RESEARCH ARTICLE

Turtle–Chicken Chimera: An ExperimentalApproach to Understanding EvolutionaryInnovation in the TurtleHiroshi Nagashima,1,2 Katsuhisa Uchida,1† Keiko Yamamoto,1 Shigehiro Kuraku,1 Ryo Usuda,1 andShigeru Kuratani1*

Turtles have a body plan unique among vertebrates in that their ribs have shifted topographically to asuperficial layer of the body and the trunk muscles are greatly reduced. Identifying the developmentalfactors that cause this pattern would further our understanding of the evolutionary origin of the turtles. Asthe first step in addressing this question, we replaced newly developed epithelial somites of the chicken atthe thoracic level with those of the Chinese soft-shelled turtle Pelodiscus sinensis (P. sinensis somites intoa chicken host) and observed the developmental patterning of the grafted somites in the chimera. The P.sinensis somites differentiated normally in the chicken embryonic environment into sclerotomes anddermomyotomes, and the myotomes differentiated further into the epaxial and hypaxial muscles withhistological morphology similar to that of normal P. sinensis embryos and not to that of the chicken. Epaxialdermis also arose from the graft. Skeletal components, however, did not differentiate from the P. sinensissclerotome, except for small fragments of cartilage associated with the host centrum and neural arches. Weconclude that chicken and P. sinensis share the developmental programs necessary for the earlydifferentiation of somites and that turtle-specific traits in muscle patterning arise mainly through acell-autonomous developmental process in the somites per se. However, the mechanism for turtle-specificcartilage patterning, including that of the ribs, is not supported by the chicken embryonic environment.Developmental Dynamics 232:149–161, 2005. © 2004 Wiley-Liss, Inc.

Key words: somites; ribs; transplantation; turtles; evolutionary innovation

Received 20 July 2004; Revised 7 September 2004; Accepted 7 September 2004

INTRODUCTIONThe turtle shell is often cited as a typ-ical example of evolutionary noveltybecause of its unusual anatomicalcomposition; the dorsal half of theshell, or the carapace, is based on ribsthat have moved to a superficial posi-tion, covering the limb girdles dorsally(reviewed by Hall, 1998; Gilbert et al.,2001; Rieppel, 2001). These changes

in the topographical relationships ofthe skeletal elements, which also cor-relate with morphological changes inthe muscles, appear to be due to thelateral growth of the ribs and not tothe descent of the girdles into the ribcage (Ruckes, 1929; Walker, 1947;Emelianov, 1936; reviewed by Burke,1989, 1991; Ewert, 1985). This shift inrib growth also appears to be the basis

of modified tissue interactions thatyield the dermal bones and expandedscales of the carapace (reviewed byHall, 1998). A small change in theplace of development (heterotopy;Haeckel, 1875) can result in a large-scale alteration in morphology. Thefunction of carapacial ridge (CR) hasbeen assumed (Burke, 1989) in the to-pographical shift of the ribs. The CR is

1Laboratory for Evolutionary Morphology, Center for Developmental Biology (CDB), RIKEN Kobe, Kobe, Japan2Graduate School of Science and Technology, Kobe University, Kobe, JapanGrant sponsor: the Ministry of Education, Science, and Culture of Japan.†Dr. Uchida’s present address is Sado Marine Biological Station, Faculty of Science, Niigata University, 87 Tassha, Sado, Niigata 952-2135,Japan.*Correspondence to: Shigeru Kuratani, Laboratory for Evolutionary Morphology, Center for Developmental Biology (CDB),RIKEN, 2-2-3 Minatojima-minami, Chuo-ku, Kobe, Hyogo 650-0047, Japan. E-mail: saizo@cdb.riken.jp

DOI 10.1002/dvdy.20235Published online 3 December 2004 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 232:149–161, 2005

© 2004 Wiley-Liss, Inc.

a structure that consists of aggregatedmesenchyme surrounded by thick-ened ectoderm and appears in theflank of the late pharyngula. By sur-gical removal of the CR, Burke (1991)showed that the CR is required fornormal rib growth of the turtle. Thus,an inductive activity similar to that inthe limb bud, has been assumed in theCR (Burke, 1989, 1991), and in thesame context, CR-specific localizationof some molecules and gene expres-sions have been reported recently(Burke, 1989; Loredo et al., 2001; Vin-cent, 2003).

In association with shell formation,the trunk muscles (both epaxial andhypaxial muscles) of turtles aregreatly reduced at the flank level. Im-portantly, both the trunk muscles andthe ribs differentiate from somites,the segmented mesodermal blocks inthe early embryo (Seno, 1961; Pinot,1969; Sweeney and Watterson, 1969;Christ et al., 1974; Christ and Wilting,1992; Huang et al., 1994, 1996, 2000;Kato and Aoyama, 1998; Evans,2003). From the perspective of evolu-tionary developmental biology, there-fore, the key to innovation in the evo-lution of the turtles is the changesintroduced into the developmentalprograms of the somite derivatives.

As for turtle somite development,Yntema (1970) showed in Chelydraserpentina that removal of somitesleads to the loss of scutes and ribs inthe carapace. He also thought that theturtle ribs are derived from the lateralpart of the somites. The developmen-tal fates and mechanisms of differen-tiation of amniote somites have beenstudied more extensively in model an-imals such as the chicken and mouse(reviewed by Christ and Ordahl, 1995;Christ et al., 2000; Dockter, 2000;Monsoro-Burq and Le Douarin, 2000;Brent and Tabin, 2002). Newly seg-mented epithelial somites undergoinitial differentiation into the ventro-medial deepithelialized part, or thesclerotome, and the rest of the somite,generally called the dermomyotome(Christ and Ordahl, 1995; Christ etal., 2000). The sclerotome then givesrise to skeletal elements, includingthe vertebrae and ribs during laterdevelopment (Christ and Wilting,1992; Huang et al., 1994, 1996; Evans,2003). It has also been suggested thatat least a part of the rib differentiates

from the dermomyotome (Kato andAoyama, 1998). In the developmentalpatterning of turtle somites, the mostintriguing questions are: Whatchanges have been introduced duringthe evolution of this animal group?Into which stage of its developmentwere they introduced, relative to thegeneralized time table of somite devel-opment in amniotes such as thechicken and mouse?

