch VII Cartilage 112 112 112 212 + + + + + Bone 217 316 515 i i + i i i + + Neural Arch VIlI Cartilage 112 - 112 112 212 i + i Bone 111 116 5/51 i i i i i i + + Neural Arch IX Cartilage 112 112 212 i 617 + Bone 5/5+ i i i + i i + Neural Arch X Cartilage 212 + + + i Bone 115 515 i + -t i- i i i Neural Arch XI Cartilage 112 112 216 116 ------115 Bone 115 415 415 415 415 515 + Neural Arch XI1 Cartilage 315 115 Bone 115 315 515 + + (continued) 38 L. TRUEB AND J. HANKEN APPENDIX 3. Schedule of axial skeletogenesis in Xenopus laevis' (continued) Developmental stages (Nieuwkoop and Faber, '56) Element 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 Centrum I Cartilage 212 + t t Bone 416 516 515 + ++++++ + Centrum I1 Cartilage 212 116 1/6 Bone 216 416 515 + ++++++ + Centrum I11 Cartilage 112 116 116 Bone 116 216 515 + ++++++ + Centrum IV Cartilage 112 116 - Bone 116 - 515 + ++++++ + Centrum V Cartilage 112 - - Bone 515 + ++++++ + Centrum VI Cartilage 112 - - Bone 515 + ++++++ + Centrum VII Cartilage Bone 515 + ++++++ + Centrum VIII Cartilage Bone 515 i ++++++ + Centrum IX Cartilage Bone 415 515 ++++++ + Hypochord Cartilage 115 215 Bone 115 315 215 515 t + Fused Urostyle Cartilage Bone 515 Fused Rib PSV I1 Cartilage 215 + + Bone 315 415 515t + + + + t Rib PSV 111 Cartilage 315 Bone 515 + ++++++ t Rib PSV IV Cartilage 215 + + Bone 315 415 5/5+ + + t + + TP PSV I1 Cartilage 515 + + Bone 515 + + TP PSV Ill Cartilage 515 + + Bone 515 + + TP PSV IV Cartilage 515 + + Bone 515 + + TP PSV V Cartilage 115 + + Bone 215 415 515 TP PSV VI Cartilage 115 + + Bone 215 415 515 TP PSV VII Cartilage 115 + + Bone 315 415 515 TP PSV VIII Cartilage 115 + Bone 415 515 + Sacral diapophysis Cartilage 215 Bone 515 + + + 'The number to the left of the solidus indicates the number of individuals in which the element is either present in cartilage or hone; the number to the right is the total number of specimens examined in that stage. A "+ " indicates that either bone or cartilage wa8 present in all individualsexamined, whereas a "-"indicates absence. PSV, presacral vertebra: TP, transverseprocess. SKELETAL DEVELOPMENT IN XENOPUS LAEVZS 39

APPENDIX C. Pectoral girdle and forelimb skeletogenesis in Xenopus laevis' Developmental stages (Nieuwkoop and Faber, '56) Element 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 Coracoid Cartilage 515 + + Bone 315 415 515 + i i + ++ Scapula Cartilage 416 515 + + + + Bone 115 215 515 i i ++ Clavicle 415 515 + i t + + ++ Epicoracoid and Procoracoid 415 515 t 415 515 + f i ++ Sternum 315 515 ?4/5 515 + Suprascapula 515t + + i + t i ++ Cleithrum 315 415 515 + + i + ++ Humerus Cartilage 2122 i i i ? ?112 ? ?617 + 215 Bone 315 515 + f + + + + ++ Radioulna Cartilage ? ? ? ?617 616 215 Bone 315 515 + f i + + + ++ Ulnare Cartilage ? ? ??5/1616+ + + f + + + + ++ Bone Postaxial centrale Cartilage ? ? ? ?2/1 516 515 + + + + + f t ++ Bone Preaxial centrale Cartilage ? ? ? ?1/7 516 515 + + + + f + i ++ Bone Distal Carpal I Cartilage ? 116 515 + i + i + + t ++ Bone Distal Carpal I1 Cartilage ? 1117 216 515 i t i- + i + I ++ Bone Distal Carpal 111 Cartilage ? ?217 516 515 i i + i + f + ++ Bone Metacarpal I Cartilage ? ?I17 416 515 + i +i Bone 115 215 515 415 515 + + ++ Metacarpal I1 Cartilage ? ?3/7 616 + i Bone 315 515 + i + i + ++ Metacarpal 111 Cartilage ? ?3/1 616 + + Bone 315 515 + + i + + i+ Metacarpal IV Cartilage ? ?3/7 616 + + Bone 315 515 + + + i i i+ Phalanx 1-1 5/5+ + + i + 215 115 215 515 + ++

