- Email: [email protected]
Patterning of the turtle shell
Available online at www.sciencedirect.com
ScienceDirect Patterning of the turtle shell Jacqueline E Moustakas-Verho1, Judith Cebra-Thomas2 and Scott F Gilbert3 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. Addresses 1 Developmental Biology Program, Institute of Biotechnology, University of Helsinki, Viikinkaari 5 (P.O. Box 56), FIN-00014 Helsinki, Finland 2 Biology Department, Millersville University, USA 3 Biology Department, Swarthmore College, USA Corresponding author: Moustakas-Verho, Jacqueline E ([email protected])
Current Opinion in Genetics & Development 2017, 45:124–131 This review comes from a themed issue on Developmental mechanisms, patterning and evolution Edited by Christian S Hardtke and Yoshiko Takahashi
http://dx.doi.org/10.1016/j.gde.2017.03.016 0959-437/ã 2017 Elsevier Ltd. All rights reserved.
Introduction The turtle is an important emerging research model in evolutionary developmental genetics. Though some questions have remained the same for almost two centuries [1], advances in molecular and imaging technologies have enabled detailed investigations into the developmental and evolutionary origins of the turtle shell (Figure 1). This structure is considered to be an evolutionary novelty based on its evolutionary history and morphology (Figure 2). It is composed of a dorsal carapace and a ventral plastron that enclose the shoulder and pelvic girdles (Figure 1). The carapace is formed from costal bones fused with ribs, neural bones fused with thoracic vertebrae, peripheral bones, and an anterior nuchal bone Current Opinion in Genetics & Development 2017, 45:124–131
(Figure 1). Developmental evidence and muscular attachments suggest that the nuchal bone is derived from the cleithra of the ancestral tetrapod pectoral girdle [2]. The plastron is formed from the suturing together of an entoplastron (interclavicle), epiplastra (clavicles), and three to five paired bones (homologous with gastralia) (Figure 1). The bones of the shell are overlain by keratinous ectodermal scutes, which are modified scales (Figure 1). The composite shell is a synapomorphy (shared, derived character state) that diagnoses turtles: Testudinata is defined as the clade originating from the first amniote with a fully developed turtle shell [3]. Extant turtles fall into two clades; Cryptodira (the more diverse group of hidden-necked turtles found throughout the temperate and tropical regions of the world) and Pleurodira (side-neck turtles of the Southern Hemisphere), and the stem lineage leading to the crown group Testudines (Figure 2). The stem group is composed of fossil turtles with diverse morphologies [4]. Species from the two main clades of living turtles have been developed as research models for developmental biology (Figure 2) [5–24,25,26,27]. The main cryptodiran species used as research models range from having a complete shell in the slider turtle (Trachemys scripta) to reductions in both the bony and ectodermal scutes in the Chinese softshell turtle (Pelodiscus sinensis); the pleurodiran model, the redbellied short-necked turtle (Emydura subglobosa), has a complete shell (Figure 2). These qualitative differences in the morphology of the shell among chelonian models further help us diagnose developmental features that are autapomorphic for certain taxa from the synapomorphies that diagnose turtles. The process of patterning in the turtle shell has been used to refer to the tessellation of the scutes into a pattern of non-overlapping shapes on the shell, as well as the development of skeletal and associated elements of the shell that produces the unique body plan of turtles. We review the history of these concepts and studies critical to our current understanding of the patterning of the turtle shell.
Turing and turtles: patterning the scutes of the turtle shell Ectodermal appendages in vertebrates include morphologically disparate organs such as hair, teeth, scales, horns, and mammary glands [28–30]; the turtle shell can be covered with scutes (large epidermal shields separated by furrows and forming a unique mosaic) or tubercles www.sciencedirect.com
Patterning of the turtle shell Moustakas-Verho, Cebra-Thomas and Gilbert 125
Figure 1
The turtle’s shell. The turtle shell is composed of a dorsal carapace and a ventral plastron. (a) Dorsal view of the carapace of a fossil turtle (Mongolemys ulensis courtesy of Igor Danilov) with the bones of the shell outlined in black (nu, nuchal bone; n, neural bone; c, costal bone; p, peripheral bone) and the overlying scutes of the shell outlined in white (m, marginal scute; v, vertebral scute; c, costal scute (costal scutes are synonymous with pleural scutes)). (b) Ventral view of the shell of the same fossil showing the plastron that is united to the carapace by an area called the bridge. The sutures of the bones are again highlighted in black (epi, epiplastron; ento, entoplastron; hyo, hyoplastron; hypo, hypoplastron; xiphi, xiphiplastron; p, peripheral bone from carapace), with the scutes outlined in white (m, marginal scute from carapace). (c) Turtle skeleton from an extant specimen (Emys orbicularis, UCMP 124448) with the plastron removed to show the incorporation of the thoracic vertebrae and ribs into the dorsal carapace, and the position of the limb girdles. Also seen are the overlying marginal scutes (m) on the carapace. Anterior is towards the top in all photos.
(numerous small epidermal bumps) [31]. Turtle species can often be recognized by the tessellation and pigmentation of scutes on their shell, and scutes have been independently lost or reduced in certain freshwater and marine taxa. We have recently shown that regulation of placodal signaling centers patterns the scutes of the turtle shell, and that two coupled reaction-diffusion systems can reproduce the natural and abnormal pattern variations of turtle scutes [25]. Although we made observations in both the carapace and plastron, we focused on the carapace because ossification is delayed compared to the plastron, where scute and bone development occur concurrently. This study is not only important for understanding the evolution of organs of the skin, but also because it establishes specific roles for genes previously hypothesized to be involved in the general growth and patterning of the carapace [18,25]. Turtle shell development begins by the formation of the carapacial ridge (CR) along the lateral trunk of the turtle embryo [32,33]. The CR is a protrusion of dermatomal mesenchyme overlain by a thickened epithelium that will form the lateral margin of the carapace [17,18,33]. We found that genes such as Shh, Bmp2, and Gremlin (also implicated in the patterning of scales and feathers [34,35,36]) were expressed in the developing scutes www.sciencedirect.com
of the hard-shelled turtle T. scripta (Figure 3). Moreover, by culturing developing turtle trunks with various inhibitors, we found that hedgehog, Bmp, and Fgf signaling were necessary for patterning the scutes [25]. The loss of scutes in ex vivo cultures resembled carapace development in the softshell turtle P. sinensis, which has lost its scutes evolutionarily [25]. Using this molecular information, we hypothesized that turtle scutes are patterned by Turing mechanisms [37] and constructed a mathematical model of scute formation based on the basic Meinhardt-Gierer model (Figure 3) [25,38]. To test our proposed activator-inhibitor dynamics in scute formation, we cultured developing turtle trunks with beads soaked in various proteins and found FGF4 to act as an inhibitor (or activator of an inhibitor) of the first reaction-diffusion system, SHH to behave as a potential inhibitor of the inhibitor of the first reactiondiffusion system, and SHH to act as an activator in the second reaction-diffusion system [25]. We further hypothesized that changing the dimensions of the turtle shell could alter the dynamics of the reaction-diffusion systems to produce natural variation in scute numbers as seen in several species of sea turtles (Figure 2), and proposed an epigenetic (environmental) role in the production of scute anomalies [25]. Testing these Current Opinion in Genetics & Development 20172017, 4545:124–131
126 Developmental mechanisms, patterning and evolution
Figure 2
A phylogenetic hypothesis of evolutionary relationships of turtles (based on [4,64]). Node 1 (blue circle) denotes the clade Testudinata, which includes stem turtles leading to the crown group Testudines (node 2). The morphology of the ribs and bridge in Odontochelys (image courtesy of Chun Li; [45]) are suggestive of the presence of a carapacial ridge (CR), though the lack of a carapace has also been hypothesized. Scutes are present on the shell of Odontochelys [65], as well as teeth in the jaws [45]. Proganochelys quenstedti has a robust, complete shell, though fewer scutes than the crown group of turtles (courtesy of the AMNH; [66]). The Proganochelys tenertesta specimen from the carapace shows what appears to be metaplastic bone fused the ribs (image courtesy of Walter Joyce, personal communication; [67]). Several representative taxa of Pleurodira (side-neck turtles; node 3) and Cryptodira (hidden-necked turtles; node 4) that have been used in developmental studies are shown at the branch tips. Emydura subglobosa is a pleurodiran taxon that has been developed for laboratory studies (image courtesy of Ingmar Werneburg; [21]). The Pelodiscus sinensis embryonic specimen shows the expression of Wnt5a in the CR; the Wnt pathway is hypothesized to be critical for the development of the turtle shell (image courtesy of Shigeru Kuratani; [26]). The embryology of Chelonia mydas (image courtesy of Jeanette Wyneken) and Caretta caretta (drawing by Tiff Shao) have been studied [9], and C. caretta shows that some species of sea turtles have a greater number of scutes in the carapace [25]. The Trachemys scripta specimens show a phylogenetically stable number of scutes in the carapace (left) and plastron (right) (images courtesy of Bob Smither) [25]. Phylogenetic tree drawn using APE [68].
