The scutes of the turtle shell are epidermal shields that begin their formation during the early stages of shell development. Like other skin appendages, turtle scutes are hypothesized to be patterned by reaction–diffusion systems. We have previously established ex vivo and in silico systems to study these mechanisms experimentally and have further shown that mathematical models can explain the dynamics of the induction of turtle scute primordia and the generation of final scute architecture. Using these foundations, we expand our current knowledge and test the roles of ectodysplasin and activin signaling in the development of turtle scutes. We find that these molecules play important roles in the prepatterning of scute primordia along the carapacial ridge and show that blocking Edar signaling may lead to a complete loss of marginal scute primordia. We show that it is possible to reproduce these observations using simple mathematical modeling, thereby suggesting a stabilizing role for ectodysplasin within the reaction–diffusion mechanisms. Finally, we argue that our findings further entrench turtle scutes within a class of developmental systems composed of hierarchically nested reaction–diffusion mechanisms, which is conserved across ectodermal organs.

The bones of the plastron, the ventral portion of the turtle shell, develop through intramembranous ossification, in the same manner as facial bones, suggesting they are also produced by neural crest cells (NCCs). Previous work has demonstrated the existence of a unique second migration of NCCs away from the neural tube of turtle (Trachemys scripta) embryos; these migrate ventrally, appear to be skeletogenic and may contribute to the bones of the plastron. The goal of this project is to examine the expression of neural crest specifiers in the premigratory NCCs located in the neural tube and in migrating NCCs using immunofluorescence and whole mount in situ hybridization. During the switch‐over period, the neural crest specifiers Sox9 and Sox10 are both expressed in premigratory NCCs. However, their expression diverges in migrating NCCs; the cells that retain Sox10 are located within the major migratory stream in the rostral somite leading to the production of the dorsal root ganglion and other peripheral nerves, consistent with a neurogenic cell fate. By contrast, cells that retain Sox9 appear to take a novel migratory pathway through the caudal portion of the somite, and are excluded from the dorsal root ganglia, consistent with a non‐neurogenic and perhaps skeletogenic cell fate. We are currently examining the expression of transcription factors that have been found to be preferentially expressed in either trunk NCCs or cranial NCCs in the well‐characterized chicken (G. gallus) system. Elucidating the molecular control of NCC specification in the unique population of trunk NCCs would further our understanding of its role in the development of the turtle shell.

Reptiles have great taxonomic diversity that is reflected in their morphology, ecology, physiology, modes of reproduction, and development. Interest in comparative and evolutionary developmental biology makes protocols for the study of reptile embryos invaluable resources. The relatively large size, seasonal breeding, and long gestation times of turtles epitomize the challenges faced by the developmental biologist. We describe protocols for the preparation of turtle embryos for ex ovo culture, electroporation, in situ hybridization, and microcomputed tomography. Because these protocols have been adapted and optimized from methods used for frog, chick, and mouse embryos, it is likely that they could be used for other reptilian species. Notes are included for alligator embryos where appropriate.

Turtle plastron bones develop by intramembranous ossification, suggesting that they are derived, like the facial bones, from neural crest cells. Using cell‐labeling and neural tube explant cultures, we have shown that cells expressing neural crest markers emerge from the trunk neural tube in the turtle Trachemys scripta in two migratory phases over a greatly extended period. These findings were confirmed by electroporation of plasmids driving expression of fluorescent proteins. The early phase of migration (comparable to the period of neural crest cell (NCC) migration in other amniotes) extends until stage G10–11. These cells give rise to typical NCC derivatives. The NCCs that emerge late (beginning in stage G15–16 turtle embryos, well beyond the stage of neural crest migration in chick or mouse embryos) appear to migrate ventrally to form an ectomesenchymal dermis that gives rise to the bones of the plastron. Thus, there appear to be two distinct migratory phases in vivo. To test the hypothesis that the second wave of trunk NCCs in turtle embryos is capable of differentiating into bone, T. scripta neural tubes were cultured. The resulting NCCs were allowed to differentiate, and the differentiated cell types produced were analyzed by immunofluorescence. A subset of the cultures produced melanocytes, a typical trunk NCC‐derived cell type, while virtually all of the cultures produced a substantial number of osteoblasts. Our results suggest that the late trunk NCCs are predisposed to differentiate into osteoblasts, and thus provide good candidates for the cells that form the plastron. Craniosynostosis is a common human developmental deformity involving premature fusion of the calvarial sutures between the bones of the skull. A better understanding of intramembranous ossification, and analysis of an enriched population of osteogenic NCCs, could result in improved treatment options.Support or Funding InformationNational Science Foundation, Millersville University, and Stowers InstituteThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ectothermal reptiles have internal pigmentation, which is not seen in endothermal birds and mammals. Here we show that the development of the dorsal neural tube-derived melanoblasts in turtle Trachemys scripta is regulated by similar mechanisms as in other amniotes, but significantly later in development, during the second phase of turtle trunk neural crest emigration. The development of melanoblasts coincided with a morphological change in the dorsal neural tube between stages mature G15 and G16. The melanoblasts delaminated and gathered in the carapacial staging area above the neural tube at G16, and differentiated into pigment-forming melanocytes during in vitro culture. The Mitf-positive melanoblasts were not restricted to the dorsolateral pathway as in birds and mammals but were also present medially through the somites similarly to ectothermal anamniotes. This matched a lack of environmental barrier dorsal and lateral to neural tube and the somites that is normally formed by PNA-binding proteins that block entry to medial pathways. PNA-binding proteins may also participate in the patterning of the carapacial pigmentation as both the migratory neural crest cells and pigment localized only to PNA-free areas.

