Cranial Neural-Crest Migration and Chondrogenic Fate In

Home , Chondrocranium, Trabecular cartilage

JOURNAL OF MORPHOLOGY 229:105-120 (1996)

Cranial Neural-Crest Migration and Chondrogenic Fate in the Oriental Fire-Bellied Toad Bombina orientalis: Defining the Ancestral Pattern of Head Development in Anuran Amphibians

LENNART OLSSON AND JAMES HANKEN Department of Environmental, Population, and Organismic Biology, University of Colorado, Boulder, Colorado 80309-0334 (L.O., J.H.); Department of Environmental and Developmental Biology, Uppsala University, S-752 36 Uppsala, Sweden (L.0.) ABSTRACT We assess cranial neural-crest cell migration and contributions to the larval chondrocranium in the phylogenetically basal and morphologically generalized anuran Bombina orientalis (Bombinatoridae). Methods used include microdissection, scanning electron microscopy, and vital dye labeling, in conjunction with confocal and fluorescence microscopy. Cranial neural-crest cells begin migrating before neural-fold closure and soon form three primary streams. These streams contribute to all cranial cartilages except two medial components of the hyobranchial skeleton (basihyal and basibranchial cartilages), the posterior portion of the trabecular plate, and the otic capsule, the embryonic origin of which is unknown. Chondrogenic fate is regionalized within the cranial neural folds, with the anterior regions contributing to anterior cartilages and the posterior regions to posterior cartilages. A neural- crest contribution also was consistently observed in several cranial nerves and the connective tissue component of many cranial muscles. Notwithstanding minor differences among species in the initial configuration of migratory streams, cranial neural-crest migration and chondrogenic potential in metamorphosing anurans seem to be highly stereotyped and evolutionarily conserva- tive. This includes a primary role for the neural crest in the evolutionary origin of the paired suprarostral and infrarostral cartilages, two prominent caenogenetic features of the rostral skull unique to anuran larvae. Our results provide a model of the ancestral pattern of embryonic head development in anuran amphibians. This model can serve as a basis for examining the ontogenetic mechanisms that underlie the diversity of cranial morphology and development displayed by living frogs, as well as the evolutionary consequences of this diversity. o 1996 wiley-Liss, Inc.

Evolutionary developmental biology is rigorous phylogenetic context, including ex- emerging as a distinct field of research, ask- plicit hypotheses of phylogenetic relationship ing such questions as “How do developmen- and the identification of primitive vs. derived tal processes and mechanisms evolve?” and character states (e.g., Northcutt, ’90; Collazo “To what extent does development bias or and Marks, ’94; Patel, ’94; Wake and Han- constrain phenotypic evolution?” Although ken, ’96). Typically, such analyses require the intellectual roots of the field are ancient, the consideration of “non-model” organ- it has experienced a renaissance in the last isms, because the model systems of develop- 15-20 years (Gould, ’77; Bonner, ’82, ’88; mental biology are a biased sample of organ- Goodwin et al., ’83; Thomson, ’88; Muller, isms generally not chosen with evolutionary ’91; Hall, ’92). Accompanying this rebirth questions in mind (Hanken, ’93; Bolker, ’95). and maturation is the widespread recogni- tion that, as in any field of comparative biology, analyses of evolutionary pattern and Address reprint requests to Dr. James Hanken, Department of process are conducted most effectively in a EPO Biology, University of Colorado, Boulder, CO 80309-0334.

D 1996 WILEY-LISS, INC. 106 L. OLSSON AND J. HANKEN The primary focus of our research is the this species is highly derived phylogenetically development and evolution of the head in (Cannatella and de Sa, '93) and has a very anuran amphibians. We are particularly inter- specialized skull morphology in both the larva ested in the unique problems regarding and the adult (Trueb and Hanken, '92); it is mechanisms of cranial pattern formation an inappropriate baseline for evolutionary posed by the group's ancestral, biphasic on- comparisons among anurans or between frogs togeny (which involves, for instance, morpho- and other vertebrates (Cannatella and Trueb, logically distinct skulls in larvae and adults), '88; Cannatella and de Sa, '93). And while as well as the effects of the evolution of important developmental parameters such derived life histories, such as direct develop- as cranial neural-crest biology have been stud- ment, on cranial patterning (Hanken, '92, ied in many other species of anurans (Table '93; Hanken et al., '92; Hanken and Thoro- 11, nearly all of these studies predate the good, '93). Examination of these topics has application of modern, improved tools to been hampered by the lack of comprehensive study cell migration and lineage, such as baseline data regarding the presumed ances- scanning electron microscopy (SEMI and fluo- tral pattern of head development in frogs. rescent cell labels (e.g., Collazo et al., '93). For example, while there is considerable, de- In this paper, we 1) document the pattern tailed knowledge about cranial development and timing of cranial neural-crest emergence in the pipid frog Xenopus laeuis (Table l), and migration during embryogenesis, and