hypotheses will elucidate the evolution of pattern formation and the role of environmental heterogeneity in reaction-diffusion dynamics.
Patterning the skeletal elements of the turtle shell: the evolution of the carapacial ridge As the ribs of the turtle grow, they fan out laterally towards the margins of the carapace, and rather than migrating ventrally towards a sternum, they remain arrested in the primaxial domain [12,17,33,39,40]. Ruckes [32] hypothesized that the turtle body plan is the result of the displacement of the ribs and their incorporation into the specialized dermis of the Current Opinion in Genetics & Development 2017, 45:124–131
embryonic turtle carapace [33]. Burke [10,33] named and further characterized the CR, showing patterns of proliferation and the distribution of morphogenetic molecules similar to other organs (feathers and limbs) that develop via epithelial–mesenchymal interactions. Following extirpation experiments, Burke [10] noted that the CR shows a remarkable degree of regeneration, but that when portions of the CR were successfully removed in the snapping turtle Chelydra serpentina, the growth of the ribs was deflected towards a neighboring rib. This same behavior by the ribs was observed following cauterization of the CR in the softshell turtle P. sinensis [17], showing that the CR patterns the ribs within the dorsal www.sciencedirect.com
Patterning of the turtle shell Moustakas-Verho, Cebra-Thomas and Gilbert 127
Figure 3
The patterning of scutes in the turtle shell. The expression of Shh is used to show the development of scutes in hard-shelled Trachemys scripta embryos. (a) Shh is shown to be expressed in the developing scutes of the carapace (m, marginal scute primordia along the CR; v, vertebral scute primordia; central arrowhead points to costal scute primordia) and plastron (p). (b) Scutes have been lost or reduced independently in several lineages of turtles, and in the softshell turtle Pelodiscus sinensis, the process of scute formation appears to be arrested at the CR. (c) In a control explant culture of T. scripta, we see a normal spacing of marginal scute primordia along the CR. (d) In explants cultured with SU5402 to disrupt FGF signaling, the periodic patterning along the developing shell is lost. (e) Beginning from a pre-pattern of marginal scute primordia along the CR, two coupled reaction-diffusion systems are able to produce the pattern of scutes on the carapace. Images from [25].
dermis of the carapace. Interestingly, when a foil barrier was placed at the border between somitic and lateral plate mesoderm before formation of the CR in the hardshell turtle, portions of the carapace were absent and the ribs entered the abaxial domain and interdigitated with the dermal bones of the plastron [10]. This suggests that the lateral plate mesoderm (including the future area where the plastron will develop) could play a role in the lateral deflection of the ribs in the turtle. There are several additional studies that support the hypothesis that the lateral plate mesoderm is a key factor in patterning the turtle skeleton. The function of hepatocyte growth factor (HGF) is essential for the development of the CR; however, HGF expression in the turtle is seen in the lateral body wall and sclerotome, but not in the CR itself [41]. A study on Hox gene expression in turtles compared with other amniotes found conserved expression of Hoxc-6 in the sclerotome and dermatome at thoracic levels of the anterior-posterior axis, and an absence of Hoxc-6 expression from the turtle somatopleure [42]. Because Hoxc-6 is thought to determine thoracic (rib-forming) identity, it has been proposed that the absence of the expression of this gene from the lateral plate mesoderm is related to the absence of ribs ventrally in turtles [42]. Consistent with the absence of ribs ventrally in turtles is the expression of genes associated with the suppression of chondrogenesis in the ventral mesenchyme of the developing turtle [43]. www.sciencedirect.com
It, therefore, appears that the morphology of the ribs in turtles requires both the derived condition of the lateral plate mesoderm and the CR [44]. This, then, suggests that the rib morphology of turtles evolved together with the shell. Going back to the fossil evidence, the prototurtle Odontochelys shows a fully developed plastron ventrally, and does not show a fully ossified carapace but rather has neural plates of bone [45]. The fanned ribs of Odontochelys (Figure 2) [45] that do not curve ventrally suggest the presence of a CR. Further hints of CR (and hence, carapace, albeit with reduced ossification of dermal elements) in Odontochelys include the presence of a lateral bridge that would connect the plastron to the carapace in other turtles [46]. The homologies of the turtle shell have been debated since at least the 19th century, with some workers hypothesizing that the bones of the turtle shell have endoskeletal origins derived from the ribs, vertebrae, and sternum, whereas others viewed it as a composite structure formed by a mixture of endoskeletal and dermal ossification [1,47]. This is an important distinction not only for understanding the developmental process, but also in distinguishing alternate hypotheses for the evolutionary origin of the turtle shell as being formed by first, a novel epithelial-mesenchymal interaction in the CR that results in the deflection of the ribs and carapace formation independent of dermal ossification patterns [44], second, the fusion of expanded ribs and neural arches with Current Opinion in Genetics & Development 20172017, 4545:124–131
128 Developmental mechanisms, patterning and evolution
overlying osteoderms (expansion of ribs and vertebrae evolving independently from occurrence of osteoderms) [48], third, the endoskeleton via periosteal expansion of the ribs and neural arches of trunk vertebrae to form the costal and neural plates with no contribution from the dermis [49], or fourth, periosteal ossification of the ribs and neural arches followed by metaplastic ossification of a specialized dermis formed by the CR and the displacement of the ribs [12,22,47,50,51]. Metaplastic ossification is the conversion of one cell type to another, and in the case of the turtle carapace, ossification of the dermis. One of the most basal known genera of turtles with a complete shell, Proganochelys, shows what appears to be ribs fused to metaplastic bone in the carapace (Figure 2), though this ossification has not been investigated histologically. The formation of independent ossification centers in the mesenchyme and dermis is clear in the plastron, as well as in the nuchal and peripheral bones of the carapace [[12,22,49]12,22,43,49]. Such independent ossification centers are also seen in the nascent dermal osteoderms of the alligator [49,50,52]. However, the neural and costal bones of the turtle carapace begin ossification by periosteal ossification of the trunk vertebrae and ribs, followed by fusion of this periosteum of the vertebrae and ribs with bony spiculae formed in the dermis [22,47,49–51]. It is interesting to note that softshell turtles (P. sinensis, for example) do not have peripheral bones in the carapace and their ribs do not extend to the margins of the carapace [17], suggesting that the ribs may have a role in inducing the peripheral bones in the marginal dermis. Because the development of the turtle shell spans months, there is a clear need for better in ovo techniques to enable longterm cell labeling, lineage mapping, and experimental manipulation. As we defined Testudinata earlier as the clade originating from the first amniote with a fully developed turtle shell, the CR is essential to this diagnosis. What induces the CR itself? The mesenchyme of the CR is derived from the dermatomal compartment of somitic mesoderm [17,18], and replacement of chick somites with turtle somites did not result in the formation of a CR or axial arrest of the ribs [16], suggesting that the epithelial component of the CR is necessary. Though it is clear that there has been a co-option of morphogenetic mechanisms and gene regulatory networks for some developmental processes in carapace development [10,18,25,33], there is also a conservation of gene expression related to cell lineage [18]. Screening for CR-specific expression, components of the canonical Wnt signaling pathway were found to be expressed in the softshell turtle: Lef-1 and APCDD1 expression in the mesenchyme of the CR with nuclear localization of b-catenin protein in the epithelium of the CR [53]. Using the draft genomes of the softshell and green sea turtle, Wnt5a was found to be the only Wnt gene expressed in the CR region [54], and the researchers have Current Opinion in Genetics & Development 2017, 45:124–131
since shown that Wnt5a, Lef-1, and APCDD1 are expressed in the CR of not only softshell turtles (Figure 2), but also the other main cryptodiran research model, T. scripta, as well as the pleurodiran E. subglobosa [26]. Furthermore, electroporation of dominant-negative Lef-1 into the epidermis of the CR resulted in a local arrest of CR growth [17].