The turtle-specific traits of somitedifferentiation and the developmentalmechanisms responsible for thesetraits should reflect the functions ofthe alleles actually selected during theevolutionary establishment of the tur-tles. Importantly, such changes wouldnot necessarily be found in the cell-autonomous mechanism of the somiteitself, because the embryonic environ-ment surrounding the somites canalso exert various signals through tis-sue interactions that determine thespecific developmental fates of somite-derived cells. Therefore, the turtle-specific developmental program canbe classified into the cell-autonomousand non–cell-autonomous (or epige-netic) factors involved in somite differ-entiation.

In the present study, as a first stepin determining the cell-autonomousfactors involved in turtle-specific pat-terning mechanisms, interspecies chi-meras were constructed betweenchicken and turtle, i.e., turtle somiteswere transplanted into chicken hostsat comparable developmental stages(Fig. 1). Although the phylogenetic po-sition of the turtles remains enig-matic, recent molecular phylogeneticanalyses have supported the close af-finity of birds, crocodiles, and turtles(Platz and Conlon, 1997; Zardoya andMeyer, 1998, 2001a,b; Hedges andPoling, 1999; Kumazawa and Nishida,1999; Mannen and Li, 1999; Cao et al.,2000), which justifies the use of Gallusgallus as an competent host for thechimera. As the donor species, we se-lected the Chinese soft-shelled turtlePelodiscus sinensis, which is commer-cially available from fish farms in Ja-pan (Fig. 1). To trace the cell lineagesof P. sinensis graft derivatives, weraised anti–P. sinensis IgY, which suc-cessfully labeled P. sinensis cells inthe unstained chicken background.Young transplanted somites from P.sinensis responded to the chicken em-

bryonic environment, differentiatinginto sclerotomes and dermomyotomes.However, although the cell-autono-mous nature of somite differentiationwas apparent in muscle differentia-tion, the skeletal differentiation of P.sinensis somites was substantially ar-rested in the chimeric environment.

RESULTS

Comparative Anatomy ofEmbryonic Thoracic Regionsand Somitic Derivatives

The morphology of the thoracic re-gions was compared between P. sinen-sis and G. gallus embryos at stageswhen species-specific anatomical fea-tures become apparent (Fig. 2A,B). Inthe chicken, myotomes differentiatedinto the epaxial muscle in the dorsalpart of the body and into the hypaxialmuscles in the lateral body wall (Fig.2A). In the chicken–quail chimera, inwhich the somites of the chicken hosthad been unilaterally replaced bythose of the quail, the somite-deriveddermis was predominantly distrib-uted in the dorsal part of the bodysurrounding the epaxial muscle,which was also of quail-somite origin.However, the lateral body wall did notcontain dermis composed of quail cells(Fig. 2C,D), as already reported(Nowicki et al., 2003; Burke andNowicki, 2003; Fig. 2A). Our experi-mental procedure for chimera con-struction allowed us to remove all thesomitic cells from the experimentalside, and half the centrum was com-pletely replaced with quail cells (Fig.2D). In the skeletal system, quail cellsdifferentiated into chondroblasts ofthe centrum, neural arch, and ribs(Fig. 2D). Quail cells also contributedto part of the mesonephros and part ofthe dorsal aorta. Dorsal root gangliawere never replaced with quail cells(Fig. 2D). Therefore, in the chickenembryo, the rib primordia and hypax-ial muscles derive from somites andsecondarily grow ventrally into thehypaxial domain, as “primaxial” ele-ments (Nowicki et al., 2003; Burkeand Nowicki, 2003; Fig. 2A).

In the P. sinensis embryo, morpho-logically normal epaxial muscle devel-oped, although its extent was lessthan that observed in the chickenhost, whereas the hypaxial muscle

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primordia appeared as a thin threadof fibrous tissue that was stained withthe monoclonal antibody, MF-20 (datanot shown; see below). The massiverib primordia grew more laterallycompared with that of the chicken(Fig. 2A,B), and it never entered thelateral body wall; the ribs remained inthe epaxial domain. Therefore, theribs and the hypaxial muscles of P.sinensis did not develop side-by-sidein the lateral body wall, as was seen inthe chicken embryo (Fig. 2A,B). Acharacteristic trait of turtle embryos,the CR, grew on the lateral aspect ofthe body wall (Fig. 2B). This ridge waslateral to the distal tip of the rib. Nosuch ridge appeared in the corre-sponding region of the chicken embryo(Fig. 2A). Because the rib never in-vaded the hypaxial domain but in-stead grew laterally toward the CR,

and the hypaxial muscles of P. sinen-sis always developed ventral to thedistal tip of the ribs (Fig. 2B; also seeFig. 3), we can assume that the CRarises in the epaxial domain adjacentto the junction of the epaxial and hy-paxial domains.

G. gallus–P. sinensisCommon DevelopmentalStages

To examine the developmental pat-tern of P. sinensis somite derivativesin the chicken embryonic environ-

Fig. 1. Pelodiscus sinensis and transplantationof somites. Top: Juvenile P. sinensis. Below:Three successive newly formed somites (somitestages �I to �III; Ordahl et al., 1993) were re-moved unilaterally from the thoracic level of anHamburger and Hamilton stage 15 chicken em-bryo. Three epithelial thoracic somites were ex-cised from a Tokita and Kuratani stage 10 P.sinensis embryo on the corresponding side andimplanted into the scar made in the host chickenembryo. Scale bar � 6 cm.

Fig. 2. Comparison of thoracic anatomy of Gallus gallus and Pelodiscus sinensis embryos.Transverse sections through the thoracic regions of Pelodiscus and Gallus stage 11 G. gallus (A)and P. sinensis (B) embryos are shown. Broken lines indicate the morphological boundariesbetween epaxial (EP) and hypaxial (HY) regions. A: In G. gallus, the rib (r) grows from the epaxialregion ventrally into the hypaxial region, within the medial part of the somatopleure. Both theepaxial (em) and hypaxial muscles (hm) are massive. B: In P. sinensis, the rib (r) stays in the epaxialdomain, growing laterally toward the carapacial ridge (cr). Epaxial (em) and hypaxial muscles (hm)are relatively smaller than those of the chicken. C: Transverse section of a chicken–quail chimera4 days after surgery, in which chicken thoracic epithelial somites had been replaced with those ofquail. Immunohistochemically stained with QCPN monoclonal antibody to identify quail cells andcounterstained with hematoxylin. Primaxial domain of the embryonic thorax is occupied byQCPN-positive quail cells. Quail somite derivatives have moved into the lateral body wall or thehypaxial domain of the chimera. D: High magnification of the box in C. Arrowheads and whitearrows indicate quail cells contributing to the mesonephros (mn) and the dorsal aortic wall (a),respectively. The black arrow indicates the interface between quail and chicken dermis. a, dorsalaorta; c, centrum; cr, carapacial ridge; d, dermis; drg, dorsal root ganglia; mn, mesonephros; n,notochord; na, neural arch; nt, neural tube; r, rib.