Cartilage 515i + + + + i i ++ Bone 315 415 415 Phalanx 11-1 Cartilage 515 i + + + Bone 315 315 515 i + ++ Phalanx 11-2 Cartilage 515i + i i i i + Bone 315 315 515 + Phalanx 111-1 Cartilage ? 116 515 i + + + + Bone 315 315 415 515 + + + (continued) 40 L. TRUEB AND J. HANKEN APPENDIX C. Pectoral girdle and forelimb skeletogenesis in Xenopus laevis' (continued) Developmental stages (Nieuwkoop and Faber, '56) Element 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 Phalanx 111-2 Cartilage 5/5+ i + i + i Bone 315 115 315 515 + i Phalanx 111-3 Cartilage 515i+++it i ++ Bone 115 215 215 415 Phalanx IV-1 Cartilage ? 116 515 + + + + + Bone 415 315 315 515 t + I Phalanx IV-2 Cartilage 515i + + + i + Bone 115 115 315 515 + i Phalanx IV-3 Cartilage 515++i+i i i + i Bone 115 215 215 415 Sesamoids 415 515 i + i

'The number to the left of the solidus indicates the number of individuals in which the element is either present in cartilage or bone; the number to the right is the total number of specimens examined in that stage. A " + " indicates that either bone or cartilage was present in all individuals examined. A "?" indicates that some or all of the specimens were damaged in preparation such that elements that might be expected to be present could not be documented. 2Limbbud.

APPENDIX D. Pelvic girdle and hind-limb skeletogenesis in Xenopus laevisl Developmental stages (Nieuwkoop and Faber, '56) Element 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 Ilium Cartilage 217 616 + Bone 1/55/5+ + + i + + + + Ischium Cartilage 215 i i Bone 115 215 215 515 i f + Pubis Cartilage 215 415 515 + + + Bone 515 Epipubis 115 215 415 515 t + + Femur Cartilage 2/22 + f i ?112 ? ?I12 717 + f Bone 415515i + + + i i + i Tibia and fibula Cartilage ? ? ?1/2 717 i Bone 5/5+iiiit t ii Tibiale and fibulare Cartilage ? ? ?I12 717 i + Bone 1/55/5+ + + + + t i + Naviculare Cartilage ? ?1/2717+ + + + i i + i t + + Bone Tarsal I Cartilage 111616+ i i i + i i + + + Bone Tarsal I1 Cartilage ? 317616 + t + + + + i f i i Bone Prehallux Cartilage 416515f i i i t i + t i Bone Metatarsal I Cartilage ? ? 417 616 + Bone 115515i + i i i i i i Metatarsal I1 Cartilage ? ?I12 717 i + Bone 115515i + i i i t t + (continued) SKELETAL DEVELOPMENT IN XENOPUS LAEVIS 41 APPENDIX D. Pelvic girdle and hind-limb skeletogenesis in Xenopus laevis' (continued) Developmental stages (Nieuwkoop and Faber, '56) Element 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 Metatarsal I11 Cartilage ? ?l/2 711 i i Bone 115515i i i f + i i + Metatarsal IV Cartilage ? ?I12 711 f f Bone 115515i f i + + i f + Metatarsal V Cartilage ? ?112 717 516 + Bone 115515+ + i i i i f t Phalanx 1-1 Cartilage 216 515 + + Bone 215 515 + i i f f i Phalanx 1-2 Cartilage 515 i f Bone 215 515 i i f i i i Phalanx 11-1 Cartilage 117 516 515 i + Bone 415 415 515 i i + i i + Phalanx 11-2 Cartilage 216 515 i + Bone 315 415 515 i i i i i i Phalanx 111-1 Cartilage 317 616 f i Bone 415 515 i i i i + + + Phalanx 111-2 Cartilage 416 515 i i Bone 215 4/51515 i i i i i i Phalanx 111-3 Cartilage 216 515 + + + Bone 115 215 515 415 515 i f f i Phalanx IV-1 Cartilage 317 616 i i Bone 315 515 + i + f + + + Phalanx IV-2 Cartilage 416 515 i i Bone 215 415 515 i f i i f i Phalanx N-3 Cartilage 116 515 + + i Bone 115 515 415 515 f i i i Phalanx N-4 Cartilage 515f f f i f f i Bone 115 115 115 415 315 515 i Phalanx V-1 Cartilage 117 516 515 i f Bone 215 415 515 i f + i f i Phalanx V-2 Cartilage 216 515 i i + Bone 115 515 415 515 i i i i Phalanx V-3 Cartilage 515i i i + i i i Bone 115 115 015 315 415 515 +

IThe number to the left of the solidus indicates the number of individuals in which the element is either present in cartilage or bone; the number to the right is the total number of speeimens examined in that stage. A "+" indicates that either bone or cartilage was present in all individuals examined. A "?" indicates that some or all ofthe specimens were damaged in preparation such that elements that might be expected to be present could not be documented. Separate entries for chondrification of parts of pelvic girdle are noted to indicate when cartilage elements were distinct from one another. $Limbbud.