Development of the Turtle Plastron, the Order-Defining Skeletal Structure

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Development of the turtle plastron, the order-defining skeletal structure

Ritva Ricea,1, Aki Kallonenb, Judith Cebra-Thomasc,d, and Scott F. Gilberta,c,1

aDevelopmental Biology, Institute of Biotechnology, University of Helsinki, Helsinki 00014, Finland; bDepartment of Physics, University of Helsinki, Helsinki 00014, Finland; cDepartment of Biology, Swarthmore College, Swarthmore, PA 19081; and dDepartment of Biology, Millersville University, Millersville, PA 17551

Edited by Clifford J. Tabin, Harvard Medical School, Boston, MA, and approved March 30, 2016 (received for review January 19, 2016) The dorsal and ventral aspects of the turtle shell, the carapace and the fontanel at the midline of the plastron. Moreover, processes extend plastron, are developmentally different entities. The carapace con- dorsally from the hyoplastron and hypoplastron to form a bridge that tains axial endochondral skeletal elements and exoskeletal dermal connects the plastron with the ribs and the carapace. In some turtles bones. The exoskeletal plastron is found in all extant and extinct (especially ancient lineages), a further set of paired plastron bones, species of crown turtles found to date and is synaptomorphic of the the mesoplastra, lie between the hyoplastra and hypoplastra (7). order Testudines. However, paleontological reconstructed transition Although the anatomy of plastron bones has been known for forms lack a fully developed carapace and show a progression of centuries, and the homology of these bones to the skeletal structures bony elements ancestral to the plastron. To understand the evolu- of other reptilian clades has been debated almost as long (3, 4, 6, 8), tionary development of the plastron, it is essential to know how it has we still know very little about how these intramembranous bones formed. Here we studied the molecular development and patterning arise within the ventral mesenchyme and why cartilage does not of plastron bones in a cryptodire turtle Trachemys scripta. We show form in this region. Both bone and cartilage arise from mesenchy- that plastron development begins at developmental stage 15 when mal condensations formed by osteochodrogenic progenitor cells, osteochondrogenic mesenchyme forms condensates for each plastron which are able to differentiate either to chondrocytes or osteoblasts bone at the lateral edges of the ventral mesenchyme. These conden- depending on their molecular regulation via key transcription fac-

sations commit to an osteogenic identity and suppress chondrogenesis. tors and signaling pathways (9). This dual potential in osteochon- BIOLOGY Their development overlaps with that of sternal cartilage develop- droprogenitor cells is largely due to coexpression of a chondrogenic DEVELOPMENTAL ment in chicks and mice. Thus, we suggest that in turtles, the transcription factor Sox9 and an osteochondrogenic transcription sternal morphogenesis is prevented in the ventral mesenchyme factor Runx2. For osteochondroprogenitor cells to commit to an by the concomitant induction of osteogenesis and the suppression osteogenic path, the chondrogenic potential must be suppressed; of chondrogenesis. The osteogenic subroutines later direct the this happens through the inhibition of expression of Sox9, while growth and patterning of plastron bones in an autonomous man- inducing or maintaining that of Runx2 (10, 11). Coexpression of ner. The initiation of plastron bone development coincides with the transcription factor Twist with Runx2 in osteoprogenitor cells that of carapacial ridge formation, suggesting that the develop- maintains these cells in a proliferative, undifferentiated state—Twist ment of dorsal and ventral shells are coordinated from the start directly binds the DNA-binding domain of Runx2 to inhibit its and that adopting an osteogenesis-inducing and chondrogenesis- function (12, 13). Once expression of Twist is switched off, Runx2 is suppressing cell fate in the ventral mesenchyme has permitted able to induce expression of Osterix (Osx) (14). This molecular