DIFFERENTIATION OF TURTLE SOMITES 151

ment, it is necessary to construct chi-meras. To reduce any possible devel-opmental arrest in the chimera thatmight arise from differences in devel-opmental rates, we initially triedto establish common developmentalstages at the thoracic level betweenthe two animal species. Histologicalsections were examined and mesoder-mal development was compared insomites (s) 15–21 of P. sinensis ands22–26 of G. gallus (corresponding tothe thoracic level in each species; Fig.3). Based on this comparison, we de-fined common developmental stages(PG stages; PG stands for Pelodiscusand Gallus), as shown in Table 1.They ranged from the newly formedepithelial somite observed at PG stage1, corresponding to HH stage 15 of G.gallus (Hamburger and Hamilton,1951) and TK stage 9 of P. sinensis(Tokita and Kuratani, 2001), to PGstage 11, when the anatomical fea-tures of the muscles and skeletal ele-ments become apparent in each spe-cies, corresponding to HH stage 30 ofG. gallus and TK stage 16 of P. sinen-sis (Fig. 3).

Based on the above staging, devel-opmental rates were compared at dif-ferent temperatures for both speciesto determine the most appropriate

conditions for the incubation of chime-ras (Fig. 4). P. sinensis developed atthe greatest rate at 34°C, but the via-bility of embryos decreased abruptlywhen incubated at temperatureshigher than 38°C. Although chickenembryos developed normally in therange of 34°C to 38°C, they did notdevelop with normal embryonic pat-terns at temperatures below 30°C(Fig. 4). We concluded that chimerasshould be incubated most appropri-ately within the range of 34–36°C.Within this window, the developmen-tal rates of both species synchronizedwell, especially between PG stages 1and 5 (Fig. 4).

Anti–P. sinensis Antiserum

An anti–P. sinensis serum was raisedas a cell-lineage marker with which totrace P. sinensis cells in the chimera.The serum obtained from chickens in-jected with homogenized P. sinensisembryos was applied to sectioned em-bryos of chicken and P. sinensis to de-termine its immunoreactivity. The an-tiserum clearly labeled all P. sinensistissues but did not stain chicken tis-sues at all (Fig. 5). The staining pat-tern on P. sinensis was ubiquitous, al-though less dense on cartilage because

the cartilage matrices were notstained (data not shown). Labelingwas predominant on cell surfaces andmost of the epitopes were probablycell-membrane molecules. This find-ing is consistent with the fact that thelabeling was lost or greatly reducedwhen high concentrations of detergent(Triton X-100) were used in the buffer(data not shown). Therefore, the anti-serum could be used as a marker forembryonic P. sinensis cells.

Somitic Chimeras

In the chimeric embryos, the host tho-racic somites were replaced with P.sinensis somites (Figs. 1, 6A). In thetotal of 93 chimeric embryos, 13 (14%)survived up to the stages when histo-logical observations were performed.Six hours after surgery, the develop-mental rates of the host and donorsomites appeared to be synchronized.

Two days after surgery, the donorsomites had differentiated into scle-rotomes and dermomyotomes withcorrect topographical orientation inthe chimeric embryo, in all chimerasexamined (Fig. 6B). Unlike the hostsomite on the control side, however,the donor sclerotome was associatedwith numerous blood vessels (Fig.

TABLE 1. Common Developmental Stages of Pelodiscus sinensis and Gallus gallusa

Common stages(PG stages)

P. sinensis(TK stages)

G. gallus(HH stages) Histological description

1 9 15 Epithelial somites.2 10 16 Ventromedial part of somites starts to be

deepithelialized to form sclerotomes.3 11� 17 Sclerotomal cells migrate toward the notochord.4 11 18 Initial appearance of myotomes.5 12� 20 Formation of myotome completed; midportion of

dermatome starts to be deepithelialized.6 12 22 Ventrolateral lip of the dermomyotome invades the

somatopleure.7 12� 23 Dermatome almost deepithelialized except for its

ventrolateral and dorsomedial lips.8 13 24 Sclerotomal cells aggregate around the notochord:

beginning of centrum formation. Carapacialridge begins to appear in P.sinensis.

9 14�–14 25–26 Ventrolateral lip of dermatome deepithelialized.10 14�–15 27–28 Appearance of rib primordia as mesenchymal

condensation.11 15�–16 29–30 Species-specific morphology established.

aHistological observations were made on transversely sectioned specimens at the levels of somites (s) 15–21 for P. sinensis ands22–26 for G. gallus (see Fig. 3). TK stages refer to the developmental stages of P. sinensis described by Tokita and Kuratani (2001),and HH stages to those described by Hamburger and Hamilton (1951) for G. gallus development. The common stages wereestablished on the basis of the developmental patterning of somites. PG, Pelodiscus and Gallus.

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6B,C). These vessels were composed ofP. sinensis endothelial cells, althoughblood cells were mostly of chicken or-igin (Fig. 6C). Mitotic cells were ob-served in both the sclerotomes anddermomyotomes on the chimeric side,although fewer in number than thoseon the control side (Fig. 6D–F). Thedifferentiation of the donor somite de-scribed above appeared to be inducedby the host embryonic environment,because the slightly older P. sinensissomites often self-differentiated intosclerotomes and dermomyotomes thatwere misoriented in the host (data notshown).