Future prospects The availability of genomic [54,55] and transcriptomic resources [23], as well as the development of in ovo [5,10,17,27] and ex vivo [25,47] laboratory protocols for studying turtles, has allowed workers to address old questions about the evolution of these organisms and to postulate novel hypotheses. Although the composite nature of the shell makes deciphering the tempo and mode of regulatory changes necessary for inferring its evolutionary origin complicated, the groundwork has already been laid for asking important questions. What is the mechanism of co-option of gene regulatory networks, such that the signaling center for the turtle scute uses some of the same proteins as feathers, hair, and teeth, but in different patterns? Did the evolution of the turtle shell require gradual, additive changes, or was there a large change (such as rib displacement) followed by secondary smaller modifications? Why is the pattern of scutes on the turtle shell stable phylogenetically, but labile in the individual? Fossil evidence still provides us with new questions about developmental mechanisms. Was the metaplastic ossification of the ribs, as seen in the proposed stem-turtles — Permian Eunotosaurus [56] and Middle Trassic Pappochelys [57], an exaptation in the evolution of the turtle shell? Is it possible developmentally to form a plastron without a carapace, as has been hypothesized (and criticized) in the Late Triassic proto turtle Odontochelys [45,46]? In addition to the trunk, there have been notable morphological changes to patterning of the cranium in turtles. The plesiomorphic presence of teeth in turtles has been extended to the Late Jurassic basal turtle Sichuanchelys palatodentata (Figure 2) [4], and the mechanism of tooth loss in turtles appears to be different from that in birds [58]. How this convergent loss of teeth and formation of a beak happens will be exciting to see, as well as the potential evolutionary developmental mechanism behind the hypothesized secondary condition of an anapsid skull (no temporal fenestration) in turtles from a primitively diapsid (two fenestrations) condition [59,60,61,62].
Conclusions The taxonomic and morphologic diversity represented by turtle species used in developmental biology currently allows hypotheses of evolutionary transformations rather than typological comparisons. The comparison of www.sciencedirect.com
Patterning of the turtle shell Moustakas-Verho, Cebra-Thomas and Gilbert 129
laboratory model species in evolutionary developmental biology is rather akin to the long-branch attraction problem [63]; the derived morphologies of model organisms obscure the phylogenetic signal of evolutionary transformations. The first step towards resolving this problem is the addition of relevant taxa to the comparison. The second step is to combine expression patterns and morphologies with an evolutionary hypothesis of relationships, and thereby deduce the polarity of the developmental transformations. Examples of this phylogenetic approach are the apomorphic expression of Wnt pathway genes in the CR tissues of turtles [26] and the patterned expression of genes that regulate scute development and loss in turtles [25]. We, therefore, see the abundance of turtle species being investigated for the origin and evolution of the turtle shell as a positive phenomenon. The elucidation of the developmental and genetic mechanisms involved in the origin and diversification of the turtle shell has raised the bar in evolutionary developmental biology and unified this field with genomics, paleontology, mathematical modeling, and ecology.
The biogeographic history of turtles and consideration of the ecological context for the origin and diversification of turtle lineages.
References and recommended reading
13. Loredo GA, Brukman A, Harris MP, Kagle D, Leclair EE, Gutman R, Denny E, Henkelman E, Murray BP, Fallon JF, Tuan RS, Gilbert SF: Development of an evolutionarily novel structure: fibroblast growth factor expression in the carapacial ridge of turtle embryos. J Exp Zool (Mol Dev Evol) 2001, 291B:274-281.
Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
Conflict of interest statement Nothing declared.
Acknowledgements
5.
Yntema CL: Procurement and use of turtle embryos for experimental procedures. Anat Rec 1964, 149:577-586.
6.
Yntema CL: A series of stages in the embryonic development of Chelydra serpentina. J Morphol 1968, 125:219-252.
7.
Yntema CL: Extirpation experiments on the embryonic rudiments of the carapace of Chelydra serpentina. J Morphol 1970, 132:235-244.
8.
Ewert MA: Embryology of turtles. In Biology of the Reptilia, 14. Edited by Gans C, Billet F, Maderson PFA . 1985:74-255.
9.
Miller JD: Embryology of marine turtles. In Biology of the Reptilia, 14. Edited by Gans C, Billet F, Maderson PFA . 1985: 271-328.
10. Burke AC: The development and evolution of the turtle body plan. Inferring intrinsic aspects of the evolutionary process from experimental embryology. Am Zool 1991, 31:616-627. 11. Beggs K, Young J, Georges A, West P: Ageing the eggs and embryos of the pig-nosed turtle. Carettochelys insculpta (Chelonia: Carettochelydidae), from northern Australia. Can J Zool 2000, 78:373-392. 12. Gilbert SF, Loredo GA, Brukman A, Burke AC: Morphogenesis of the turtle shell: the development of a novel structure in tetrapod evolution. Evol Dev 2001, 3:47-58.
14. Tokita M, Kuratani S: Normal embryonic stages of the Chinese softshelled turtle Pelodiscus sinensis (Trionychidae). Zool Sci 2001, 18:705-715. 15. Vincent C, Bontoux M, Le Douarin NM, Pieau C, MonsoroBurq AH: Msx genes are expressed in the carapacial ridge of turtle shell: a study of the European pond turtle, Emys orbicularis. Dev Genes Evol 2003, 213:464-469. 16. Nagashima H, Uchida K, Yamamoto K, Kuraku S, Usuda R, Kuratani S: Turtle-chicken chimera: an experimental approach to understanding evolutionary innovation in the turtle. Dev Dynam 2005, 232:149-161.
We thank Anne Burke, Jason Head, Walter Joyce, Torsten Scheyer, and James Parham for discussion, the American Museum of Natural History, Walter Joyce, Shigeru Kuratani, Chun Li, and Ingmar Werneburg for the use of images from published figures, Igor Danilov for the use of images from an unpublished specimen, Patricia Holroyd for the image of the University of California Museum of Paleontology (UCMP) specimen, and Fabien Lafuma for help with APE. This work was funded by the Academy of Finland, NSF grant IOS-145177, and Millersville University and Swarthmore College Faculty Research Funds.
17. Nagashima H, Kuraku S, Uchida K, Kawashima-Ohya Y, Narita Y, Kuratani S: On the carapacial ridge in turtle embryos: its developmental origin, function and the chelonian body plan. Development 2007, 134:2219-2226.