turtles to develop their order-specific ventral morphology. regulation ensures osteogenic differentiation and maturation. Commitment to an osteogenic cell lineage also requires the se- turtle | plastron | osteogenesis | development quential activation of Hedgehog and canonical Wnt/β-catenin signaling in osteoprogenitor cells (15–18). Other critical regulatory he ventrum of turtles is covered by a protective shell, the plas- Ttron, composed of bony plates that are themselves covered by Significance keratinous scutes. The plastron is thought to be the oldest part of turtle’s shell as the earliest known turtles in the fossil record to Theplastron,theorder-defining skeletal structure for turtles, date—Odontochelys and Pappachelys—had plastron-like ventral provides a bony exoskeleton for the ventral side of the turtle. We bones, but only a partial carapace (1, 2). Most modern turtles have provide here the first molecular analysis of plastron bone forma- nine bones in the plastron that develop within the ventral mesen- tion. We show that plastron bone morphogenesis in the ventral chyme in between the ectodermal scutes and the visceral organs mesenchyme employs a program of bone formation that usually (Fig. 1). The anterior of the plastron contains a medial rostral characterizes the vertebrate face and skull. The plastron bones, entoplastron bone and two lateral rostral epiplastron bones that will however, have a preliminary step that is not included in head grow and suture together to form an anterior bone plate. The formation: They must suppress the usual chondrogenic programs entoplastron is thought to be derived from the interclavicle bone, that would create the sternum cartilage. We suggest that the early whereas the paired epiplastra are thought to be homologous to the osteogenic fate adopted by the ventral mesenchyme prevents the clavicles (3–5). In the primitive turtle Proganochelys a dorsal process chondrogenic sternal development in turtles and that this was a extending from the epiplastron bone to the carapace acted as a critical step in forming the ossification centers for this new type of clavicle but in modern turtles this dorsal process is missing. Instead, vertebrate structure. a modified acromion process of the scapula blade provides contact between the carapace and the entoplastron (6). Three pairs of lat- Author contributions: R.R. designed research; R.R. and A.K. performed research; R.R., A.K., — and J.C.-T. contributed new reagents/analytic tools; R.R., A.K., and S.F.G. analyzed data; eral plastron bones the centrally located hyoplastra and hypo- and R.R., A.K., J.C.-T., and S.F.G. wrote the paper. — plastra and the posterior xiphiplastron bones are thought to be The authors declare no conflict of interest. derived from the paired gastralia seen as floating ventral ribs in This article is a PNAS Direct Submission. numerous reptiles groups. This position is made stronger by the 1To whom correspondence may be addressed. Email: [email protected] or sgilber1@ paired gastralia seen forming a plastron-like structure in the stem swarthmore.edu. turtle Pappochelys (1). The posterior ends of the hyoplastron bones, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. together with the anterior ends of hypoplastra, form an umbilical 1073/pnas.1600958113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1600958113 PNAS | May 10, 2016 | vol. 113 | no. 19 | 5317–5322 Downloaded by guest on September 26, 2021 bone at the expense of cartilage in the same manner as calvarial and facial ectomesenchyme, the tissue most studied for the molecular regulation of intramembranous ossification. In turtles, from developmental Greenbaum stage (G)15 onwards, we observed a series of mesenchymal condensations corresponding to each plastron bone forming within the lateral edges of the ventral mesenchyme below the fore- and hindlimb buds. Each condensation followed the transcriptional code leading to osteogenic lineage commitment; initially Sox9 expressing osteochondrogenic precursor Fig. 1. The plastron of the hard-shelled turtle T. scripta.