TABLE 1. Published studies of cranial neural-crest biology in anuran amphibians' Species Analytical method(s) Reference Bombinatoridae Bombina orientalis Histology of normal development, SEM, This study vital labeling B. pachypus Homospecific and heterospecific grafts Andres, '46, '49; Wagner, '48, '49, '55, '59; Baltzer, '50, '52; Chen and Baltzer, '54; Henzen, '57 B. variegata Organ culture of tissue explants Petricioni, '64 Discoglossidae Discoglossus pictus Homospecific grafts Fagone, '59; Cusimano et al., '62; Cusi- mano-Carollo, '63, '69, '72 Alytes obstetricans Histology of normal development Reisinger, '33 Pipidae Xenopus laevis Histology of normal development Nieuwkoop and Faber, '56 Ablation, homospecific grafts Seufert and Hall, '90 SEM, heterospecific grafts Sadaghiani and ThiBbaud, '87 Heterospecific grafts Wagner, '49 In situ hybridization Bradley et al., '92; Dirksen et al., '93; Essex et al., '93; Papalopulu and Kintner, '93; Ho et al., '94; Winning and Sargent, '94; Brandli and Kirschner, '95 In situ hybridization, immunocytochem- Song and Slack, '94 istry Lectin and thiodigalactoside treatment Varma et al., '94 Bufonidae Bufo uiridis Homospecificgrafts Raunich, '57 Leptodactylidae Eleutherodactylus coqui SEM Moury and Hanken, '95 Ranidae Rana sp. Histology of normal development Corning, 1899 R. esculenta Homospecific grafts, organ culture of tissue Raunich, '57, '58 explants R. fusca Heterospecific grafts Raven. '33 R. japonica Histology of normal development, ablation, Ichikawa, '35, '37; Okada and Ichikawa, organ culture of tissue explants, '56; Okada, '57 homospecific and heterospecific grafts R. palustris Histology of normal development, ablation, Stone, '22, '27, '29, '32 vital labeling, homospecific grafts R. pipiens Histology of normal development JSnouff, '27 R. temporaria Histology of normal development, ablation, Lundborg, 1899; Reisinger, '33 homospecific grafts

*Only those studies are listed that provide data on patterns of crest emergence, migration, or gene expression, or on cranial skeletal derivatives and differentiation. Species are listed according to family (Ford and Cannatella, '93). CRANIAL NEURAL-CREST MIGRATIONiFATE IN ANURANS 107 2) assess the contribution of neural crest to ization to metamorphosed froglet. In B. orien- the larval chondrocranium in the Oriental talis, larvae hatch at around stage 22 (Han- fire-bellied toad Bombina orientalis. This is a ken, personal observations). phylogenetically basal and morphologically generalized species that is among the most SEM appropriate living taxa for inferring ances- At least eight embryos each of stages 14 tral features of anurans (Cannatella and de (neural plate) to 19 (beginning of heart beat) Sa, '93). Previous studies of this, and closely were prepared for SEM. Embryos were dejel- related, species have documented cranial mor- lied either chemically (0.63 g cysteine HC1, phology in larvae and adults, as well as de- 0.12 g NaC1, 24 ml HzO, buffered to pH 8.0 tails of cranial embryogenesis and metamor- with 5 N NaOH) or manually with watchmak- phosis (Stadtmiiller, '31; Ramaswami, '42; er's forceps; they were decapsulated manu- Slabbert, '45;Sokol, '81; Hanken and Hall, ally and fixed at 4°C in modified Karnovsky '84, '88; Hanken and Summers, '88; Hanken fixative (1.5% glutaraldehydell.5% parafor- et al., '89; Smirnov, '89; Viertel, '91). Here maldehyde in 0.1 M cacodylate buffer; Karno- we focus on the neural crest because of its vsky, '65) overnight or longer. The embryos prominent role in head development in verte- were transferred to ice-cold 0.1 M phosphate brates generally, including amphibians (re- buffer (PB), where the cranial epidermis was viewed in Hall and Horstadius, '88), and be- carefully removed with tungsten needles. Be- cause of its likely role in mediating many cause neural-crest cells are dark and stand aspects of the evolution of cranial patterning out against the lighter background of adja- (Hunt et al., '91; Hanken and Thorogood, cent tissues in such preparations, camera- '93; Langille and Hall, '93). Our analysis is lucida drawings and photomicrographs were based on a variety of techniques, including made at this stage as an aid to identifying microdissection, SEM, and vital labeling. neural crest later using SEM. Specimens then While Bombina has figured prominently in were postfixed in 1% OsO4 in cacodylate many classical studies of amphibian neural- buffer for 1 h. After thorough rinsing in PB, crest biology (Table l), neither patterns of specimens were dehydrated in a graded series cranial crest migration nor crest contribu- of ethanol and transferred to liquid COz in a tions to the entire larval skull have been critical-point dryer. Dried specimens were reported for any species. Our primary aim is mounted, sputtercoated with gold/palladium, to derive a model of the ancestral pattern of and examined in a Philips CM 10 scanning head development in anuran amphibians. electron microscope at 10 kV. Such a model can serve as a basis for examining the ontogenetic mechanisms that under- Vital labeling lie the pronounced diversity of cranial morphology and development displayed by living Eighty-two live embryos were labeled with taxa, as well as the evolutionary conse- vital dye at stage 14 (neural plate; Fig. l), quences of this diversity. immediately before the onset of cranial neural-crest migration. After being dejellied and MATERIALS AND METHODS decapsulated, embryos were immobilized in Embryos shallow trenches cut into 2% agar gelled in Eggs were obtained from laboratory mat- the bottom of Petri dishes. A 0.5% stock ings among wild-caught adults (Charles D. solution of the lipophilic dye DiI (1,l'-diocta- Sullivan Co., Inc., Nashville, TN) that are decyl-3,3,3', 3 '-tetramethylindocarbocya- maintained as a breeding colony at the Uni- nine, perchlorate; Molecular Probes D-282, versity of Colorado. Methods for breeding Eugene, OR) was prepared in 100% ethanol and husbandry followed established proce- and stored at 4°C. Immediately before injec- dures (Carlson and Ellinger, '80; Frost, '82). tion, it was diluted in 0.3 M sucrose to work- Adults were injected subcutaneously with hu- ing concentrations of 0.1 and 0.05%. Micro- man chorionic gonadotropin (Sigma Chemi- pipets pulled from thin-walled, 1.2 mm cal Co., St. Louis, MO) and allowed to spawn diameter glass microfilaments were filled with overnight in the dark at room temperature. dye and attached to a Picospritzer I1 (Gen- Fertilized eggs were reared in 10% Holt- eral Valve Corp., Fairfield, NJ). Micropipet freter's solution at 10-25°C. Embryos and tips were broken to a diameter of about 20 tadpoles were staged from external morphol- Fm. DiI was injected into one of six different ogy according to the scheme of Gosner ('601, sites in the left neural fold (Fig. 1) by insert- which defines a total of 46 stages from fertil- ing a micropipet and expelling a small amount 108 L. OLSSON AN ID J. HANKEN 1 tographed using a Wild MPS55 photoau- tomat and Kodak T-h4AX P3200 or Ekta- chrome P1600 film, or digital images were recorded with a Panasonic WV-CL350 video camera and processed using Adobe Photo- shop 3.0 on an Apple Macintosh 7100 com- puter. Photographic prints were produced with a Polaroid Palette on Kodak Ekta- chrome Elite 100 film. Fifty-nine labeled tadpoles (stage 26-hind- limb bud appears) were prepared as cryostat sections. The specimens were killed in 30% aqueous chloretone (1,1,l-trichloro-2-methyl- 2-propanol; Sigma T-5138) and fixed overnight or longer in 4% paraformaldehydel 0.25% glutaraldehyde in PB at 4°C. They were then washed thoroughly in PB, soaked in 15% sucrose solution overnight, transferred through two changes of a 15% sucrose/ 7.5% gelatin solution at 37°C for a total of at least 5 h, and embedded in fresh sucrose1 gelatin solution at 4°C. Once set, the specimens were rapidly frozen in liquid nitrogen and stored at -40°C. Approximately 20 pm Fig. 1. Stage 14 (neural plate) embryo of Bombzna orientalis. The left side depicts the six regions of the sections were cut using an International Har- cranial neural fold that were injected with DiI. The right ris cryostat (International Equipment Co., side depicts the approximate origins of the neural-crest Needham Heights, MA). Sections were viewed cells that contribute to the three cranial migratory streams-mandibular (M), hyoid (HI, and branchial (B). unmounted using fluorescence microscopy Dorsal view, anterior at the top. and documented as described above.