References
19. Nagashima H, Sugahara F, Takechi M, Ericsson R, KawashimaOhya Y, Narita Y, Kuratani S: Evolution of the turtle body plan by the folding and creation of new muscle connections. Science 2009, 325:193-196.
1.
MacCord K, Caniglia G, Moustakas-Verho JE, Burke AC: The dawn of chelonian research: turtles between comparative anatomy and embryology in the 19th century. J Exp Zool (Mol Dev Evol) 2015, 324B:169-180. Historical dissection of the evidence and context for theories regarding the evolutionary and developmental origin of the turtle shell. 2.
Lyson TR, Bhullar BAS, Bever GS, Joyce WG, de Queiroz K, Abzhanov A, Gauthier JA: Homology of the enigmatic nuchal bone reveals novel reorganization of the shoulder girdle in the evolution of the turtle shell. Evol Dev 2013, 15:317-325.
3.
Joyce WG, Parham JF, Gauthier JA: Developing a protocol for the conversion of rank-based taxon names to phylogenetically defined clade names, as exemplified by turtles. J Paleontol 2004, 78:989-1013.
4.
Joyce WG, Rabi M, Clark JM, Xu X: A toothed turtle from the Late Jurassic of China and the global biogeographic history of turtles. BMC Evol Biol 2016, 16:236.
www.sciencedirect.com
18. Moustakas JE: Development of the carapacial ridge: implications for the evolution of genetic networks in turtle shell development. Evol Dev 2008, 10:29-36.
20. Valenzuela N: The painted turtle, Chrysemys picta: a model system for vertebrate evolution, ecology, and human health. Cold Spring Harb Protoc 2009, 4:1-9. 21. Werneburg I, Hugi J, Mu¨ller J, Sanchez-Villagra MR: Embryogenesis and ossification of Emydura subglobosa (Testudines, Pleurodira, Chelidae) and patterns of turtle development. Dev Dynam 2009, 238:2770-2786. 22. Lima FC, Santos ALQ, Vieira LG, Da Silva-Junior LM, Roma˜o MF, De Simone SBS, Hirano LQL, Silva JMM, Montelo KM, Malvasio A: Ontogeny of the shell bones of embryos of Podocnemis unifilis (Troschel, 1848) (Testudines, Podocnemididae). Anat Rec 2011, 294:621-632. 23. Kaplinsky NJ, Gilbert SF, Cebra-Thomas J, Lillevali K, Saare M, Chang EY, Edelman HE, Frick MA, Guan Y, Hammond RM et al.: The embryonic transcriptome of the red-eared slider turtle (Trachemys scripta). PLoS ONE 2013, 8:e66357. Current Opinion in Genetics & Development 20172017, 4545:124–131
130 Developmental mechanisms, patterning and evolution
24. Cordero GA, Janzen FJ: An enhanced developmental staging table for the painted turtle, Chrysemys picta (Testudines: Emydidae). J Morphol 2014, 275:442-455. 25. Moustakas-Verho JE, Zimm R, Cebra-Thomas J, Lempia¨inen NK, Kallonen A, Mitchell KL, Ha¨ma¨la¨inen K, Salazar-Ciudad I, Jernvall J, Gilbert SF: The origin and loss of periodic patterning in the turtle shell. Development 2014, 141:3033-3039. The molecular and developmental basis of scute evolution in turtles that combine evolutionary, laboratory, and mathematical experiments to describe the diversification of the skin of the turtle shell. 26. Pascual-Anaya J, Hirasawa T, Sato I, Kuraku S, Kuratani S: Comparative analysis of pleurodiran and cryptodiran turtle embryos depicts the molecular ground pattern of the turtle carapacial ridge. Int J Dev Biol 2014, 58:743-750. Consideration of differences reported in the developmental expression of turtle shell development from different species, combined with hypothesis testing using additional species of turtle and newly published genomic resources. 27. Nomura T, Yamashita W, Gotoh H, Ono K: Genetic manipulation of reptilian embryos: toward an understanding of cortical development and evolution. Front Neurosci 2015, 9:1-11. Development of techniques forin ovo electroporation and short-term ex vivo culture of turtle embryos. 28. Chang C, Wu P, Baker RE, Maini PK, Alibardi L, Chuong CM: Reptile scale paradigm: Evo-Devo, pattern formation, and regeneration. Int J Dev Biol 2009, 53:813-826. 29. Dhouailly D: A new scenario for the evolutionary origin of hair, feather, and avian scales. J Anat 2009, 214:587-606. 30. Thesleff I, Mikkola ML: Development of ectodermal organs. Semin Cell Dev Biol 2014, 25–26:1-2. 31. Moustakas-Verho JE, Cherepanov GO: The integumental appendages of the turtle shell: an evo-devo perspective. J Exp Zool (Mol Dev Evol) 2015, 324B:221-229. A perspective on the evolution of the organs of the skin of the turtle shell. 32. Ruckes H: Studies in chelonian osteology. Part II. The morphological relationships between the girdles, ribs and carapace. Ann N Y Acad Sci 1929, 31:81-120. 33. Burke AC: Development of the turtle carapace: implications for the evolution of a novel bauplan. J Morphol 1989, 199:363-378. 34. Harris MP, Fallon JF, Prum RO: Shh-Bmp2 signaling module and the evolutionary origin and diversification of feathers. J Exp Zool 2002, 294:160-176. 35. Bardot B, Lecoin L, Fliniaux I, Huillard E, Marx M, Viallet JP: Drm/ Gremlin, a BMP antagonist, defines the interbud region during feather development. Int J Dev Biol 2004, 48:149-156. 36. Di-Poı¨ N, Milinkovitch MC: The anatomical placode in reptile scale morphogenesis indicates shared ancestry among skin appendages in amniotes. Sci Adv 2016, 2:e1600708. This work establishes that the skin appendages of reptiles share not only molecular signals in their developmental program, but also the anatomical placode with other amniotes. 37. Turing AM: The chemical basis of morphogenesis. Philos Trans R Soc Lond B Biol Sci 1952, 237:37-72. 38. Gierer A, Meinhardt H: A theory of biological pattern formation. Kybernetik 1972, 12:30-39. 39. Shearman RM, Burke AC: Ontogeny and phylogeny of the lateral somatic frontier. J Exp Zool (Mol Dev Evol) 2009, 312B:603-612. 40. Rice R, Gilbert SF, Riccio P, Cebra-Thomas J: Emerging from the rib: resolving turtle controversies. J Exp Zool (Mol Dev Evol) 2015, 324B:208-220. Consideration of differences in the developmental expression of turtle shell development in hard-shelled versus soft-shelled species. 41. Kawashima-Ohya Y, Narita Y, Nagashima H, Usuda R, Kuratani S: Hepatocyte growth factor is crucial for development of the carapace in turtles. Evol Dev 2011, 13:260-268. 42. Ohya KY, Kuraku S, Kuratani S: Hox code in embryos of Chinese soft-shelled turtle Pelodiscus sinensis correlates with the evolutionary innovation in the turtle. J Exp Zool (Mol Dev Evol) 2005, 304B:107-118. Current Opinion in Genetics & Development 2017, 45:124–131
43. Rice R, Kallonen A, Cebra-Thomas J, Gilbert SF: Development of the turtle plastron, the order-defining skeletal structure. PNAS 2016, 113:5317-5322. This work describes the molecular and histological development of the mesenchymal condensation in turtle plastron development, and proposes a developmental program for the induction of osteogenesis and comcomitant suppression of chondrogenesis in the evolution of the plastron. 44. Burke AC: Turtles . . .
. . . again. Evol Dev 2009, 11:622-624.