Five days after surgery, the chimerahad developed to approximately PGstage 10 (Fig. 7G–I). By this stage, thegraft-derived dermis had grown lessextensively than that of the host, butwas distributed in a pattern similar tothe epaxial dermis observed in thechicken-quail chimera (Fig. 2D). Thelateral limit of the graft-derived der-mis did not correspond to the host ec-todermal notch (Fig. 7H); this lack ofcorrespondence was due to torsion inthe chimeric embryo during postsurgi-cal healing and does not necessarilyrepresent a discrepancy with thenotch that develops in the normalchicken embryos (Fig. 2A). This P. si-nensis dermis was also limited epaxi-ally and formed a rather clear bound-ary against the more ventral hypaxialdermis of host origin (Fig. 7H). As inthe chicken, P. sinensis somite deriv-atives invaded the hypaxial region,where the P. sinensis cells differenti-ated into blood-vessel endothelial cellsas well as into hypaxial muscle fibers(Fig. 7H,I). Except for the epaxial der-mis, P. sinensis cells also formed partof the dorsal aorta, mesonephros, ep-axial muscle, and the dense mesen-

Fig. 3. Comparison of embryonic development inPelodiscus sinensis and Gallus gallus. Fixed em-bryos of G. gallus (left) and P. sinensis (right) areshown on either side of the figure, and histologicalimages of comparable stages are shown in themiddle. Compare with Table 1. Numbers indicatethe common (Pelodiscus and Gallus) stages, andspecies-specific stages are prefixed by HH (Ham-burger and Hamilton) for chicken and TK (T okitaand Kuratani) for P. sinensis. Not all the stages areshown here. Bars on the bottom right � 5 mm(each notch indicating 1 mm) for whole-mountembryos; and all the bars on histological sec-tions � 100 �m

DIFFERENTIATION OF TURTLE SOMITES 153

chyme at a site apparently corre-sponding to the site of neural-archdifferentiation (Fig. 7H,I). Pelodiscussinensis cells were never found in thedorsal root ganglia, which are neural-crest derivatives (data not shown; seebelow). We did not determine whether

Fig. 4. Comparison of developmental rates. Developmental stages were plotted along a time scale between Pelodiscus and Gallus stages 1 to 11for Gallus gallus and Pelodiscus sinensis raised at various temperatures. Dots and lines indicate developmental stages with standard deviations for P.sinensis (blue) and G. gallus (red). Gallus gallus stopped developing at temperatures below 30°C, and P. sinensis did not develop at temperatures above38°C. Both species develop between 34°C and 36°C.

Fig. 5.

Fig. 6.

Fig. 5. Species-specific immunoreactivity of an-ti–Pelodiscus sinensis IgY. A,B: Transversesections of Gallus gallus (A) and P. sinensis (B)at the thoracic level stained with anti–P. sinen-sis antiserum (Ab), counterstained with hema-toxylin (H). Note the species-specific immuno-reactivity of the antiserum. C–F: Highmagnification of boxes in B, showing histologyof the epaxial muscle (C), rib cartilage (D), sym-pathetic ganglion (E), and carapacial ridge (F) inP. sinensis. Scale bars � 500 �m in A,B, 50 �min F (applies to C–F).Fig. 6. Transplanted Pelodiscus sinensissomites. A: After 6 hr incubation after somitegrafting between P. sinensis and Gallus gallus.A transverse section stained immunohisto-chemically with anti–P. sinensis antiserum andcounterstained with hematoxylin. Note that thesomite of the host is unilaterally replaced with aP. sinensis somite (s on the left). B–F: Two daysafter implantation of the P. sinensis somite inthe chicken, shown on the right side. B and Care stained with anti–P. sinensis antiserum. Thegrafted somite has differentiated into a dermo-myotome (dm) and sclerotome (sc), and numer-ous blood vessels (bv) have appeared on theoperated side. C. High magnification of the boxin B. Note that the endothelium of the bloodvessels (bvw) is composed of P. sinensis cells,whereas the blood cells (bc) are chicken cells.D–F: Hematoxylin and eosin–stained sectionsfrom the specimen shown in B and C. D. Animage of a mitotic epithelial cell in the P. sinen-sis somite-derived dermomyotome (arrow). E,F:Mitotic mesenchymal cells in the P. sinensissomite-derived sclerotome (arrows). a, dorsalaorta; n, notochord; nt, neural tube. Scalebars � 100 �m in A,B, 50 �m in C, 10 �m inD–F.

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P. sinensis cells formed any other skel-etal tissues, although P. sinensis mes-enchyme tended to be aggregatedaround the host notochord and at thesite of prospective neural-arch devel-opment (Fig. 7H,I).

Seven days after surgery, the P. si-nensis somites contributed to thesame repertoire of embryonic tissuesas observed in the 5-day chimeras(Fig. 7J,M,N). Dorsal root gangliawere never populated by P. sinensiscells (Fig. 7M). The MF-20–positiveepaxial and hypaxial muscles werealso derived from P. sinensis somites(Fig. 7B,C,E,F), and by this stage, thelatter muscle had differentiated into athin sheet of muscle fibers (Fig.7M,N). The overall morphology of thismuscle more closely resembled the P.sinensis hypaxial muscle shown inFigure 2B than that of the host (Fig.2A). A few cartilage nodules were com-posed of P. sinensis cells, associatedwith host cartilage, but no larger P.sinensis cartilage was found with thenormal morphology of ribs or verte-brae (Fig. 7M,N). Those small piecesof P. sinensis cartilage had no exten-sive extracellular matrix that could bestained with Alcian blue, as did thehost cartilage (Fig. 7K,L). No ribswere derived from P. sinensis somitesat this stage. In chimeras that hadreceived somites and the overlyingsurface ectoderm of P. sinensis, ribsderived from the graft were not ob-served at even 7 days after the sur-gery (3 embryos survived of 21 chime-ras constructed; data not shown).

Expression of Shh and Pax9Orthologues

To determine whether the differencesin cartilage differentiation between P.sinensis and host somites in the chi-mera were due to differences in induc-tive signaling derived from the noto-chord, the expression of Shh wasexamined (Fig. 8A–D). At PG stages 4to 8, Shh orthologues were specificallyand strongly up-regulated in both spe-cies in the floor plate of the neuraltube and in the notochord, suggestingthat the gene is involved in the pat-terning of the mesoderm at similarstages of embryonic development inboth species, with shared embryonictopography.

To examine whether the implanted

P. sinensis somites responded to thesignals derived from Shh, expressionof Pax9 was observed in chimeras thathad been incubated for 3 days aftersurgery (Fig. 8E). An antisense probefor P. sinensis Pax9 detected high lev-els of transcripts in the sclerotomes ofboth chicken and P. sinensis (theprobe also recognized chicken Pax9transcripts).