Confocal microscopy of dye solution; the right side was not injected and served as a control. Injections were Twenty-three living, labeled embryos made by hand or with a micromanipulator. (stages 15-20) were mounted in agar on brass The total of six injection sites represents the slides and scanned with an inverted, laser- maximum number of discrete regions of the scanning confocal microscope (Molecular Dy- cranial neural fold that could be injected with namics, Sunnyvale, CA). Images were re- both a high degree of repeatability among corded and processed with a 3000 Silicon embryos and a minimum of overlap with Graphics Iris workstation using Imagespace adjacent sites. All neural-crest and presump- software (Molecular Dynamics). They were tive neural-tube cells within each site ap- stored on a Sony Magneto-optical disk and peared to be labeled following injection. photographed with Screenstar (Presentation Technologies, Sunnyvale, CA) on Kodak Ekta- Fluorescence microscopy of whole embryos chrome Elite 100 film. and cryostat sections Injected embryos were reared in 10% Holt- RESULTS freter's solution containing 50 mgll gentami- Cranial neural-crest cell migration cin sulfate (Sigma G-1264). They were maintained individually in 24-well tissue culture Cranial neural-crest cell emergence and plates at 25°C. For fluorescence imaging, liv- migration begin early in stage 15, before the ing embryos were temporarily mounted in neural folds fuse; most cells have begun mi- rectangles cut into agar gelled at the bottom grating away from the neural tube by the end of custom-made brass slides with coverslip of that stage. At least initially, the cells mi- floors (provided by M. Klymkovsky, Univer- grate in dense streams. Two streams form sity of Colorado) and observed with a Leitz first (Fig. 2A,B). The mandibular (or rostral) Dialux 20 epifluorescence microscope with a stream originates at the level of the prospec- rhodamine (N2) filter block. They were pho- tive mesencephalon and migrates anteriorly CRANIAL NEURAL-CREST MIGRATIONiFATE IN ANURANS 109 toward the optic vesicle. A second, posterior Neural-crest contribution to the larval skull stream emerges soon after at the level of the Contributions of DiI-injected cranial neu- prospective rhombencephalon; it is separated ral-crest cells to the cartilaginous larval skull from the mandibular stream by a narrow, were determined from cryosectioned tad- crest-free zone (Fig. 2B). poles at stage 26. Only a small portion of the At stage 16, the mandibular stream is mi- skull was labeled in any given embryo; re- grating rostrally around the optic vesicle. sults from all embryos were combined to give Most cells migrate along a ventral pathway, a composite image of the extent of crest con- but some follow a dorsal route (Figs. 3A,B, tribution to each individual cartilage (Table 4A,B). The second stream divides into an 2, Fig. 5). anterior hyoid (rostral otic) stream and a Cells from all six injection sites except site posterior branchial (caudal otic) stream (Fig. 1 (transverse neural folds) contributed to 2C). Later in stage 16, the branchial stream cranial cartilages. Cells derived from the man- further subdivides into two parallel cell dibular stream (sites 2 and 3) were found in masses (Figs. 2D, 3C,D). the upper and lower jaws (suprarostral, infrarostral, and Meckel’s cartilages; Fig. 6A-C), At stage 17, the mandibular stream com- the jaw suspensorium (palatoquadrate carti- pletely surrounds the optic vesicle. The hyoid lage; Fig. 6E), and the rostral portion of the stream migrates ventrally faster than either neurocranium (trabecular horns; Fig. 6D). cell mass of the branchial stream and over- The hyoid stream (sites 3-5) contributed to takes them (cf. Figs. 2D,E, 3E, 40. Even at the ceratohyal cartilage, a prominent, antero- this relatively late stage, some branchial ventral component of the hyobranchial (gill) stream cells are present within the zone that skeleton, and to the anterior portion of the separates its two cell masses (Fig. 3F). trabecular plate, which constitutes the floor By stage 18, the mandibular stream sur- of the neurocranium ventral to the brain rounds and extends rostral to the optic cup. (Fig. 6F,G). The branchial stream contrib- The hyoid stream has migrated farther ven- uted to nearly all of the remainder of the trally and nearly reaches the anlagen of the hyobranchial skeleton, with cells from the cement glands (Figs. 2E, 4C). After further anterior cell mass (site 5) contributing to the subdivision, the branchial stream comprises first ceratobranchial cartilage (CB I) and four cell masses, each within a different bran- those from the posterior cell masses (site 6) chial arch (Fig. 2F). After this stage, when contributing to CB 11-IV (Figs. 6H, 7). DiI- cranial neural-crest cells begin to disperse labeled neural crest from posterior portions from within their respective streams into the of site 6 did not contribute to any cranial adjacent mesoderm, their migration and fates cartilages. There is no evidence of a cranial neural- no longer can be followed by dissection and crest contribution to either the basihyal or SEM. With confocal microscopy, labeled neu- the basibranchial (two median, ventral carti- ral-crest cells are seen to migrate into the lages in the hyobranchial skeleton), to the transient epidermal (external) gills that ex- posterior portion of the trabecular plate, or tend from branchial arches 1and 2 (Fig. 4D). to any portion of the cartilaginous otic cap- Relative contributions of the six different sule. The boundary between crest and non- injection sites (Fig. 1) to each cranial migra- crest-derived portions of the trabecular plate tory stream were determined from DiI-la- lies approximately at the level of the ascend- beled cells in living embryos examined using ingprocess of the palatoquadrate (Fig. 5). confocal and conventional fluorescence microscopy (Table 2). The mandibular stream Other neural-crest derivatives was derived from cells originating within in- The presence of DiI-labeled cells was noted jection sites 2 and 3. The hyoid stream was in several cranial tissues in addition to carti- derived almost exclusively from site 4; addi- lage, although these additional tissues were tional, minor contributions from sites 3 and not surveyed comprehensively (data not 5 were observed in a few embryos. The bran- shown). Labeling was consistently observed chial stream was derived from sites 5 and 6; in cranial nerves V, VII, IX, and X; in the site 5 contributed to the most anterior cell connective tissue (non-contractile) elements mass and site 6 to the posterior masses. No of cranial muscles (especially the complex labeled cells from injection site 1 contributed jaw abductor and adductors, e.g., orbitohyoi- to any migratory stream. deus); in specific portions of the eye (cornea, 110 L. OLSSON AND J. HANKEN