(A) Alizarin red stained embryonic plastron at G25. (B) At G22, the bridge bone from cells suppressed their chondrogenic potential and began to express the hyoplastron extended underneath the second rib (r2), and the tip of the genes directing osteogenesis. We also show that transient paired bridge bone grew past the rib anteriorly. The bridge extension of the mesoplaston osteogenic condensations formed between the hyo- hypoplastron had grown underneath the seventh rib (r7), and the tip of and hypoplastron condensations at the early stages of plastron bone the bridge bone extended past the rib posteriorly. Movie S1 demonstrates development. Characterization of plastron bone growth dem- the bridge extensions. (C) Hematoxylin–eosin-stained sagittal section onstrated that the formation of bone spicules is intrinsic to plastron showing an osteogenic front (arrow) of a hyoplastron bridge bone adjacent bone development and patterning, and that their growth is driven to a rib (r) at G19. e, epiplastron; en, entoplastron; fl, forelimb; hp, hypo- by the osteogenic signaling. The timing and location of early de- plastron; hy, hyoplastron; x, xiphiplastron; *, bridge extensions of hyo- and velopment and osteogenic commitment of plastron bone conden- hypoplastron;. (Scale bar in A, ∼0.5 cm.) sations seems to overlap with that of paired chondrogenic sternal condensations in birds and mice (28, 30). Thus, we suggest that the systems in intramembranous bone formation include the BMP- order-specific ventral morphology in turtles is created by intrinsic induced transcription factor Msx2, which promotes the proliferation patterning information in ventral mesenchyme that suppresses of preosteoblasts, while suppressing differentiation (19–21). chondrogenesis and drives osteogenesis. The plastron bones appear to have taken over the role of the Results ventral portion of the ribs and the sternum that are missing in turtles. In most vertebrates, the ribs develop from the somitic Osteogenic Fate of Plastron Bones and Ventral Mesenchyme. Turtle mesenchyme within the primaxial domain of the embryo. As they skeleton formation has been studied in multiple species from hard- expand, the ribs cross the lateral somitic frontier and enter into the shelled to soft-shelled turtles and from side-necked (Pleurodira)to – abaxial domain of the embryo (22). The sternum develops fully in hidden-neck turtles (Cryptodira) (33, 36 40). These studies have the lateral plate mesenchyme. In turtles, the rib development is emphasized the development of the carapacial skeleton, following affected by the turtle-specific signaling/organizing center called the the order of mineralization of skeletal elements during late em- carapacial ridge (23), and the ribs remain as primaxial structures bryogenesis and comparing carapace development between differ- that do not cross the lateral somitic frontier (24–27). In birds and ent species of turtles. Here we studied plastron bone development Trachemys scripta mice, the sternum develops in the lateral plate mesenchyme as in the hard-shelled red-eared pond slider from its earliest stages of development. We used X-ray microtomography paired rod-like cartilage anlagen under the posterior end of the (μCT) imaging to detect the mesenchymal condensations that will wing/forelimb bud, extending some distance posteriorly at the lateral become plastron bones, and phosphotungstic acid (PTA) was used edge of the ventral mesenchyme. The sternal cartilage anlagen de- as a contrast stain to show regions of higher tissue density within velop independently of the ribs or clavicles, and they migrate toward the turtle embryonic mesenchyme (41). At developmental stage the ventral midline where they eventually fuse (28–31). The sternal G17, nine definite areas encircling the periphery of the ventral Runx1 anlagen begin as mesenchymal condensations that express mesenchyme were seen to be denser (i.e., more condensed) than and Runx2. These transcription factors cooperate to induce the the surrounding mesenchyme (Fig. 2A). The locations of these expression of Sox5 and Sox6. Sox5 and Sox6 initiate chondrogenesis condensations matched the location of the nine future plastron in the condensed cells and subsequently lead to the induction of type