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 sig- naling 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 mo- lecular regulation of intramembranous ossification. In turtles, from developmental Greenbaum stage (G)15 onwards, we observed a series of mesenchymal condensations corresponding to each plas- tron bone forming within the lateral edges of the ventral mesen- chyme 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