Fig. 2. Cranial neural-crest migration in Bornbzna anterior (hyoid, H)and posterior (branchial, B) streams. orientalis. Preserved embryos are photographed in lat- Scale = 0.25 mm. D: Early stage 17. The branchial stream eral view, anterior to the left, with the cranial epidermis is further subdivided into two distinct cell masses. removed to reveal the underlying migratory streams. A Scale = 0.25 mm. E: Late stage 17. All three streams Early in stage 15, crest cells migrate within anterior have migrated further ventrally. Scale = 0.25 mm. F: (mandibular, M) and posterior (arrow) streams. Stage 18. The branchial stream comprises four parallel Scale = 0.2 mm. B: Later in stage 15, these two streams cell masses. Boundaries of all cranial streams are less are separated by a crest-free zone (arrow). Scale = 0.5 distinct as neural-crest cells begin to disperse within the mm. C: Stage 16. The posterior stream has divided into head. Scale = 0.25 mm. CRANIAL NEURAL-CREST MIGRATIONiFATE IN ANURANS 111

Fig. 3. Scanning electron micrographs of embryonic (H), and branchial streams (B) are all prominent. Bombina orientalis depicting migratory streams of cra- Scale = 0.2 mm. D: Close-up of inset in C. The branchial nial neural crest. All are lateral views (anterior to the stream is subdividing to form anterior and posterior cell left) unless indicated otherwise; overlying epidermis has masses; crest cells in the separation zone (arrowheads) been removed. A: Anterolateral view of the head at stage are moving to either side. Scale = 0.1 mm. E: Stage 17. 16. Crest cells within the mandibular stream migrate Mandibular stream, hyoid stream, and two cell masses of rostrally around the optic vesicle (OV) via either dorsal or the branchial stream (B) are distinct. Scale = 0.2 mm. F: ventral pathways (arrowheads). Scale = 0.2 mm. B: Close-up of inset in E. A few crest cells remaining in the Close-up of inset in A showing crest cells migrating separation zone within the branchial stream (arrow- around optic vesicle via dorsal pathway (arrowhead). heads) are moving horizontally to join either cell mass. Scale = 0.05 mm. C: Stage 16. Mandibular (MI, hyoid Scale = 0.05 mm. 112 L. OLSSON AND J. HANKEN

Fig. 4. Confocal microscopic images of heads of living deeper level. C: Stage 17 embryo injected at site 4. Most embryos of Bombina orientalis, showing migration of labeled crest cells are within the hyoid stream (H); a few cranial neural-crest cells labeled with DiI at stage 14. are within the anterior portion of the branchial stream Lateral views, anterior to the left. A Stage 16 embryo (B). CG, cement gland. D Stage 19 embryo injected at injected at site 2 (black arrow). Cells in the mandibular site 6. Labeled crest cells derived from the anterior two stream that are close to the epidermis migrate dorsally cell masses of the branchial stream are populating the (left arrow) and ventrally (right arrow) around the optic transient epidermal (external) gills CViertel, '91) on bran- vesicle (OV).B: In the same embryo at the same time, chid arches 1 and 2. Scales = 0.05 mm. only cells in the thicker, ventral pathway migrate at a choroid, and sclera); and in undifferentiated have begun to migrate away from the develop- head mesenchyme. These results will be pre- ing neural tube by the end of this stage. The sented in a future paper. cells initially migrate in two streams (Fig. 2A,B), although the posterior stream soon DISCUSSION divides in two (stage 16; Fig. 2C). The result- Defining the ancestral pattern of head ing three streams are the mandibular (or development in anurans rostral), which supplies the first visceral arch; Basic features of cranial neural-crest cell the hyoid (rostral otic), which supplies the emergence and migration in Bombina orien- second arch; and the branchial (caudal otic), talis may be summarized as follows. Crest which comprises a series of parallel cell cell emergence begins early in stage 15, be- masses that supply arches 3-6. These three fore neural-fold closure, and most crest cells streams or their component cell masses can CRANIAL NEURAL-CREST MIGRATIONiFATE IN ANURANS 113 TAJ3LE2. Derivation of cranial neural-crest streams tions of the cranial neural folds are chondro- and their contribution to larval cranial cartilages in B. orientalis' genic exceptfor the most anterior (trans- - verse) region (Fig. 1, site 1). The posterior Neural-crest boundary between cranial (chondrogenic)and stream Injection site Cartilage trunk (non-chondrogenic) neural crest lies - 1 - within the caudal portion of site 6. Skeleto- Mandibular 233 (Part) Suprarostral, infra- genic fate is at least broadly regionalized rostral, Meckel's, palatoquadrate, within the neural folds, with the anterior trabecular horns regions ke., mandibular stream) contribut- Hyoid 3 (part), 4, Anterior trabecular ing to anterior cartilages and the posterior 5 (part) plates ceratohyal regions (branchial stream) to posterior carti- Branchial Anterior mass 5 (part) Ceratobranchi&I lages (Fig. 1, Table 2). Because we labeled Posterior masses 6 Ceratobranchials premigratory neural-crest Cells as groups 11-IV rather than individuallv. we cannot assess 'Locations of injection sites 1 (anterior) to 6 (posterior)within the segment restrictions as" finely as has been cranial neural folds are depicted in Figure 1. done for some other vertebrates. In the zebrafish, for example, segment restrictions within premigratory branchial crest are, at be recognized as discrete entities as late as least initially, much less precise than those of stage 18, when the individual cells begin to more anterior crest; individual crest-cell pro- disperse throughout the head. genitors of branchial segments can contrib- Cranial neural crest is the predominant ute to as many as three adjacent arches embryonic source of progenitor cells for the (Schilling and Kimmel, '94). Our data, how- larval chondrocranium, which is first visible ever, are at least consistent with this observa- at stage 20. Indeed, the only larval cranial tion; thus, the same phenomenon may apply cartilages not derived from neural crest are to anurans. Our data also do not allow us to two medial components of the hyobranchial assess neural-crest contributions to the osteo- skeleton (basihyal and basibranchial carti- cranium, which is first visible at stage 31 but lages), the posterior portion of the trabecular not fully formed until after metamorphosis plate, and the otic capsule (Fig. 5). All por- (stage 46; Hanken and Hall, '88), well after