bones (compare with Fig. 1A). A transverse digital section of a μCT II collagen expression, a chondrocyte-specific extracellular matrix imaged embryo and a hematoxylin–eosin-stained section of the component (31, 32). When only Runx2 expression is up-regulated in hyoplastron condensation showed extracellular matrix in the center the lateral plate mesenchyme before sternal morphogenesis, it in- and condensed mesenchymal cells surrounding it, indicating that terferes with chondrogenesis and promotes formation of ectopic the developing plastron bones are osteoids at this stage (Fig. 2 B intramembranous bone in the ventral mesenchyme (32). and C). Nothing resembling rod-like sternal cartilage anlagen was Reptilian gastralia, the homologs of the posterior plastron bones, seen in μCT imaged samples (Fig. 2A), indicating that sternum develop similarly to plastron bones in the ventral mesenchyme development was not initiated in the turtle. through intramembranous ossification. Despite their apparent Osteochondrogenic marker gene expression patterns allowed us similarities, they are not equal. Each gastralial bone consists of a to establish a developmental timeline and the locations of osteo- lateral and a medial bone element that articulate with each other, genesis in the developing plastron. The initiation of plastron bones and gastralial bones do not form bone spicules. Whereas plastron was seen at G15 when osteochondrogenic mesenchymal conden- bones start to mineralize from the periphery of the ventrum in a sations for the hyo-, hypo-, and xiphiplastron were expressing Sox9 slight anterior-to-posterior preference (33), gastralia mineralize in in distinct regions at lateral edges of ventral mesenchyme between a posterior-to-anterior sequence such that the posteriormost row of the fore- and hindlimb but were not yet expressing Runx2 (Fig. 3 A paired lateral elements of gastralia mineralize first. Once the lateral and B). By G16, the expression patterns of Sox9 and Runx2 were elements have mineralized, the medial elements begin to miner- reversed (Fig. 3 C and D), indicating that chondrogenic potential alize, starting again at the posterior end (34). Other exoskeletal had been suppressed and the condensations had committed to an dermal bones found in various reptilian taxa include osteoderms, osteogenic fate. Lack of chondrocytes was confirmed by lack of which differ from plastron bones in that they ossify via the meta- collagen II expression (Fig. 3D). Osteogenic plastron bone con- plasia of fibrous connective tissue into bone (34). Unlike the de- densations expressed Runx2 throughout the condensations (Fig. veloping plastron bones, which develop intramembranously, these 3E). Twist was expressed at the osteogenic fronts of the conden- osteoderms lack osteoblasts, osteoid, and periosteum (35). sations and only minimally within them (Fig. 3F). The expression of Here we demonstrate that the ventral mesenchyme of the Msx2 overlapped with that of Runx2 in the developing plastron Emydid cryptodire turtle Trachemys scripta elegans is biased to form bones except for the Twist-positive osteogenic fronts (Fig. 3G), and

5318 | www.pnas.org/cgi/doi/10.1073/pnas.1600958113 Rice et al. Downloaded by guest on September 26, 2021 Developmental Patterning of Plastron Bones. To connect the dorsal and ventral exoskeletons, the hyo- and hypoplastron bones grow extensions from their lateral edges that reach underneath the rib cage in the carapace (Fig. 1, Movie S1). To follow bridge bone growth, we cultured G18 plastron explants in vitro with fluorescent

Fig. 2. Plastron bone condensations. (A) PTA-stained G17 embryo showing nine developing plastron bones. (B) Digital transverse section of hyoplastron shown in A.(C) Hematoxylin–eosin-stained transverse section of forming hyoplastron consisting of an extracellular matrix-filled center surrounded by condensed preosteoblastic cells. Color code: red, epiplastron; yellow, hyoplastron; green, hypoplastron; blue, xiphiplastron. These have pairs on the opposite side. Orange ^ indicates the entoplastron as barely visible linear bar at midline. fl, forelimb. (Scale bar in histological section, ∼0.5 mm.)