Interest in the origin and evolution of the turtle shell has resulted in a most unlikely clade becoming an important research group for investigating morphological diversity in developmental biology. Many turtles generate a two-component shell that nearly surrounds the body in a bony exoskeleton. The ectoderm covering the shell produces epidermal scutes that form a phylogenetically stable pattern. In some lineages, the bones of the shell and their ectodermal covering become reduced or lost, and this is generally associated with different ecological habits. The similarity and diversity of turtles allows research into how changes in development create evolutionary novelty, interacting modules, and adaptive physiology and anatomy.

The dorsal and ventral aspects of the turtle shell, the carapace and the plastron, are developmentally different entities. The carapace contains axial endochondral skeletal elements and exoskeletal dermal bones. The exoskeletal plastron is found in all extant and extinct species of crown turtles found to date and is synaptomorphic of the order Testudines. However, paleontological reconstructed transition forms lack a fully developed carapace and show a progression of bony elements ancestral to the plastron. To understand the evolutionary development of the plastron, it is essential to know how it has formed. Here we studied the molecular development and patterning of plastron bones in a cryptodire turtle Trachemys scripta. We show that plastron development begins at developmental stage 15 when osteochondrogenic mesenchyme forms condensates for each plastron bone at the lateral edges of the ventral mesenchyme. These condensations commit to an osteogenic identity and suppress chondrogenesis. Their development overlaps with that of sternal cartilage development in chicks and mice. Thus, we suggest that in turtles, the sternal morphogenesis is prevented in the ventral mesenchyme by the concomitant induction of osteogenesis and the suppression of chondrogenesis. The osteogenic subroutines later direct the growth and patterning of plastron bones in an autonomous manner. The initiation of plastron bone development coincides with that of carapacial ridge formation, suggesting that the development of dorsal and ventral shells are coordinated from the start and that adopting an osteogenesis-inducing and chondrogenesis-suppressing cell fate in the ventral mesenchyme has permitted turtles to develop their order-specific ventral morphology.

Two of the major controversies in the present study of turtle shell development involve the mechanism by which the carapacial ridge initiates shell formation and the mechanism by which each rib forms the costal bones adjacent to it. This paper claims that both sides of each debate might be correct-but within the species examined. Mechanism is more properly "mechanisms," and there is more than one single way to initiate carapace formation and to form the costal bones. In the initiation of the shell, the rib precursors may be kept dorsal by either "axial displacement" (in the hard-shell turtles) or "axial arrest" (in the soft-shell turtle Pelodiscus), or by a combination of these. The former process would deflect the rib into the dorsal dermis and allow it to continue its growth there, while the latter process would truncate rib growth. In both instances, though, the result is to keep the ribs from extending into the ventral body wall. Our recent work has shown that the properties of the carapacial ridge, a key evolutionary innovation of turtles, differ greatly between these two groups. Similarly, the mechanism of costal bone formation may differ between soft-shell and hard-shell turtles, in that the hard-shell species may have both periosteal flattening as well as dermal bone induction, while the soft-shelled turtles may have only the first of these processes.

Turtle plastron bones develop by intramembranous ossification, suggesting that they are derived, like the facial bones, from neural crest cells. Using cell‐labeling and neural tube explant cultures, we have shown that cells expressing neural crest markers emerge from the trunk neural tube in the turtle Trachemys scripta for a greatly extended period compared other model amniotes. The neural crest cells that emerge in a second wave, well beyond the stage of neural crest emigration in chick or mouse embryos, appear to migrate ventrally to form an ectomesenchymal dermis that gives rise to the bones of the plastron. The specification of premigratory neural crest cells and the epithelial‐mesenchymal transition that produces migratory neural crest cell is controlled by a gene regulatory network including the transcription factors Snail2, FoxD3, Sox9, and Sox10. We are currently examining the expression of markers of premigratory and early migratory neural crest cells to examine whether the premigratory domain persists during the period in between the early and late migratory phases. If the expression of these markers persist throughout this period, it will suggest that the premigratory region is maintained, and that the lack of neural crest cell migration may be due to the lack of a supportive environment. In contrast, if these genes are only expressed during the periods of active neural crest cell emigration, then the second wave of neural crest cell migration would require a second inductive signal not found in chick embryos.Grant Funding Source: NSF