SH -I MC - BH - a

=Mandibular Stream Hyoid Stream Branchial Streams

Fig. 5. Neural-crest contribution to the larval skull in horn); IR, infrarostral; MC, Meckel's cartilage; OC, otic Bombina orientalis. Crest-derivedcomponents are shaded capsule; PQ, palatoquadrate; SR, suprarostral; TP, tra- according to migratory stream (see also Figs. 1, 7); non- becular plate. Skulls (stage 36) are redrawn from Hanken crest-derived components are gray. Cartilage abbrevia- and Summers ('88) and depicted in dorsal (left) and tions: BB, hasibranchid, BH, hasihyal; CB, ceratobranchi- ventral views. als I-IV CH, ceratohyal; CT, cornua trabecula (trabecular Fig. 6. Fluorescence microscopic images of cryosedions of individual cartilages.A Infrmstral (lower jaw) cartilage, showingDil-labeled cranial cartilages in Bombina orientalis. labeled along its ventral margin. B Suprarostral cartilage Transverse sections, dorsal is up; all hut D and F depict the (upper jaw). C: Meckel's cartilage (lower jaw). D: Median left side, in which lateral is to the right. Arrowheads denote section depictingpaired cornu trabeculae (trabecularhorns). regions of labeled cells, which are seen as small bright dots of Only the left side is labeled. E: Palatoquadrate cartilage. F: crystallized dye. Unlabeled chondrocytes appear as red circles; Median section depicting the trabecular plate, which under- extracellular matrix is black. Areas of solid red or pink result lies the brain (faint red tissue dorsally).G Ceratohyal carti- from aubfluorexence within adjacent connective tissues lage. H First ceratohranchial cartilage (CB I). Scales = 0.05 (e.g.,muscles, perichondrium). See Figure 5 for the location mm, except in G (0.2mm). CRANIAL NEURAL-CREST MIGRATIONiFATE IN ANURANS 115