Osx expression was found in the core of each of the developing BIOLOGY

plastron bones (Fig. 3H). Thus, the plastron bones displayed the DEVELOPMENTAL same combinations and sequence of osteogenic transcription factors found in any developing intramembranous bones: the centers of the condensations expressed osteogenic factors that drive the osteogenesis toward differentiation (Msx2, Runx2,andOsx), whereas the osteogenic fronts of the condensations were maintained as proliferative osteoprogenitors (Twist1 and Runx2) to allow further growth. Surprisingly, the thin ventral mesenchyme between the opposing lateral plastron bone condensations was not undifferentiated but also showed osteogenic potential by expressing Runx2 by G15, and Runx2, Msx2,andTwist1 by G16 (Fig. 3 B and E–G,whitear- rows). The central area expressed the chondrogenic marker gene Sox9 at a minimal level at G15 but not at G16 (Fig. 3 A and C), and it did not express Collagen II (Fig. 3D). Preskeletal mesenchyme, before osteogenic or chondrogenic identity, expresses Prrx1 (42). Prrx1 regulates the formation of osteogenic and chondrogenic condensations, and it is found broadly expressed in the ventral mesenchyme in chicks at Hamburger Hamilton (HH)25 and in mice at embryonic day (E)12; but no expression of the osteogenic marker genes Runx2, Twist,orMsx2 are seen at this stage (32, 42–44). In turtles, at developmental stage G16, both the central ventral mesenchyme and the lateral regions of the ventral mesenchyme that correspond to plastron bone initiation sites expressed Prrx1 (Fig. 3I). Fig. 3. Osteogenic fate of plastron bone condensations and ventral mesenchyme in turtles. (A and B) At G15, the plastron bone condensations and ventral Transient Mesoplaston. The molecular analysis of osteogenic mark- mesenchyme exhibited osteochondrogenic potential by being Sox9 positive ers revealed that T. scripta embryos had a transient plastron bone (circled in A) with minimal Runx2 expression (B). (C and D) At G16, the chon- condensation between the hyo- and hypoplastron condensations drogenic potential of both the plastron bone condensations (orange circle in C) – reminiscent of the mesoplastron (Fig. 3 E–H, black arrows). At G16, and the ventral mesenchyme had been suppressed. (E H) By G16, an osteogenic the potential mesoplastron primordium transiently expressed Msx2, condensation for each plastron bone had formed at the lateral edges of the developing plastron and the central area of the ventral mesenchyme had an Runx2, and minimal levels of Osx.AlowlevelofTwist expression osteogenic identity. Runx2 was expressed throughout the condensations (E). Its extended across the mesenchyme between the hyo- and hypo- suppressor Twist1 was expressed by osteoprogenitor and preosteoblast cells in plaston. However, by G18, the mesoplastron condensation was no osteogenic fronts at the periphery of the condensations (F). Low level of Twist longer visible as a Runx2-positive area between hyo- and hypo- expression located between hyo- and hypoplastral condensations, and Msx2 plastron (Fig. S1). The μCT image of the PTA-stained G17 embryo was expressed by preosteoblasts in the center of the condensations (G). The did not reveal any condensation for mesoplastron, indicating that it ventral mesenchyme had osteogenic identity (white arrows in E–G). Osterix does not secrete any significant amounts of extracellular matrix or (Osx) was expressed in the core of the condensations, indicating osteogenic differentiation (H). A transient osteogenic condensation (black arrows in E–H) that it has not reached a critical density that would render it de- may represent the mesoplaston. (I) Prrx1 was expressed in preskeletal mesen- tectable (Fig. 2A). The site of a transient mesoplaston was not chyme at G16 in both the osteogenic plastron condensates and ventral mes- aligned with the location of the scutes; rather it was positioned enchyme. Asterisks are adjacent to condensations. Yellow, hyoplastron; green, between pectoral and abdominal scutes (Fig. 4 C and E). hypoplastron; blue, xiphiplastron. (Scale bar, ∼250 μm.)