45. Li C, Wu X-C, Rieppel O, Wang L-T, Zhao L-J: An ancestral turtle from the Late Triassic of southwestern China. Nature 2008, 456:497-501. 46. Reisz RR, Head JJ: Palaeontology: turtle origins out to sea. Nature 2008, 456:450-451. 47. Cebra-Thomas J, Tan F, Sistla S, Estes E, Bender G, Kim C, Riccio P, Gilbert SF: How the turtle forms its shell: a paracrine hypothesis of carapace formation. J Exp Zool (Mol Dev Evol) 2005, 304B:558-569. 48. Lee MSY: Correlated progression and the origin of turtles. Nature 1993, 379:812-815. 49. Hirasawa T, Nagashima H, Kuratani S: The endoskeletal origin of the turtle carapace. Nat Commun 2013, 4:2107 http://dx.doi.org/ 10.1038/ncomms3107. 50. Cherepanov GO: The origin of the bony shell of turtles as a unique evolutionary model in reptiles. Russ J Herpetol 1997, 4:155-156. 51. Scheyer TM, Bru¨llmann B, Sanchez-Villagra MR: The ontogeny of the shell in side-necked turtles, with emphasis on the homologies of costal and neural bones. J Morphol 2008, 269:1008-1021. 52. Vickaryous MK, Hall BK: Development of the dermal skeleton in Alligator mississippiensis (Archosauria, Crocodylia) with comments on the homology of osteoderms. J Morphol 2008, 269:398-422. 53. Kuraku S, Usuda R, Kuratani S: Comprehensive survey of carapacial ridge-specific genes in turtle implies co-option of some regulatory genes in carapace evolution. Evol Dev 2005, 7:3-17. 54. Wang Z, Pascual-Anaya J, Zadissa A, Li W, Niimura Y, Huang Z, Li C, White S, Xiong Z, Fang D et al.: The draft genomes of softshell turtle and green sea turtle yield insights into the development and evolution of the turtle-specific body plan. Nat Genet 2013, 45:701-706. 55. Schaffer HB, Minx P, Warren DW, Shedlock AM, Thomson RC, Valenzuela N, Abramyan J, Amemiya CT, Badenhorst D, Biggar KK et al.: The western painted turtle genome, a model for the evolution of extreme physiological adaptations in a slowly evolving lineage. Genome Biol 2013, 14:R28. 56. Lyson TR, Bever GS, Scheyer TM, Hsiang AY, Gauthier JA: Evolutionary origin of the turtle shell. Curr Biol 2013, 23: 1113-1119. 57. Schoch RR, Sues H-D: A Middle Triassic stem-turtle and the evolution of the turtle body plan. Nature 2015, 523:584-587. A stem-turtle assignment to a fossil that shows similarities in the morphology of the ribs with other stem turtles, paired gastralia proposed to be homologous with the plastron of turtles, and a diapsid condition of the skull, which supports a diapsid origin for turtles. 58. Lainoff AJ, Moustakas-Verho JE, Hu D, Kallonen A, Marcucio RS, Hlusko LJ: A comparative examination of odontogenic gene expression in both toothed and toothless amniotes. J Exp Zool (Mol Dev Evol) 2015, 324B:255-269. A developmental genetic investigation of the evolutionary convergence of edentulism in amniotes. 59. Rieppel O, deBraga M: Turtles as diapsid reptiles. Nature 1996, 384:453-455. 60. Bever GS, Lyson TR, Field DJ, Bhullar BAS: Evolutionary origin of the turtle skull. Nature 2015, 525:239-242. www.sciencedirect.com
Patterning of the turtle shell Moustakas-Verho, Cebra-Thomas and Gilbert 131
61. Bever GS, Lyson TR, Field DJ, Bhullar BAS: The amniote temporal roof and the diapsid origin of the turtle skull. Zoology 2016, 119:471-473. A discussion of the developmental stage of fossil taxa proposed to be stem turtles, and the possible convergence of the anapsid condition from diapsid skulls.
65. Lyson TR, Schachner ER, Botha-Brink J, Scheyer TM, Lambertz M, Bever GS, Rubidge BS, de Queiroz K: Origin of the unique ventilatory apparatus of turtles. Nat Commun 2014, 5:5211 http://dx.doi.org/10.1038/ncomms6211. This work considers the origin of the ventilation mechanism in turtles, and re-diagnoses the presence of scutes in the stem turtleOdontochelys.
62. Schoch RR, Sues H-D: The diapsid origin of turtles. Zoology 2016, 119:159-161.
66. Gaffney ES: The comparative osteology of the Triassic turtle Proganochelys. Bull Am Mus Nat Hist 1990, 194:1-263.
63. Felsenstein J: Cases in which parsimony or compatibility methods will be positively misleading. Syst Biol 1978, 27:401-410.
67. Joyce WG, Lucas SG, Scheyer TM, Heckert AB, Hunt AP: A thinshelled reptile from the Late Triassic of North America and the origin of the turtle shell. Proc R Soc B 2009, 276:507-513.
64. Crawford NG, Parham JF, Sellas AB, Faircloth BC, Glenn TC, Papenfuss TJ, Henderson JB, Hansen MH, Simison WB: A phylogenomic analysis of turtles. Mol Phylogenet Evol 2015, 83:250-257. A genome-scale phylogeny of turtles produced using ultra-conserved elements and several algorithms, with discussion of the temporal biogeographic origin of turtle clades.
www.sciencedirect.com
68. Paradis E, Claude J, Strimmer K: APE: analyses of phylogenetics and evolution in R language. Bioinformatics 2004, 20:289-290.
Current Opinion in Genetics & Development 20172017, 4545:124–131
ScienceDirect Patterning of the turtle shell Jacqueline E Moustakas-Verho1, Judith Cebra-Thomas2 and Scott F Gilbert3 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. Addresses 1 Developmental Biology Program, Institute of Biotechnology, University of Helsinki, Viikinkaari 5 (P.O. Box 56), FIN-00014 Helsinki, Finland 2 Biology Department, Millersville University, USA 3 Biology Department, Swarthmore College, USA Corresponding author: Moustakas-Verho, Jacqueline E ([email protected])
Current Opinion in Genetics & Development 2017, 45:124–131 This review comes from a themed issue on Developmental mechanisms, patterning and evolution Edited by Christian S Hardtke and Yoshiko Takahashi
http://dx.doi.org/10.1016/j.gde.2017.03.016 0959-437/ã 2017 Elsevier Ltd. All rights reserved.
Introduction The turtle is an important emerging research model in evolutionary developmental genetics. Though some questions have remained the same for almost two centuries [1], advances in molecular and imaging technologies have enabled detailed investigations into the developmental and evolutionary origins of the turtle shell (Figure 1). This structure is considered to be an evolutionary novelty based on its evolutionary history and morphology (Figure 2). It is composed of a dorsal carapace and a ventral plastron that enclose the shoulder and pelvic girdles (Figure 1). The carapace is formed from costal bones fused with ribs, neural bones fused with thoracic vertebrae, peripheral bones, and an anterior nuchal bone Current Opinion in Genetics & Development 2017, 45:124–131
(Figure 1). Developmental evidence and muscular attachments suggest that the nuchal bone is derived from the cleithra of the ancestral tetrapod pectoral girdle [2]. The plastron is formed from the suturing together of an entoplastron (interclavicle), epiplastra (clavicles), and three to five paired bones (homologous with gastralia) (Figure 1). The bones of the shell are overlain by keratinous ectodermal scutes, which are modified scales (Figure 1). The composite shell is a synapomorphy (shared, derived character state) that diagnoses turtles: Testudinata is defined as the clade originating from the first amniote with a fully developed turtle shell [3]. Extant turtles fall into two clades; Cryptodira (the more diverse group of hidden-necked turtles found throughout the temperate and tropical regions of the world) and Pleurodira (side-neck turtles of the Southern Hemisphere), and the stem lineage leading to the crown group Testudines (Figure 2). The stem group is composed of fossil turtles with diverse morphologies [4]. Species from the two main clades of living turtles have been developed as research models for developmental biology (Figure 2) [5–24,25,26,27]. The main cryptodiran species used as research models range from having a complete shell in the slider turtle (Trachemys scripta) to reductions in both the bony and ectodermal scutes in the Chinese softshell turtle (Pelodiscus sinensis); the pleurodiran model, the redbellied short-necked turtle (Emydura subglobosa), has a complete shell (Figure 2). These qualitative differences in the morphology of the shell among chelonian models further help us diagnose developmental features that are autapomorphic for certain taxa from the synapomorphies that diagnose turtles. The process of patterning in the turtle shell has been used to refer to the tessellation of the scutes into a pattern of non-overlapping shapes on the shell, as well as the development of skeletal and associated elements of the shell that produces the unique body plan of turtles. We review the history of these concepts and studies critical to our current understanding of the patterning of the turtle shell.