Bornbina Xenopus Ranu Eleutheroductvlus

Fig. 7. Evolutionary conservation of gross patterns of cal phylogenetic relationships are represented according cranial neural-crest migration in extant frogs that have to Ford and Cannatella ('93).Depictions of cranial neural- been studied. The number and configuration of cranial crest migration are based on the following sources: Bom- neural-crest streams are remarkably constant among bina-this study; Xenopus-Sadaghiani and Thiebaud, some distantly related anurans, including some with '87; Raiaa-Stone, '29; Eleutherodaetylus-Moury and direct development (e.g., EZeutherodactylus). Hypotheti- Hanken, '95. our labeled larvae were preserved. Resolution of Precocious onset of neural-crest emergence DiI-labeled cells is technically much more diffi- and migration relative to neural-fold closure cult in a calcified tissue (such as bone) than in may represent a fourth primitive character cartilage. This analysis, however, currently for anurans, but variation both among frogs is underway in our laboratories. and among other vertebrates makes this de- Several of the above features of cranial termination much more difficult. For ex- neural-crest cell biology in B. orientalis are ample, whereas precocious crest emergence shared by all other anurans (Table 1); we and migration are also characteristic of mam- regard these features as plesiomorphic among mals (Tan and Morriss-Kay, '85, '86; Ni- all living taxa. These features include the chols, '86, '87; Chan and Tam, '881, neither final number and configuration of principal feature occurs in urodeles, birds, or turtles. migratory streams (Fig. 7), non-chondro- In the latter three taxa, crest emergence and genic fate of the transverse neural fold, and migration commence much later, after neu- the extensive contribution to the embryonic ral-fold closure (Tosney, '78; Le Douarin, chondrocranium (e.g., Stone, '29; Sadaghiani '82; Jacobson and Meier, '84; Hou and Takeu- and Thiebaud, '87; Seufert and Hall, '90). All chi, '94). And while precocious crest emer- three features are characteristic of verte- gence and migration long have been regarded brates generally (Hall and Horstadius, '88); as characteristic features of all living frogs, presumably they were retained by the earli- recent evidence suggests that, at least in est anurans from their immediate ancestors. Xenopus, cranial neural-crest emergence does 116 L. OLSSON AND J. HANKEN not begin until after neural-fold closure (Col- of the rostral skull unique to larval anurans. lazo et al., '94). Lack of sufficient compara- The suprarostrals, which constitute the skel- tive data precludes determination of the likely eton of the larval upper jaw, likely evolved via evolutionary transitions in this character elaboration of the neural crest-derived ante- among these taxa, or even its evolutionary rior trabeculae typical of the neurocranium polarity. Thus, it is not known whether preco- of all vertebrates and from which the supra- cious emergence and migration represent an rostrals extend anteriorly (Fig. 5). The infra- evolutionary innovation that occurred one or rostrals, together with the laterally adjacent more times within the Anura (independent of Meckel's cartilages, represent a doubling of mammals), or if they were inherited from an the number of independent cartilages in the ancestral taxon and lost at least once in living crest-derived mandibular (lower jaw) skel- frogs (i.e., Xenopus). eton. Developmental mechanisms underly- Apparent variation in three additional char- ing these changes in skeletal patterning are acters suggests minor evolutionary trends unknown. While neural-crest derivation of affecting neural-crest migration among liv- both cartilages has been claimed in earlier ing anurans (Fig. 7). First, unlike Bombina, studies based on crest ablation (Stone, '29; in which two initial cranial migratory streams Seufert and Hall, '901, on transplantations subdivide to form three streams (Fig. ZA), X. between anurans and urodeles (Wagner, '49, laeuis (Pipidae), four species of Rana (R. '591, or on tissue explants (Raunich, '57; japonica, R. palustris, R. pipiens, and R. Cusimano et al., '621, reliability of these temporaria; Ranidae), and Eleutherodacty- claims has been difficult to assess because of lus cogui (Leptodactylidae) display three the technical problems or artifacts frequently streams virtually from the inception of migra- associated with such methods. Sadaghiani tion (Knouff, '27; Stone, '29; Reisinger, '33; and Thibbaud ('87) used a more refined tech- Ichikawa, '37; Sadaghiani and Thiebaud, '87; nique for assessing embryonic derivation in Moury and Hanken, '95; Table 1). If Bom- Xenopus (interspecific grafts involving a per- bina displays the primitive character state, manent cell marker). These authors, how- then the occurrence of a derived state in ever, provided only limited evidence of a neu- representative pipid, ranid, and leptodactylid ral-crest contribution to the anterior portion taxa may evidence a heterochronic shift (pre- of the "ethmoid-trabecular cartilage" (= su- displacement) that resulted in a much