DISCUSSION

Despite its evolutionary importance,experimental embryological studies ofthe turtle are rare (Yntema, 1970; Fal-lon and Crosby, 1977; Burke, 1991),partly due to the difficulties in thecollection and handling of turtle eggsand to a lack of cell-lineage markers.In the present study, we raised an-ti–P. sinensis polyclonal IgY for thefirst time. It specifically recognizedtissues of P. sinensis, allowing us todetermine the developmental fates ofP. sinensis grafts in chimeras at thecellular level (Figs. 5–7). Anotherproblem associated specifically withinterspecific chimeras is the incom-patibility of developmental time ta-bles; the development of different an-imals proceeds at different rates atdifferent temperatures. A well-knownexample is the chimera between Xeno-pus and axolotl, which cannot begrown to advanced stages (Armstrongand Muneoka, 1989). The same prob-lem was encountered in the presentstudy. In chicken–turtle chimera, Fal-lon and Crosby (1977) reported thatthe zone of polarizing activity (ZPA)from the limb buds of Cherydra ser-pentina and Chrysemys picta was ableto function in the chicken host. Inthese experiments, however, the tis-sue differentiation of the graft was notobserved, and only the ZPA-derivedsignaling molecules appeared to haveacted on the chicken limb buds. Actu-ally, when incubated at 38°C, the P.sinensis somite could form only asmall mesenchymal cell population inthe chicken host environment even 5days after the surgery (data notshown). To overcome species-specificdifferences in the developmental timetables, we first identified the commondevelopmental stages based on the de-velopmental patterning of somite de-rivatives at the histological level andidentified 34–36°C as the appropriate

range of temperatures at which bothspecies could grow to histogeneticstages (Fig. 4). The incompatibility oftissue interactions will be discussedbelow, especially in the context of car-tilage development in the chimera.

Chimeric surgery was apparentlysuccessful in the present study. Inboth the G. gallus–C. coturnix and G.gallus–P. sinensis chimeras, graft-de-rived cells occupied the space lateralto the notochord, showing that thehost tissue to be replaced had beenremoved properly (Figs. 2, 6A,B,7H,K,M). Furthermore, tissues knownto be derived from somites were re-placed with grafted cells (Figs. 6, 7): inthe chimeras constructed betweenavian embryos, grafted somites con-tribute to the endothelium of bloodvessels, including the dorsal aorta andthose in the mesonephros (Wilting etal., 1995; Pardanaud et al., 1996; Am-bler et al., 2001). In quail–mouse chi-meras, however, mouse somite (graft)-derived cells never contributed to theendothelium (Ambler et al., 2001).The occasional presence of donor cellsin the dorsal aorta and mesonephrosin the present study is reminiscent ofinter-avian species chimera citedabove, showing a possibility that P.sinensis somites could respond toavian vascular patterning signals. Ex-tensive development of blood vesselson the experimental side of the chi-mera could be due to the wound-heal-ing after the surgery: during the re-moval of host somites, the hostembryo often bled.

Appearance of P. sinensis cells inthe mesonephric ducts of the chimera(Fig. 7H,I), on the other hand, may notreflect the normal differentiation ofthe somites in either animal, but thismight be explained by the induction ofintermediate mesoderm in the graftedtissue by the host environment. Thehost intermediate mesoderm wasdamaged during surgery, altering thedevelopmental fates of the graftedsomites. Lateral mesoderm is capableof inducing Pax-2 expression, amarker for intermediate mesoderm, inthe somites when these mesodermaltissues are cocultures (James andSchultheiss, 2003).

One of the most conspicuous ele-ments in the turtle embryo of turtle-specific morphology is the presence ofthe CR in the embryonic trunk (Figs.

DIFFERENTIATION OF TURTLE SOMITES 155

2, 3); no such structure is apparent inthe chicken embryo. By comparativemorphology and construction ofchicken–quail chimeras, we deter-mined that the CR probably develops

at the junction of the epaxial and hy-paxial regions (or at the dorsal limit ofthe lateral body wall; Fig. 2). The mes-enchyme of the CR itself appears to becomposed of somite-derived cells,

Fig. 7.

Fig. 8. Comparison of Shh and Pax9 geneexpression patterns. A–E: Expression patternsof Shh homologues were compared at the tho-racic levels of Pelodiscus and Gallus stage 4(A,B) and stage 8 (C,D) embryos of Gallus gallus(A,C) and Pelodiscus sinensis (B,D), and Pax9expression was examined in the chimera inwhich the left somite had been replaced with asomite of P. sinensis (E). An antisense ribo-probe for P. sinensis Pax9 also recognizedchicken Pax9 transcripts in the chicken host.A–D: The Shh genes are highly up-regulated inthe floor plate (fp) of the neural tube and in thenotochord (n) in both species. E: In the chimera,somites have differentiated into dermomyo-tomes and sclerotomes on both sides, andPax9 is up-regulated specifically in the scle-rotomes. The disorganized histology of the P.sinensis sclerotome reflects the hypertrophicdevelopment of blood vessels derived from thegraft (also see Fig. 6B,C). Histological observa-tions were made on transversely sectionedspecimens at the levels of somites (s) 15–21 forP. sinensis and s22–26 for G. gallus (see Fig. 3).TK stages refer to the developmental stages ofP. sinensis described by Tokita and Kuratani(2001), and HH stages to those described byHamburger and Hamilton (1951) for G. gallusdevelopment. The common stages were estab-lished on the basis of the developmental pat-terning of somites. Scale bars � 100 �m in B(applies to A,B), in D (applies to C,D), in E.

156 NAGASHIMA ET AL.

namely the epaxial dermis (Fig. 3). Inthe present study, P. sinensis somitesdifferentiated into the epaxial dermis,although not as extensively as in thehost dermis (Fig. 7H). However,whether the P. sinensis dermis couldself-differentiate or induce the CR inthe chicken host remains unansweredin the present study, mainly becausethe morphology of the chimeras wasdistorted (Fig. 7G–I). This finding ispartly caused by the differential de-velopmental rates of the dermis, aninherent problem specifically associ-ated with the construction of interspe-cific chimeras. To address this ques-tion, CR-specific marker genes(Loredo et al., 2001; Vincent et al.,2003) should be isolated and their ex-pression (or the expression of theirchicken homologues) should be ana-lyzed in the chimeras, which we areundertaking in a future project.