Turing and turtles: patterning the scutes of the turtle shell Ectodermal appendages in vertebrates include morphologically disparate organs such as hair, teeth, scales, horns, and mammary glands [28–30]; the turtle shell can be covered with scutes (large epidermal shields separated by furrows and forming a unique mosaic) or tubercles www.sciencedirect.com
Patterning of the turtle shell Moustakas-Verho, Cebra-Thomas and Gilbert 125
Figure 1
The turtle’s shell. The turtle shell is composed of a dorsal carapace and a ventral plastron. (a) Dorsal view of the carapace of a fossil turtle (Mongolemys ulensis courtesy of Igor Danilov) with the bones of the shell outlined in black (nu, nuchal bone; n, neural bone; c, costal bone; p, peripheral bone) and the overlying scutes of the shell outlined in white (m, marginal scute; v, vertebral scute; c, costal scute (costal scutes are synonymous with pleural scutes)). (b) Ventral view of the shell of the same fossil showing the plastron that is united to the carapace by an area called the bridge. The sutures of the bones are again highlighted in black (epi, epiplastron; ento, entoplastron; hyo, hyoplastron; hypo, hypoplastron; xiphi, xiphiplastron; p, peripheral bone from carapace), with the scutes outlined in white (m, marginal scute from carapace). (c) Turtle skeleton from an extant specimen (Emys orbicularis, UCMP 124448) with the plastron removed to show the incorporation of the thoracic vertebrae and ribs into the dorsal carapace, and the position of the limb girdles. Also seen are the overlying marginal scutes (m) on the carapace. Anterior is towards the top in all photos.
(numerous small epidermal bumps) [31]. Turtle species can often be recognized by the tessellation and pigmentation of scutes on their shell, and scutes have been independently lost or reduced in certain freshwater and marine taxa. We have recently shown that regulation of placodal signaling centers patterns the scutes of the turtle shell, and that two coupled reaction-diffusion systems can reproduce the natural and abnormal pattern variations of turtle scutes [25]. Although we made observations in both the carapace and plastron, we focused on the carapace because ossification is delayed compared to the plastron, where scute and bone development occur concurrently. This study is not only important for understanding the evolution of organs of the skin, but also because it establishes specific roles for genes previously hypothesized to be involved in the general growth and patterning of the carapace [18,25]. Turtle shell development begins by the formation of the carapacial ridge (CR) along the lateral trunk of the turtle embryo [32,33]. The CR is a protrusion of dermatomal mesenchyme overlain by a thickened epithelium that will form the lateral margin of the carapace [17,18,33]. We found that genes such as Shh, Bmp2, and Gremlin (also implicated in the patterning of scales and feathers [34,35,36]) were expressed in the developing scutes www.sciencedirect.com
of the hard-shelled turtle T. scripta (Figure 3). Moreover, by culturing developing turtle trunks with various inhibitors, we found that hedgehog, Bmp, and Fgf signaling were necessary for patterning the scutes [25]. The loss of scutes in ex vivo cultures resembled carapace development in the softshell turtle P. sinensis, which has lost its scutes evolutionarily [25]. Using this molecular information, we hypothesized that turtle scutes are patterned by Turing mechanisms [37] and constructed a mathematical model of scute formation based on the basic Meinhardt-Gierer model (Figure 3) [25,38]. To test our proposed activator-inhibitor dynamics in scute formation, we cultured developing turtle trunks with beads soaked in various proteins and found FGF4 to act as an inhibitor (or activator of an inhibitor) of the first reaction-diffusion system, SHH to behave as a potential inhibitor of the inhibitor of the first reactiondiffusion system, and SHH to act as an activator in the second reaction-diffusion system [25]. We further hypothesized that changing the dimensions of the turtle shell could alter the dynamics of the reaction-diffusion systems to produce natural variation in scute numbers as seen in several species of sea turtles (Figure 2), and proposed an epigenetic (environmental) role in the production of scute anomalies [25]. Testing these Current Opinion in Genetics & Development 20172017, 4545:124–131
126 Developmental mechanisms, patterning and evolution
Figure 2
A phylogenetic hypothesis of evolutionary relationships of turtles (based on [4,64]). Node 1 (blue circle) denotes the clade Testudinata, which includes stem turtles leading to the crown group Testudines (node 2). The morphology of the ribs and bridge in Odontochelys (image courtesy of Chun Li; [45]) are suggestive of the presence of a carapacial ridge (CR), though the lack of a carapace has also been hypothesized. Scutes are present on the shell of Odontochelys [65], as well as teeth in the jaws [45]. Proganochelys quenstedti has a robust, complete shell, though fewer scutes than the crown group of turtles (courtesy of the AMNH; [66]). The Proganochelys tenertesta specimen from the carapace shows what appears to be metaplastic bone fused the ribs (image courtesy of Walter Joyce, personal communication; [67]). Several representative taxa of Pleurodira (side-neck turtles; node 3) and Cryptodira (hidden-necked turtles; node 4) that have been used in developmental studies are shown at the branch tips. Emydura subglobosa is a pleurodiran taxon that has been developed for laboratory studies (image courtesy of Ingmar Werneburg; [21]). The Pelodiscus sinensis embryonic specimen shows the expression of Wnt5a in the CR; the Wnt pathway is hypothesized to be critical for the development of the turtle shell (image courtesy of Shigeru Kuratani; [26]). The embryology of Chelonia mydas (image courtesy of Jeanette Wyneken) and Caretta caretta (drawing by Tiff Shao) have been studied [9], and C. caretta shows that some species of sea turtles have a greater number of scutes in the carapace [25]. The Trachemys scripta specimens show a phylogenetically stable number of scutes in the carapace (left) and plastron (right) (images courtesy of Bob Smither) [25]. Phylogenetic tree drawn using APE [68].
hypotheses will elucidate the evolution of pattern formation and the role of environmental heterogeneity in reaction-diffusion dynamics.