earlier prarostral plate; Trueb and Hanken, '92), a subdivision of hyoid and branchial streams. likely homologue of the suprarostrals, and Second, the large gap, or crest-free zone, that did not evaluate the derivation of the infraros- separates mandibular and hyoid streams in trals. We find no support for Wagner's ('49, Bornbina (Fig. 2B) is much smaller in Xeno- '59) suggestion that the suprarostrals form pus and Eleutherodactylus and absent, or from crest cells that migrate dorsally from nearly so, in Rana (Fig. 7). Finally, whereas the stomodeum distinct from those that form the mandibular stream is substantially more the anterior trabeculae. Similarly, we do not massive than the other two cranial-crest corroborate the claims based on tissue-ex- streams in Bombina, in Rana, and in at least plant experiments in Discoglossus that su- one bufonid (Bufo bufo; Olsson et al., unpub- pra- and infrarostral cartilages are derived lished data), all cranial streams are approxi- from crest cells originating within the trans- mately equal in size in Xenopus. There is no verse neural fold (Fagone, '59; Cusimano et evidence that variation in these features cor- al., '62; Cusimano-Carollo, '63, '69, '72). Our relates with interspecific differences in neu- results are fully consistent with the interpre- ral-crest differentiation or fate. Neverthe- tation that, at least in anurans, neural-crest less, it is both interesting and somewhat cells within the transverse neural fold have disconcerting that X. laeuis, the anuran spe- chondrogenic potential that is not expressed cies most widely used to study neural-crest during normal development (Seufert and biology in these vertebrates, has an appar- Hall, '90; Graveson, '93). The prominent role ently derived pattern of variation in several of supra- and infrarostral cartilages in larval characters and is arguably the most atypical mouth formation is well documented (Fa- species of frog considered so far. gone, '59; Cusimano et al., '62; Cusimano- These results confirm a prominent role for Carollo, '63, '69, '72; reviewed in Hall and the neural crest in the evolutionary origin of Horstadius, '88). the paired suprarostral and infrarostral carti- Conversely, our results are consistent with lages, two prominent caenogenetic features earlier claims regarding the lack of neural- CRANIAL NEURAL-CREST MIGRATIONiFATE IN ANURANS 117 crest contribution to the basihyal and basi- ancestor no later than the Jurassic period, at branchial cartilages, to the posterior trabecu- least 144 million years ago. Indeed, the spe- lar cartilages, and to the otic capsule (e.g., cies B. orientalis may have differentiated as Stone, '29). Thus, the resulting composite early as the Miocene (Maxson and Szymura, embryological origin of the chondrocranium '79). is firmly established in several species of an- Interestingly, one situation in which this urans and by the use of several alternate otherwise highly conserved pattern of neural- experimental methods, ranging from abla- crest development has been perturbed is in tion (Stone, '291, to heterospecific chimeras the evolution of the alternate reproductive (Sadaghiani and Thihbaud, '871, to vital label- mode, direct development, as seen in the ing (this study); it is a characteristic feature leptodactylid frog E. coqui. While many ba- of the skull in all frogs examined to date. The sic, ancestral features of cranial neural-crest particular complement of non-crest-derived cell emergence and migration have been re- cartilages in anurans differs somewhat from tained (Moury and Hanken, '951, the appar- that in other vertebrates. In birds, for ex- ently stereotyped pattern of crest derivation ample, neural crest contributes to a greater of the chondrocranium seen in metamorphos- proportion of the neurocranium, including at ing taxa has been altered considerably; many least part of the otic capsule, and to the larval-specific cranial cartilages do not form entire hyobranchial skeleton (Noden, '83a,b, during embryogenesis, and the initial pattern- '86b; Hall and Horstadius, '88; Couly et al., ing of others is highly modified (Hanken et '92, '93). The embryonic origin of the non- al., '92). Detailed aspects of this evolutionary crest-derived components in anurans is un- loss of chondrogenic fate and repatterning known. Two likely and obvious candidates currently are under investigation (Olsson and are paraxial cephalic mesoderm and somitic Hanken, in preparation). mesoderm, which are the source of non-crest- derived portions of the skull in chick-quail Implications for cranial pattern formation chimeras (Noden, '83b, '86b, '88; Couly et al., The composite embryonic origin of the '92, '93), but the relative contribution of chondrocranium as seen in anurans is a char- either cell population in anurans remains to acteristic feature of skull development in ver- be assessed. Sadaghiani and Thihbaud ('87; tebrates (Noden, '83b, '86b, '88; Hall and Table 1) indicated a contribution of "meso- Horstadius, '88; Couly et al., '92, '93). In- derm" to the ethmoid-trabecular, cerato- deed, this seemingly anomalous, but never- hyal, basihyal, and