The present chimeric study was ex-pected to identify the turtle-specificdevelopmental program that proceedswithin the somite-derived cells them-selves—the cell-autonomous pro-grams in somite derivatives, such asthe muscles and ribs. These struc-tures show species-specific differencesin morphology in chicken and turtle(Fig. 2). The key question is whetherthis specific morphology is inherent tothe somite-derived cells (predeter-mined in the somites) or if it requiresspecies-specific interactions with theembryonic environment. The develop-ment and differentiation of P. sinensis

somites, when exposed to the chickenenvironment, might identify the partof the turtle-specific developmentalprogram that is predetermined withinthe somites, as will be discussed be-low.

The amniote somite undergoes hier-archical steps in its differentiation inresponse to signals derived from theembryonic environment. For the ini-tial differentiation of the epithelialsomite into the sclerotome and dermo-myotome, the signal from the noto-chord that induces deepithelializationof the ventromedial part of the somiteto form the sclerotome is indispens-able (Brand-Saberi et al., 1993; Goul-ding et al., 1994; Ebensperger et al.,1995; also see: Fan and Tessier-Lavigne, 1994; Christ and Ordahl,1995). In the present study, the P. si-nensis somite, which at the stage oftransplantation is epithelial, was ca-pable of responding to such a signal,because it always differentiated intothe sclerotome and dermomyotomewith the appropriate polarity; withdermomyotome beneath the ectoderm,and sclerotome near the notochord(Fig. 6B). Slightly older P. sinensissomites often self-differentiated intothose subdivisions with an inappro-priate polarity (the dermomyotomelateral to the neural tube, for exam-ple). This finding suggests that earlyP. sinensis somites (somite stages �Ito �III) are perfectly competent to re-spond to the signals derived from thechicken embryonic environment, con-

sistent with the ability of young somites(before stage II) to reorientate, as re-ported for chicken embryos (Aoyamaand Asamoto, 1988).

Further differentiation of thesomite involves the formation of sev-eral cell types, such as the dermis,chondrocytes, and myofibrils, whichagain requires signals derived fromthe embryonic environment (Koseki etal., 1993; Goulding et al., 1994;Ebensperger et al., 1995). We deter-mined histologically that the P. sinen-sis somites in the chimera gave rise toall these expected cell types (Figs. 6,7). Moreover, in muscle differentia-tion, the histogenesis of P. sinensissomite derivatives appeared to follow,not the pattern of the chicken, butthat of P. sinensis. This differentiationwas especially apparent in the hypax-ial part (Figs. 2A,B, 7M,N). Therefore,it seems very reasonable to assumethat the turtle-specific patterning ofthe trunk muscles is more or less gov-erned by a cell-autonomous develop-mental process, although it requirespreceding inductive events controlledby the host environment. Similar cell-autonomous differentiation of myo-fibrils has also been reported inchicken–quail chimeric experiments(Nikovits et al., 2001). Alternatively,the poor development of the hypaxialmuscles in the chimera could havebeen due to the inability of the turtlemyoblasts to respond to the host em-bryonic environment, which in turncould be one of the changes introducedto the developmental program of themuscles in the turtle ancestors. Spe-cies-specific morphogenesis of musclesmay be more reminiscent of crest cell-autonomous craniofacial patterningas demonstrated by Schneider andHelms (2003), who exchanged the ce-phalic neural crest between quail andduck and obtained graft-specificcraniofacial morphology in the chi-meric animals.

In contrast, the skeletogenesis ofthe P. sinensis somite was stronglyarrested in the chimera describedhere (Fig. 7). It is hardly conceivablethat the chimeras were not incubatedlong enough for P. sinensis cells to pro-ceed chondrification: when TK stage 9embryos were incubated at 34 to 36°Cfor 7 days, they reach PG stage 10,when cartilage primordia can bereadily seen as overt condensations of

Fig. 7. Histogenesis of P. sinensis somite in the chimera. Transverse sections of a Pelodiscussinensis–Gallus gallus chimera. Sections are cut at the site of graft and either stained with anti–P.sinensis antiserum and counterstained with hematoxylin (A,B,D,E,G,H,J,K,M), immunostainedwith MF-20 antibody and counterstained with hematoxylin and Alcian blue (C,F), or simply stainedwith hematoxylin and eosin and then Alcian blue (I,L,N). A–F: P. sinensis somite-derived musclesin 7-day chimera. B and C are adjacent sections at higher magnification corresponding to the boxin A, showing P. sinensis somite-derived epaxial muscle (arrows in B and C). At this level, theneural arch (na) and ribs are of host tissue. E and F are adjacent sections at higher magnificationcorresponding to the box in D, similarly showing the hypaxial muscle (arrows in E and F). Only themuscle fibers are of P. sinensis somite origin. G: Transverse section of a chimera incubated for 5days. H,I: Adjacent sections corresponding to the box in G at high magnification. Pelodiscussinensis somite-derived cells are observed in the dermis (d), dorsal wall of the dorsal aorta (a),mesenchyme, corresponding to the site of neural arch development (na), epaxial muscle primor-dium (em), prospective centrum (c), putative hypaxial muscle (hm), and mesonephros (mn).J–N: Seven days after surgery. K is a higher magnification of the box in M, which is a highermagnification of the box in J. L and N are sections adjacent to K and M, respectively. P. sinensiscells occur in tissues known to be somite derivatives (M and N). Neural crest-derived dorsal rootganglion (drg) is composed of chicken cells (M). Note that only a small population of P. sinensiscells on the right side of the host notochord (n; K) has differentiated into a small cartilage, stainedwith Alcian blue (L). Also note that the P. sinensis somite-derived hypaxial muscle (hm) morestrongly resembles the muscle in the normal P. sinensis embryo than that in the chicken embryo(compare Fig. 2A and 2B). nt, neural tube. Scale bars � 500 �m in A (applies to A,D), in G, in J,100 �m in B (applies to B–F), in H (applies to H,I), in K (applies to K,L), in M (applies to M,N).