Patterning the skeletal elements of the turtle shell: the evolution of the carapacial ridge As the ribs of the turtle grow, they fan out laterally towards the margins of the carapace, and rather than migrating ventrally towards a sternum, they remain arrested in the primaxial domain [12,17,33,39,40]. Ruckes [32] hypothesized that the turtle body plan is the result of the displacement of the ribs and their incorporation into the specialized dermis of the Current Opinion in Genetics & Development 2017, 45:124–131
embryonic turtle carapace [33]. Burke [10,33] named and further characterized the CR, showing patterns of proliferation and the distribution of morphogenetic molecules similar to other organs (feathers and limbs) that develop via epithelial–mesenchymal interactions. Following extirpation experiments, Burke [10] noted that the CR shows a remarkable degree of regeneration, but that when portions of the CR were successfully removed in the snapping turtle Chelydra serpentina, the growth of the ribs was deflected towards a neighboring rib. This same behavior by the ribs was observed following cauterization of the CR in the softshell turtle P. sinensis [17], showing that the CR patterns the ribs within the dorsal www.sciencedirect.com
Patterning of the turtle shell Moustakas-Verho, Cebra-Thomas and Gilbert 127
Figure 3
The patterning of scutes in the turtle shell. The expression of Shh is used to show the development of scutes in hard-shelled Trachemys scripta embryos. (a) Shh is shown to be expressed in the developing scutes of the carapace (m, marginal scute primordia along the CR; v, vertebral scute primordia; central arrowhead points to costal scute primordia) and plastron (p). (b) Scutes have been lost or reduced independently in several lineages of turtles, and in the softshell turtle Pelodiscus sinensis, the process of scute formation appears to be arrested at the CR. (c) In a control explant culture of T. scripta, we see a normal spacing of marginal scute primordia along the CR. (d) In explants cultured with SU5402 to disrupt FGF signaling, the periodic patterning along the developing shell is lost. (e) Beginning from a pre-pattern of marginal scute primordia along the CR, two coupled reaction-diffusion systems are able to produce the pattern of scutes on the carapace. Images from [25].
dermis of the carapace. Interestingly, when a foil barrier was placed at the border between somitic and lateral plate mesoderm before formation of the CR in the hardshell turtle, portions of the carapace were absent and the ribs entered the abaxial domain and interdigitated with the dermal bones of the plastron [10]. This suggests that the lateral plate mesoderm (including the future area where the plastron will develop) could play a role in the lateral deflection of the ribs in the turtle. There are several additional studies that support the hypothesis that the lateral plate mesoderm is a key factor in patterning the turtle skeleton. The function of hepatocyte growth factor (HGF) is essential for the development of the CR; however, HGF expression in the turtle is seen in the lateral body wall and sclerotome, but not in the CR itself [41]. A study on Hox gene expression in turtles compared with other amniotes found conserved expression of Hoxc-6 in the sclerotome and dermatome at thoracic levels of the anterior-posterior axis, and an absence of Hoxc-6 expression from the turtle somatopleure [42]. Because Hoxc-6 is thought to determine thoracic (rib-forming) identity, it has been proposed that the absence of the expression of this gene from the lateral plate mesoderm is related to the absence of ribs ventrally in turtles [42]. Consistent with the absence of ribs ventrally in turtles is the expression of genes associated with the suppression of chondrogenesis in the ventral mesenchyme of the developing turtle [43]. www.sciencedirect.com
It, therefore, appears that the morphology of the ribs in turtles requires both the derived condition of the lateral plate mesoderm and the CR [44]. This, then, suggests that the rib morphology of turtles evolved together with the shell. Going back to the fossil evidence, the prototurtle Odontochelys shows a fully developed plastron ventrally, and does not show a fully ossified carapace but rather has neural plates of bone [45]. The fanned ribs of Odontochelys (Figure 2) [45] that do not curve ventrally suggest the presence of a CR. Further hints of CR (and hence, carapace, albeit with reduced ossification of dermal elements) in Odontochelys include the presence of a lateral bridge that would connect the plastron to the carapace in other turtles [46]. The homologies of the turtle shell have been debated since at least the 19th century, with some workers hypothesizing that the bones of the turtle shell have endoskeletal origins derived from the ribs, vertebrae, and sternum, whereas others viewed it as a composite structure formed by a mixture of endoskeletal and dermal ossification [1,47]. This is an important distinction not only for understanding the developmental process, but also in distinguishing alternate hypotheses for the evolutionary origin of the turtle shell as being formed by first, a novel epithelial-mesenchymal interaction in the CR that results in the deflection of the ribs and carapace formation independent of dermal ossification patterns [44], second, the fusion of expanded ribs and neural arches with Current Opinion in Genetics & Development 20172017, 4545:124–131
128 Developmental mechanisms, patterning and evolution
overlying osteoderms (expansion of ribs and vertebrae evolving independently from occurrence of osteoderms) [48], third, the endoskeleton via periosteal expansion of the ribs and neural arches of trunk vertebrae to form the costal and neural plates with no contribution from the dermis [49], or fourth, periosteal ossification of the ribs and neural arches followed by metaplastic ossification of a specialized dermis formed by the CR and the displacement of the ribs [12,22,47,50,51]. Metaplastic ossification is the conversion of one cell type to another, and in the case of the turtle carapace, ossification of the dermis. One of the most basal known genera of turtles with a complete shell, Proganochelys, shows what appears to be ribs fused to metaplastic bone in the carapace (Figure 2), though this ossification has not been investigated histologically. The formation of independent ossification centers in the mesenchyme and dermis is clear in the plastron, as well as in the nuchal and peripheral bones of the carapace [[12,22,49]12,22,43,49]. Such independent ossification centers are also seen in the nascent dermal osteoderms of the alligator [49,50,52]. However, the neural and costal bones of the turtle carapace begin ossification by periosteal ossification of the trunk vertebrae and ribs, followed by fusion of this periosteum of the vertebrae and ribs with bony spiculae formed in the dermis [22,47,49–51]. It is interesting to note that softshell turtles (P. sinensis, for example) do not have peripheral bones in the carapace and their ribs do not extend to the margins of the carapace [17], suggesting that the ribs may have a role in inducing the peripheral bones in the marginal dermis. Because the development of the turtle shell spans months, there is a clear need for better in ovo techniques to enable longterm cell labeling, lineage mapping, and experimental manipulation. As we defined Testudinata earlier as the clade originating from the first amniote with a fully developed turtle shell, the CR is essential to this diagnosis. What induces the CR itself? The mesenchyme of the CR is derived from the dermatomal compartment of somitic mesoderm [17,18], and replacement of chick somites with turtle somites did not result in the formation of a CR or axial arrest of the ribs [16], suggesting that the epithelial component of the CR is necessary. Though it is clear that there has been a co-option of morphogenetic mechanisms and gene regulatory networks for some developmental processes in carapace development [10,18,25,33], there is also a conservation of gene expression related to cell lineage [18]. Screening for CR-specific expression, components of the canonical Wnt signaling pathway were found to be expressed in the softshell turtle: Lef-1 and APCDD1 expression in the mesenchyme of the CR with nuclear localization of b-catenin protein in the epithelium of the CR [53]. Using the draft genomes of the softshell and green sea turtle, Wnt5a was found to be the only Wnt gene expressed in the CR region [54], and the researchers have Current Opinion in Genetics & Development 2017, 45:124–131
since shown that Wnt5a, Lef-1, and APCDD1 are expressed in the CR of not only softshell turtles (Figure 2), but also the other main cryptodiran research model, T. scripta, as well as the pleurodiran E. subglobosa [26]. Furthermore, electroporation of dominant-negative Lef-1 into the epidermis of the CR resulted in a local arrest of CR growth [17].
Future prospects The availability of genomic [54,55] and transcriptomic resources [23], as well as the development of in ovo [5,10,17,27] and ex vivo [25,47] laboratory protocols for studying turtles, has allowed workers to address old questions about the evolution of these organisms and to postulate novel hypotheses. Although the composite nature of the shell makes deciphering the tempo and mode of regulatory changes necessary for inferring its evolutionary origin complicated, the groundwork has already been laid for asking important questions. What is the mechanism of co-option of gene regulatory networks, such that the signaling center for the turtle scute uses some of the same proteins as feathers, hair, and teeth, but in different patterns? Did the evolution of the turtle shell require gradual, additive changes, or was there a large change (such as rib displacement) followed by secondary smaller modifications? Why is the pattern of scutes on the turtle shell stable phylogenetically, but labile in the individual? Fossil evidence still provides us with new questions about developmental mechanisms. Was the metaplastic ossification of the ribs, as seen in the proposed stem-turtles — Permian Eunotosaurus [56] and Middle Trassic Pappochelys [57], an exaptation in the evolution of the turtle shell? Is it possible developmentally to form a plastron without a carapace, as has been hypothesized (and criticized) in the Late Triassic proto turtle Odontochelys [45,46]? In addition to the trunk, there have been notable morphological changes to patterning of the cranium in turtles. The plesiomorphic presence of teeth in turtles has been extended to the Late Jurassic basal turtle Sichuanchelys palatodentata (Figure 2) [4], and the mechanism of tooth loss in turtles appears to be different from that in birds [58]. How this convergent loss of teeth and formation of a beak happens will be exciting to see, as well as the potential evolutionary developmental mechanism behind the hypothesized secondary condition of an anapsid skull (no temporal fenestration) in turtles from a primitively diapsid (two fenestrations) condition [59,60,61,62].