Rice et al. PNAS | May 10, 2016 | vol. 113 | no. 19 | 5319 Downloaded by guest on September 26, 2021 traversed the midline of the plastron and crossed paths with their contralateral counterparts (hyo-, hypo, and xiphiplastron bones), and they also crossed paths ipsilaterally with ones from neighboring plastron bones (epi- and hyoplastron, hyo- and hypoplastron, and hypo- and xiphiplastron). The bone spicules organized themselves so as not to touch one another, and they often alternated with the ones from neighboring/opposing bone plates (Figs. 1A and 4 D and E). Although the recruitment of the ossification pathways used in other intramembranous bones seems evident in the turtle plastron, there is no similar long and alternating bone spicule organization during avian or mammalian intramembranous ossification. To follow the development of bone spicules, we dissected G18 plastrons for organ culture supplemented with fluorescent calcein in the culture medium; after 24 h in culture, de novo bone deposition was seen mostly in the core of the plastron bone and no bone spicules were labeled. After a 48-h culture, short bone spicules were visible. By 72 h in culture, the mineralization was uniform throughout the bone plate but bone spicules had not grown longer. Short bone spicules radiated at even intervals along the medial edge of the cultured hyoplastron toward the midline (Fig. 4 F–H). The same pattern of plastron bone growth was seen in vivo at G19—short bone spicules radiating toward the midline from the medial edge of the uniformly mineralized bone plate (Fig. 4 I and J). Thus, G18 explants matured to G19 during the 3-d in vitro organ culture. The developing bone spicules were Runx2 positive, which was expressed in the osteogenic fronts surrounding the spicules. These osteogenic fronts surrounding the spicules were shown to be proliferative by BrdU incorporation (Fig. 4 I–K). Similarly to bridge bone extensions, bone spicules appeared to be part of the autonomous, intrinsic development and patterning of plastron bones driven by the osteogenic signaling. Sequential and overlapping Hedgehog and canonical Wnt signaling direct osteochondrogenic cells to choose an osteogenic path and prevent them from transdifferentiating into chondrocytes (15–17). Turtle preosteoblasts surround the plastron bone plate at G18, a stage that is before mineral deposition, and bone spicules at G22 showed nuclear localization of β-catenin (Fig. S2), indicating active canonical Wnt signaling. Patched protein was localized at the oste- Fig. 4. Development and patterning of bridge bones and bone spicules. (A and ogenic fronts of plastron bones and bone spicules (Fig. S2), repre- B) Incorporation of fluorescent calcein into cultured G18 plastron explants senting active Hedgehog signaling. showed newly grown bone from the hyo- and hypoplastron bone plates (Upper Thus, the molecular regulation responsible for intramembranous and Lower, respectively) after a 24-h culture (A)anda72-hculture(B). (C) μCT bone formation and patterning in other vertebrates also drives image of PTA-stained G19 embryo showed that bridge bones reached un- plastron bone development in turtles. The critical difference appears derneath the rib cage (white arrows). (D) G21 alizarin red staining and (E) μCT to be the suppression of chondrogenesis in the ventral mesenchyme imaging of PTA-stained plastra demonstrated the extent of mineralized and of the turtle, preventing abaxial cartilage structures from forming. unmineralized bone spicules. (F–H) G18 plastron explants cultured in vitro with fluorescent calcein. The hyoplastron explant after 24 h (F), 48 h (G), and 72 h Discussion (H) in culture showed the initiation of bone spicules at regular intervals toward the midline of the explant. The 72-h–cultured G18 hyoplastron resembled a G19 All turtles have a unique body plan: an autapomorphic carapace on hyoplastron. (I) Bone spicules expressed Runx2.(J and K)Bonespiculesgrewby the dorsal side that contains dorsal ribs and carapacial bony plates, proliferative osteogenic fronts throughout their development; Runx2-positive and a ventral shell of intramembranous bones that form a Chelonia- osteogenic fronts at G21 (J) and BrdU incorporation into osteogenic fronts and specific plastron. The dorsal and ventral shells develop as separate the tips of the bone spicules at G21 (K). f, fontanelle. and different entities in turtles. The carapace contains endochondral axial skeletal elements, and during evolution its development has required multiple skeletal and muscular changes, including calcein added to the culture medium to follow de novo bone de- broadening of the ribs, shortening the trunk to nine dorsal verte- position. After 24 h in