DIFFERENTIATION OF TURTLE SOMITES 157

mesenchyme (see Figs. 4, 7M,N). Fur-thermore, as seen in chimeras incu-bated for 5 days, development of P.sinensis somite-derivatives and hosttissues synchronized well when com-pared with the chick–quail chimerasgrown for the same period (Figs. 2C,D, 7G–I). It is likely, thus, that thelength of incubation of the turtle–chick chimeras is sufficient to allowturtle cells to chondrify. Cartilage con-densations are readily visible in turtleembryos that have been similarly in-cubated.

It has long been known that chon-drogenesis is dependent also upon theembryonic environment (Hoadley,1925; Williams, 1942; Fowler andWatterson, 1953; Watterson et al.,1954; Avery et al., 1955, 1956; Kenny-Mobbs and Thorogood, 1987) and ap-parently proceeds in hierarchicalinteractive steps. For example, chon-drification of the centrum involves ac-tive migration of the early sclerotome-derived cells toward the notochord(Williams, 1910; Jacob et al., 1975;Chernoff and Lash, 1981), where theextracellular matrix (ECM) plays animportant role (Newgreen et al., 1986;Lash et al., 1987; Sanders et al.,1988). In the chimeras of the presentstudy, this process also seems to havetaken place, because P. sinensis cellsoften aggregated normally, lateral tothe notochord (Figs. 6A,B, 7H,M). Anaggregation of P. sinensis cells corre-sponding to the shape of the prospec-tive neural arch was also observed(Fig. 7H,I). However, these cells didnot show normal chondrification. A se-ries of experiments in chicken em-bryos showed that there are certaindevelopmental stages at which scle-rotome-derived cells are able to chon-drify without signals from the embry-onic environment (Fowler andWatterson, 1953; Watterson et al.,1954; Avery et al., 1956; O’Hare, 1972;Kenny-Mobbs and Thorogood, 1987).Therefore, it appears that P. sinensiscells failed to reach those particularstages in the present chimera. The en-vironmental factors required to bringP. sinensis cells to these stages remainunknown.

As described above, the reduction inP. sinensis somite-derived cartilagedid not arise from an incapacity forchondrocyte differentiation of the P.sinensis somitic cells per se in the host

environment, because P. sinensischondrocytes were observed (Fig. 7K–M). These minor cartilaginous nod-ules were always associated with hostcartilage (Fig. 7K,M), suggesting thatthey were induced through local, cell–cell contact-dependent homogeneticinduction, which has been reportedpreviously in chondrogenesis (Cooper,1965). Although we transplanted theP. sinensis somites together with thecovering surface ectoderm that hasbeen assumed to function in inducingrib differentiation (Huang et al., 2000and references therein), no ribs werefound in these chimeras, either.

The general absence of graft-de-rived cartilage in the chimera is prob-ably due to an incompatibility be-tween the chicken environment andthe chondrogenic mesenchyme de-rived from the P. sinensis somites. Wedo not know if this incompatibility isdue to the signaling system or to dif-ferences between these species in themolecules themselves that are in-volved in the same signaling mecha-nism or act as components of theECMs. Nor is it known whether suchdifferences are relevant to the turtle-specific pattern of rib growth. Therewas no clear difference in the expres-sion patterns of Shh beyond the stageof sclerotome formation (Fig. 8A–D),although the gene products may func-tion differently in the two species.Furthermore, up-regulation of Pax9 inthe graft-derived sclerotome showsthat the mesodermal cells of P. sinen-sis could normally respond to some ofthe signals derived from G. gallus, in-cluding the product of the Shh gene(Fig. 8E). In this context, it would beworthwhile to consider the anatomicaldifference of ribs between turtles andother amniotes. Namely, as found byGoette (1899), the development of theribs and neural arches of turtles hasshifted rostrally by a half segmentwhen compared with other amniotes,and segmental patterns of muscular,nervous, and skeletal systems havebeen accordingly reorganized second-arily in the turtle lineage (Hoffstetterand Rage, 1969). Such a differencemight have disturbed the normal pat-terning of the ribs in the chimera.Even in that circumstance, however,the middle segment of the trans-planted three somites could have gen-erated a normal turtle rib. However,

this does not explain the occasionalappearance of P. sinensis-derived neu-ral arch in the chimera or the specificloss of ribs. Lastly, the poor develop-ment of cartilage in the chimera mightbe related to the widely recognizedfact that maintenance of cartilage dif-ferentiation in a culture system tendsto require finely tuned extracellularconditions (Daniels and Solursh,1991, and references therein), inmarked contrast to muscle differenti-ation. An investigation of the differ-ences between the embryonic factorsof P. sinensis and G. gallus will beincluded in our future project.

EXPERIMENTALPROCEDURES

Embryos

Fertilized eggs of P. sinensis were ob-tained from several local fish farms inJapan during the breeding seasons(June to September) of 2002 and 2003.The eggs were allowed to grow in ahumidified incubator (for tempera-tures, see below). Developmentalstages (TK stages) were determinedbased on the previous description byTokita and Kuratani (2001). Fertil-ized eggs of the chicken G. gallus andthe Japanese quail Coturnix coturnixwere also purchased from a local farm,and the eggs were incubated in a hu-mid temperature-controlled chamber.The embryos were staged according toHamburger and Hamilton (1951). Toestablish the common stages betweenchicken and P. sinensis, hematoxylinand eosin (H&E) -stained sectionswere analyzed histologically (see be-low). Based on this developmentaltime table, the eggs of both specieswere incubated at different tempera-tures to determine the appropriateconditions under which chimerasshould be incubated (see below).

Construction of Chimeras

Chick embryos incubated for 2.5 daysat 38°C (24–26 somites, HH stage 15;Hamburger and Hamilton, 1951) wereused as the host. A window was madein the shell and the embryo was visu-alized with Indian ink diluted with0.9% NaCl/distilled water (1:5) in-jected into the subgerminal cavity.With a sharpened tungsten needle,

158 NAGASHIMA ET AL.

the surface ectoderm over the threenewly developed somites was peeledunilaterally (somite stages �I to �IIIat the thoracic level; Roman numeralsindicate the positions of somitescounted rostrally from the most newlyformed one that is called somite �I;Ordahl, 1993), and a few drops of Dis-pase (500 IU/mL in Tyrode’s solution;Godo Shusei Co., Ltd., Tokyo, Japan)were applied to the scar. After 5 min,the three somites were removed usinga tungsten needle and a glass capil-lary pipette. From the thoracic level ofa P. sinensis embryo that had beenincubated for 2 days at 30°C (17–21somites, TK stage 9), three somites atstages �I to �III were excised fromthe corresponding side with Dispase,rinsed in 0.9% NaCl/distilled water,and placed into the scar of the chickenhost along the same anteroposterioraxis (Fig. 1). Surgery was always per-formed unilaterally, and the nonoper-ated side was used as the control.Thirteen successful chimeras wereused for analysis. Similar transplan-tation of somites was also carried outbetween chicken and quail embryos(quail somites into chicken hosts) forcomparison.