The origin of the turtle shell over 200 million years ago greatly modified the amniote body plan, and the morphological plasticity of the shell has promoted the adaptive radiation of turtles. The shell, comprising a dorsal carapace and a ventral plastron, is a layered structure formed by basal endochondral axial skeletal elements (ribs, vertebrae) and plates of bone, which are overlain by keratinous ectodermal scutes. Studies of turtle development have mostly focused on the bones of the shell; however, the genetic regulation of the epidermal scutes has not been investigated. Here, we show that scutes develop from an array of patterned placodes and that these placodes are absent from a soft-shelled turtle in which scutes were lost secondarily. Experimentally inhibiting Shh, Bmp or Fgf signaling results in the disruption of the placodal pattern. Finally, a computational model is used to show how two coupled reaction-diffusion systems reproduce both natural and abnormal variation in turtle scutes. Taken together, these placodal signaling centers are likely to represent developmental modules that are responsible for the evolution of scutes in turtles, and the regulation of these centers has allowed for the diversification of the turtle shell.

Background: The turtle plastron is composed of a keratinized epidermis overlying nine dermal bones. Its developmental origin has been controversial; recent evidence suggests that the plastral bones derive from trunk neural crest cells (NCCs).Results:This study extends the observations that there is a turtle‐specific, second wave of trunk NCC delamination and migration, after the original NCCs have reached their destination and differentiated. This second wave was confirmed by immunohistochemistry in whole‐mounts and serial sections, by injecting DiI (1,1′, di‐octadecyl‐3,3,3′,3′,‐tetramethylindo‐carbocyanine perchlorate) into the lumen of the neural tube and tracing labeled cells into the plastron, and by isolating neural tubes from older turtle embryos and observing delaminating NCCs. This later migration gives rise to a plastral ectomesenchyme that expresses NCC markers and can be induced to initiate bone formation.Conclusions:The NCCs of this second migration have properties similar to those of the earlier NCCs, but also express markers characteristic ofcranialNCCs. The majority of the cells of the plastron mesenchyme express neural crest markers, and have osteogenic differentiation capabilities that are similar or identical to craniofacial ectomesenchyme. Our evidence supports the contention that turtle plastron bones are derived from a late emigrating population of cells derived from the trunk neural crest.Developmental Dynamics 242:1223–1235, 2013. © 2013 Wiley Periodicals, Inc.

The bony shell of the turtle is an evolutionary novelty not found in any other group of animals, however, research into its formation has suggested that it has evolved through modification of conserved developmental mechanisms. Although these mechanisms have been extensively characterized in model organisms, the tools for characterizing them in non-model organisms such as turtles have been limited by a lack of genomic resources. We have used a next generation sequencing approach to generate and assemble a transcriptome from stage 14 and 17 Trachemys scripta embryos, stages during which important events in shell development are known to take place. The transcriptome consists of 231,876 sequences with an N50 of 1,166 bp. GO terms and EC codes were assigned to the 61,643 unique predicted proteins identified in the transcriptome sequences. All major GO categories and metabolic pathways are represented in the transcriptome. Transcriptome sequences were used to amplify several cDNA fragments designed for use as RNA in situ probes. One of these, BMP5, was hybridized to a T. scripta embryo and exhibits both conserved and novel expression patterns. The transcriptome sequences should be of broad use for understanding the evolution and development of the turtle shell and for annotating any future T. scripta genome sequences.

Turtle plastron bones develop by intramembranous ossification, suggesting that they are derived, like the facial bones, from neural crest cells. Well after the initial wave of neural crest migration, cells expressing HNK1 and the early neural crest marker FoxD3, begin accumulating in the thickened dermis of the carapace and migrating to the developing plastron. This second, later wave of HNK1+ cells can also be observed migrating away from cultured neural tubes from St. 17 embryos. These late emerging neural crest cells also express PDGFRα, which is typically expressed by cranial neural crest cells. When the lipophilic dye DiI was injected into the lumen of the neural tube, DiI-positive cells were observed in the neural crest “staging area” within a day. After several days, DiI-positive cells reached the ventral mesenchyme. Plastron mesenchyme cells have a gene expression pattern similar to cranial skeletogenic neural crest cells, and appear to have functional similarities to cranial neural crest cells as they differentiate readily in culture to form clusters of collagen I-positive cells. These data support our hypothesis that the plastron of the turtle is formed by a late emerging population of neural crest cells that collect dorsally in the carapace, migrate ventrally to the plastron, and undergo intramembranous osification. Research support: NSF, PASSHE, Millersville University and Swarthmore College.

Nature 461, 95–98 (2009) In this Letter, author Laurel Beck was incorrectly listed as Laural Beck.