culture, bridge bones had extended from the brates, dermal bone outgrowth from the perichondral collar, en- lateral edge of the plastron bone plates, and by 72 h they had grown capsulation of the scapula ventral to ribcage, and loss of intercostal to resemble the bridge bones seen in vivo at G19 (Fig. 4 A–C). In muscles and reorganization of respiratory muscles (45, 46). Emer- plastron explants, the carapace and ribs were not present to provide gence of plastron bones in turtles has been thought to result from directional cue(s), yet the morphology resembled that of a bridge modifications of the pectoral girdle and gastralial bones (3, 4, 6, 8, bone forming in vivo, indicating that the hyo- and hypoplastron 45). Meanwhile, the lack of cartilaginous sternum on the ventral bones rely on autonomous, intrinsic patterning information to grow side of turtles has not been previously considered from an evolu- bridge extensions. tionary or molecular perspective. By G21, the plastron bones have grown to cover a significant All crown turtles discovered to date have had a full plastron. portion of the forming plastron. Each plastron bone plate was Several recent studies have shown turtles to be the sister group to formed of a trabecular bone core, with extensive bone spicules ra- the archosaurs (47–51), and a possible ancestral lineage has been diating away from it and mineralizing (Figs. 1A and 4 D and E). proposed (45). The turtle plastron, its possible origin (52, 53) and MicroCT imaging showed that the bone spicules were much longer homologies (6, 45) to skeletal elements found in other tetrapods than alizarin red staining indicated; nonmineralized bone spicules have been studied and discussed for decades, yet this is the first

5320 | www.pnas.org/cgi/doi/10.1073/pnas.1600958113 Rice et al. Downloaded by guest on September 26, 2021 study to our knowledge that has established a developmental timeline and location for plastron bone development from their early uncommitted mesenchymal condensations to the development and growth of bone extensions. This study also investigates the patterning informationusedinplastronbonegrowth. The first sign of a turtle being a turtle during development is the formation of a signaling/organizing center called the carapacial ridge (CR) forming along the flank between fore- and hindlimbs at developmental stage G14 (26). By G16, the CR has grown to circle the dorsal carapacial disk that includes the ribs. The CR is essential for the turtle-specific rib phenotype—it attracts or patterns the spacing between ribs, and it is thought to be responsible for the lack of ventral ribs (25, 27, 33). This leads to the unusual situation in the turtle where the abaxial domain, the lateral plate mesenchyme, including elements that have invaded into it or migrated into it, is Fig. 5. Hypothesis of sternum, plastron, and gastralia development. The devoid of these endochondral skeletal elements. Instead, exoskeletal turtle is depicted on the Top and the alligator at the Bottom of the sche- plastron bones have formed to provide support and protection to the matic ventral side of an embryo. Anterior is to the Right. Chondrogenic ventral side of the body, and a muscular sheath encloses the lungs. sternal morphogenesis begins on lateral sides of the embryo extending from Here we have shown that plastron bone elements began to develop the posterior end of the forelimb posteriorly, based on chick and mouse as individual mesenchymal condensations at developmental stage studies (black dashed rectangle), but it does not form in turtles. This may be due to the osteogenic field of plastron bone condensations (circles) over- G15 at the periphery of the ventral mesenchyme (Fig. 3). At this lapping the sternal morphogenic area during the same developmental stage, the first turtle-specific structure, the CR, has been established window. In alligators, it is not known when or where sternal morphogenesis and rib growth will be limited to the primaxial domain of the embryo. begins. The gastralia begin to develop one to two developmental stages Thus, just as the development of the carapace has begun, so has the later than the plastron, and they develop in posterior-to-anterior sequence development of the plastron. The mesenchymal condensations of (black arrow); the lateral parts of the gastralial bones (orange) form first, intramembranous bones, such as those in the cranium, are Sox9- and this is followed by the formation of the medial parts (blue). Gastralia lie posterior to the sternum. The osteogenic gastralia development is unlikely expressing osteochondrogenic condensations (10, 54). Soon after BIOLOGY to coincide with chondrogenic sternal development. Diagram is not to scale.