Anti–P. sinensis Antiserum

P. sinensis embryos were collected atTK stage 14, homogenized, and in-jected into an adult female chicken.Serum was collected approximately 3months later (Sawady Technology, To-kyo, Japan). For immunohistochemis-try, embryos were fixed in either 4%paraformaldehyde (PFA) in phos-phate-buffered saline (PBS; pH 7.4) orBouin’s fixative, then dehydrated, andembedded in paraffin. Deparaffinizedsections (6 �m) were incubated withthe anti–P. sinensis antiserum (1:300)for 2 hr at room temperature. Horse-radish peroxidase (HRP) -conjugatedanti-chicken IgY (Bethyl Laborato-ries, Montgomery, TX) was used asthe secondary antibody. HRP reactiv-ity was visualized with the diamino-benzidine reaction.

Immunohistochemistry andHistochemistry

Histological observations were mademainly on H&E-stained sections,which were often further stained with

0.1% Alcian blue/distilled water, ac-cording to the method of Nowicki et al.(2003), to show the cartilage in olderembryos. To detect quail cells in chick-en–quail chimeras, the quail-specificantibody, QCPN (1:5; DevelopmentalStudies Hybridoma Bank [DSHB])was used on embryos fixed withSerra’s fixative (Serra, 1946). Forimmunohistochemistry with MF-20antibody (DSHB), which recognizestropomyosin, embryos were fixed with4% PFA/PBS, and 10-�m-thick sec-tions were prepared with a cryostat.Staining was performed by using themethod described by Kuratani andWall (1992). Biotin-conjugated anti-mouse IgG1 (Zymed, South San Fran-cisco, CA) was used as the secondaryantibody. Vectastain ABC Elite kit(Vector Laboratories, Burlingame,CA) was used to visualize the immu-noreaction. All histological imageswere taken using a CoolSNAP camera(RS Photometrics, Tucson, AZ) at-tached to a light microscope.

Isolation and Sequencing ofShh and Pax9 cDNAs and InSitu Hybridization

Total RNAs isolated from TK stage 14P. sinensis embryos were reverse-tran-scribed into cDNAs by using oligo(dT)primer with SuperScript III (Invitro-gen, Carlsbad, CA). These cDNAs wereused as templates for polymerase chainreaction (PCR) amplification with theExpand High Fidelity PCR System(Roche, Penzberg, Germany). Degener-ate primers were designed based onconserved amino acid residues as fol-lows (corresponding amino acids inparentheses): 5�-CTGACGCCNYT-NGCNTAYAARCARTT-3� (PLAYKQF)and 5�-TTCGCAGCTCANSWRTTYTC-NGCYTT-3� (KAENSVA) for Shh; and5�-GATCCNGGNATHTTYGCNTGG-GAR AT-3� (GIFAWEI) and 5�-GTAT-ACGGCATRTANGGNSWNACYTG-3�(QVSPYM) for Pax9. PCR was per-formed as follows: 2 min denaturationstep at 94°C; then 10 cycles of 94°C for15 sec, 48°C for 30 sec, and 72°C for 2min; followed by 30 cycles of 94°C for 15sec, 56°C for 30 sec, and 72°C for 2 min.The PCR products were purified by us-ing MinElute (Qiagen, Hilden, Ger-many) and cloned into the pT7Blue vec-tor (Novagen, Madison, WI). More thanthree independent clones were isolated

and sequenced with the 3100 GeneticAnalyzer (Applied Biosystems, FosterCity, CA). The orthology of P. sinensisShh and Pax9 cognates, including theamino acid sequences deduced from thecDNAs isolated and sequenced as de-scribed above, were confirmed by com-parison with those reported for othervertebrates using analysis of phyloge-netic trees (these sequences were depos-ited in GenBank with accession nos.AB181135 and AB181136).

For in situ hybridization, embryoswere embedded in paraffin after fixa-tion, sections (6 �m) were cut, and thedeparaffinized sections were treatedwith 0.1 M triethanolamine/1.0% HCl/0.2% acetic anhydride. After incuba-tion in hybridization buffer (50% for-mamide, 5 � standard saline citrate(SSC), 1% sodium dodecyl sulfate, 0.05mg/ml total yeast RNA, 50 mg/ml hep-arin sulfate, 5 mM ethylenediaminetet-raacetic acid–Na2, 0.1% CHAPS; Mu-rakami et al., 2001) for 2 hr at 65°C,slides were incubated in hybridizationbuffer containing digoxigenin-labeledRNA probe (0.1 �g/�l) at 65°C over-night. Sections were washed in 0.2�SSC at 65°C and at room temperaturefor 30 min each, incubated in 1% Block-ing Reagent (Roche) for 60 min, andthen incubated with Anti–Digoxige-nin-AP Fab Fragment (Roche) dilutedin 1% Blocking Reagent (1:4,000) atroom temperature for 2 hr. The anti-body detection reaction was performedas previously described (Murakami etal., 2001).

ACKNOWLEDGMENTSWe thank Raj Ladher, Kiyokazu Agata,and Yoshie Kawashima Ohya for criti-cal reading of the manuscript, andTakeshi Inoue for technical advice. Themonoclonal antibodies (MF20 devel-oped by Donald A. Fischman; QCPN byBruce M. Carlson and Jean A. Carlson)were obtained from the DevelopmentalStudies Hybridoma Bank developed un-der the auspices of the NICHD andmaintained by the Department of Bio-logical Sciences, University of Iowa,Iowa City, IA 52242. S.K was funded byGrants-in-Aid from the Ministry of Ed-ucation, Science and Culture of Japan(Specially Promoted Research).

DIFFERENTIATION OF TURTLE SOMITES 159

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