posterior branchial carti- theless consistent, mode of development has lages in Xenopus, but mesoderm was neither formed the basis for radical claims regarding labeled nor ablated in their experiments, and the evolutionary origin of different parts of no direct evidence for this assertion was pro- the skull, such as the origin of portions of the vided. crest-derived anterior trabeculae from one or more ancestral anterior visceral arches (Bjer- Evolutionary conservatism of neural-crest ring, '77). Regardless of its implications for pathways and fates head evolution, a composite origin also poses Notwithstanding the above minor differ- numerous intriguing problems regarding the ences in the initial configuration of migra- mechanisms that specify cranial pattern tory streams, cranial neural-crest develop- (Thorogood, '93). There is, for example, over- ment in metamorphosing anurans seems to whelming evidence of pattern specificity be highly stereotyped and evolutionarily con- within premigratory neural crest that contrib- servative; both migratory pathways and chon- utes to the visceral skeleton (Noden, '83a, drogenic derivatives are nearly invariant '86a,b; Prince and Lumsden, '94). Is the pat- among the 14 species studied (Table 1). For terning of skeletal elements derived from example, except for slight differences in the other sources also determined intrinsically, origin of the trabecular plate, the pattern of or do they instead receive their patterning neural-crest derivation of the larval chondro- cues extrinsically? One potential extrinsic cue cranium that we have produced for Bombina is epithelially derived macromolecules such is virtually identical to that for Rana pro- as type I1 collagen, which has been proposed duced more than 65 years ago (Stone, '29; to form a cranial prepattern that mediates Fig. 5). These taxa represent clades that are skeletogenic cell migration or differentiation among the most phylogenetically distant of (Thorogood, '88, '93). While epithelially de- all living frogs (Duellman and Trueb, '86: fig. rived type I1 collagen is present in the head of 17-3); they likely diverged from a common embryonicXenopus, it is expressed too late to 118 L. OLSSON AND J. HANKEN affect the gross patterning of neural crest expression in rhombomeres and neural crest. Mech. (Seufert et al., '94). The role, if any, of this or Dev. 40:73-84. Briindli, A.W., and M.W. Kirschner (1995) Molecular other macromolecules in patterning non- cloning of tyrosine kinases in the early Xenopus em- crest-derived cartilages in anurans is yet to bryo: Identification of Eck-related genes expressed in be evaluated. These challenges to models of cranial neural crest cells of the second (hyoid) arch. cranial pattern formation are magnified in Dev. Dyn. 203:119-140. Cannatella, D.C., and R.O. de Sa (1993)Xenopuslaeuis as cases of composite origin that involve inte- a model organism. Syst. Biol. 42476-507. grated functional systems that are also phylo- Cannatella, D.C., and L. Trueb (1988) Evolution of pipoid genetically diverse (e.g., the hyobranchial frogs: Intergeneric relationships of the aquatic frog skeleton), or even individual elements such family Pipidae (Anura). Zool. J. Linn. SOC.94:l-38. Carlson, J.T., and M.S. Ellinger (1980) The reproductive as the trabecular plate. How is patterning biology of Bombina orientalis, with notes on care. coordinated between crest-derived and non- Herpetol. Rev. 11:ll-12. crest-derived portions of such components, Chan, W.Y., and P.P.L. Tam (1988) A morphological and both during ontogeny and during evolution? experimental study of the mesencephalic neural crest We hope to address these and similar ques- cells in the mouse embryo usingwheat germ agglutinin- gold conjugate as the cell marker. Development 102: tions in future studies. 427442. Chen, P.S., and F. Baltzer (1954) Chimarische Haftfaden ACKNOWLEDGMENTS nach xenoplastichem Ektodermaustausch zwischen Tri- ton und Bombinator. Roux's Arch. Entw. Mech. 147: We thank Robert Eaton, Todd Gleeson, 2 14-258. and Richard Jones for providing ready access Collazo, A., and S.M. Marks (1994) Development of Gyri- nophilus porphyriticus: Identification of the ancestral to their laboratory equipment, Mike Klym- developmental pattern in the salamander family Pleth- kowsky and Pat Davis for assistance with odontidae. J. Exp. Zool. 268:239-258. confocal microscopy, Olle HBstad for help in Collazo, A., M. Bronner-Fraser, and S.E. Fraser (1993) preparing illustrations, and Drew Noden for Vital dye labelling of Xenopus laeuis trunk neural crest reveals multipotency and novel pathways of migration. English summaries of Wagner's papers. An- Development 118:363-376. dres Collazo, Tim Carl, Brian Hall, and David Collazo, A,, J. Rubero, M. Bronner-Fraser, P.M. Mabee, Jennings provided helpful comments on the and S.E. Fraser (1994) Cell migration and novel deriva- manuscript. Research support was provided tives of cranial neural crest and placodes in Xenopus by NSF (IBN 94-19407 to J.H.), by the Swe- laeuis. SOC.Neurosci, Abstr. 20:1672. Corning, H.K. 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