their formation, the chondrogenic potential is suppressed, and the DEVELOPMENTAL condensed cells commit to osteogenesis. This appears to also be true Circles: red, epiplastron; yellow, hyoplastron; magenta, transient mesoplastron; green, hypoplastron; blue, xiphiplastron; f, fontanelle. in the turtle plastron: the early osteochondrogenic condensations at G15 suppressed their chondrogenic potential by G16 and committed to osteogenesis (Fig. 3). The plastron bone condensations contained row at FS21 in alligator. Developmental stage FS19 in Alligator the intrinsic patterning information necessary to grow into fully pat- mississippiensis is approximately the same as G17 in T. scripta (56, ternedplastronbones(Figs.3,4,and5).Boththebridgeboneex- 57). Thus, gastralial development begins at approximately one to tensions from hyo- and hypoplastron and the bone spicules grew in two developmental stages later than plastron development. Also, plastron explants in vitro to resemble those formed in vivo. Similarly, in the mouse, intramembranous bone plates have been shown to be the adult alligator gastralia remain posterior to sternum. Thus, it is autonomous from the surrounding mesenchyme during their growth possible that the development of gastralial elements does not andpatterning,andnoneighboringmesenchymalcellsarerecruited overlap with the timing or location of sternal development in alli- to the growing bone plates (55). Also, no chondrogenesis is allowed gators (Fig. 5). inthemesenchymeonceosteogeniccommitmentismade(54). We suggest that in turtles, the suppression of chondrogenesis and In turtles, both the lateral regions of the ventral mesenchyme and the induction of osteogenesis in the ventral mesenchyme prohibit the central region in between them were found to be osteogenic the formation of the sternum. In other words, turtles choose (Fig. 3). This osteogenic potential is maintained in the ventral plastron bones. mesenchyme later in development: mesenchymal cells isolated at G19 differentiated readily into osteoblasts in cell culture (53). At Methods this stage bone spicules begin to form (Fig. 4) and osteogenic po- Material. T. scripta elegans eggs were purchased from the Kliebert Turtle and tential of the surrounding mesenchyme may support their growth. Alligator Farm. Animal work was performed in accordance with guidelines and The timing and location of the initial plastron bone condensations approval from Finnish National Board of Animal Experimentation. Eggs were in the lateral regions of the ventral mesenchyme at G15 matched the incubated in a humidified incubator at 30 °C. Embryos were staged according to developmental timing and location of sternal cartilage anlagen Greenbaum (G) (57). T. scripta elegans’s total RNA was isolated from de- formation in avians (HH24 onwards) and mice (E12 onwards) (28, velopmental stage G14 and G17 embryos and reverse transcribed into cDNAs, 30). Mutant mice that express ectopic Runx2 in the central ventral which were used as templates to clone T. scripta Osx, Prrx1, Runx2,andSox9. mesenchyme have no sternal cartilages at E15.5 and only mal- μ formed and unfused sternal cartilages at E18.5. This phenotype is X-Ray CT Imaging. The 4% (wt/vol) PFA-fixed and 0.3% phosphotungstic caused by formation of interfering ectopic intramembranous bones acid (PTA)-stained samples were imaged with a custom-built phoenix X-ray Nanotom 180 NF (GE Measurement and Control Solutions) with X-ray tube in the ventral mesenchyme (32). In these mutant animals, the Prrx1 voltage of 80 kV and tube current of 180 μA. promoter was used to drive ectopic Runx2 expression in the central ventral mesenchyme starting at E9, which is before sternal mor- Organ Culture. Dissected plastra were placed skin-side up on Nuclepore filters phogenesis at E12. The chondrogenic potential is not suppressed in supported by grids in the Trowell type organ culture system (58). Details for these mutant mice and sternal cartilage anlagen are allowed to form. all methods are available in SI Methods. Reptiles with intramembranous gastralia also have an endochondral sternum. Gastralia development begins by forming the posteri- ACKNOWLEDGMENTS. We thank Agnès Viherä, Eija Koivunen, and Raija ormost row of the paired lateral elements of gastralia first. This is Savolainen for technical assistance and Profs. David Rice and Jukka Jernvall followed by the development of the medial elements of the gastralia for comments on the manuscript. Funding was provided by the Academy of Finland (S.F.G.), the National Science Foundation (S.F.G. and J.C.-T.), Swarth- originating from the caudal end (34). As the first row of paired more College Faculty Research awards (to S.F.G.), Millersville University fac- lateral parts of the gastralia form at developmental Ferguson stage ulty research grants (to J.C.-T.), and the Pennsylvania State System of Higher (FS)19 and the first pair of medial parts appear in the caudalmost Education (J.C.-T.).

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