Conclusions The taxonomic and morphologic diversity represented by turtle species used in developmental biology currently allows hypotheses of evolutionary transformations rather than typological comparisons. The comparison of www.sciencedirect.com
Patterning of the turtle shell Moustakas-Verho, Cebra-Thomas and Gilbert 129
laboratory model species in evolutionary developmental biology is rather akin to the long-branch attraction problem [63]; the derived morphologies of model organisms obscure the phylogenetic signal of evolutionary transformations. The first step towards resolving this problem is the addition of relevant taxa to the comparison. The second step is to combine expression patterns and morphologies with an evolutionary hypothesis of relationships, and thereby deduce the polarity of the developmental transformations. Examples of this phylogenetic approach are the apomorphic expression of Wnt pathway genes in the CR tissues of turtles [26] and the patterned expression of genes that regulate scute development and loss in turtles [25]. We, therefore, see the abundance of turtle species being investigated for the origin and evolution of the turtle shell as a positive phenomenon. The elucidation of the developmental and genetic mechanisms involved in the origin and diversification of the turtle shell has raised the bar in evolutionary developmental biology and unified this field with genomics, paleontology, mathematical modeling, and ecology.
The biogeographic history of turtles and consideration of the ecological context for the origin and diversification of turtle lineages.
References and recommended reading
13. Loredo GA, Brukman A, Harris MP, Kagle D, Leclair EE, Gutman R, Denny E, Henkelman E, Murray BP, Fallon JF, Tuan RS, Gilbert SF: Development of an evolutionarily novel structure: fibroblast growth factor expression in the carapacial ridge of turtle embryos. J Exp Zool (Mol Dev Evol) 2001, 291B:274-281.
Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
Conflict of interest statement Nothing declared.
Acknowledgements
5.
Yntema CL: Procurement and use of turtle embryos for experimental procedures. Anat Rec 1964, 149:577-586.
6.
Yntema CL: A series of stages in the embryonic development of Chelydra serpentina. J Morphol 1968, 125:219-252.
7.
Yntema CL: Extirpation experiments on the embryonic rudiments of the carapace of Chelydra serpentina. J Morphol 1970, 132:235-244.
8.
Ewert MA: Embryology of turtles. In Biology of the Reptilia, 14. Edited by Gans C, Billet F, Maderson PFA . 1985:74-255.
9.
Miller JD: Embryology of marine turtles. In Biology of the Reptilia, 14. Edited by Gans C, Billet F, Maderson PFA . 1985: 271-328.
10. Burke AC: The development and evolution of the turtle body plan. Inferring intrinsic aspects of the evolutionary process from experimental embryology. Am Zool 1991, 31:616-627. 11. Beggs K, Young J, Georges A, West P: Ageing the eggs and embryos of the pig-nosed turtle. Carettochelys insculpta (Chelonia: Carettochelydidae), from northern Australia. Can J Zool 2000, 78:373-392. 12. Gilbert SF, Loredo GA, Brukman A, Burke AC: Morphogenesis of the turtle shell: the development of a novel structure in tetrapod evolution. Evol Dev 2001, 3:47-58.
14. Tokita M, Kuratani S: Normal embryonic stages of the Chinese softshelled turtle Pelodiscus sinensis (Trionychidae). Zool Sci 2001, 18:705-715. 15. Vincent C, Bontoux M, Le Douarin NM, Pieau C, MonsoroBurq AH: Msx genes are expressed in the carapacial ridge of turtle shell: a study of the European pond turtle, Emys orbicularis. Dev Genes Evol 2003, 213:464-469. 16. Nagashima H, Uchida K, Yamamoto K, Kuraku S, Usuda R, Kuratani S: Turtle-chicken chimera: an experimental approach to understanding evolutionary innovation in the turtle. Dev Dynam 2005, 232:149-161.
We thank Anne Burke, Jason Head, Walter Joyce, Torsten Scheyer, and James Parham for discussion, the American Museum of Natural History, Walter Joyce, Shigeru Kuratani, Chun Li, and Ingmar Werneburg for the use of images from published figures, Igor Danilov for the use of images from an unpublished specimen, Patricia Holroyd for the image of the University of California Museum of Paleontology (UCMP) specimen, and Fabien Lafuma for help with APE. This work was funded by the Academy of Finland, NSF grant IOS-145177, and Millersville University and Swarthmore College Faculty Research Funds.
17. Nagashima H, Kuraku S, Uchida K, Kawashima-Ohya Y, Narita Y, Kuratani S: On the carapacial ridge in turtle embryos: its developmental origin, function and the chelonian body plan. Development 2007, 134:2219-2226.
References
19. Nagashima H, Sugahara F, Takechi M, Ericsson R, KawashimaOhya Y, Narita Y, Kuratani S: Evolution of the turtle body plan by the folding and creation of new muscle connections. Science 2009, 325:193-196.
1.
MacCord K, Caniglia G, Moustakas-Verho JE, Burke AC: The dawn of chelonian research: turtles between comparative anatomy and embryology in the 19th century. J Exp Zool (Mol Dev Evol) 2015, 324B:169-180. Historical dissection of the evidence and context for theories regarding the evolutionary and developmental origin of the turtle shell. 2.
Lyson TR, Bhullar BAS, Bever GS, Joyce WG, de Queiroz K, Abzhanov A, Gauthier JA: Homology of the enigmatic nuchal bone reveals novel reorganization of the shoulder girdle in the evolution of the turtle shell. Evol Dev 2013, 15:317-325.
3.
Joyce WG, Parham JF, Gauthier JA: Developing a protocol for the conversion of rank-based taxon names to phylogenetically defined clade names, as exemplified by turtles. J Paleontol 2004, 78:989-1013.
4.
Joyce WG, Rabi M, Clark JM, Xu X: A toothed turtle from the Late Jurassic of China and the global biogeographic history of turtles. BMC Evol Biol 2016, 16:236.
www.sciencedirect.com
18. Moustakas JE: Development of the carapacial ridge: implications for the evolution of genetic networks in turtle shell development. Evol Dev 2008, 10:29-36.
20. Valenzuela N: The painted turtle, Chrysemys picta: a model system for vertebrate evolution, ecology, and human health. Cold Spring Harb Protoc 2009, 4:1-9. 21. Werneburg I, Hugi J, Mu¨ller J, Sanchez-Villagra MR: Embryogenesis and ossification of Emydura subglobosa (Testudines, Pleurodira, Chelidae) and patterns of turtle development. Dev Dynam 2009, 238:2770-2786. 22. Lima FC, Santos ALQ, Vieira LG, Da Silva-Junior LM, Roma˜o MF, De Simone SBS, Hirano LQL, Silva JMM, Montelo KM, Malvasio A: Ontogeny of the shell bones of embryos of Podocnemis unifilis (Troschel, 1848) (Testudines, Podocnemididae). Anat Rec 2011, 294:621-632. 23. Kaplinsky NJ, Gilbert SF, Cebra-Thomas J, Lillevali K, Saare M, Chang EY, Edelman HE, Frick MA, Guan Y, Hammond RM et al.: The embryonic transcriptome of the red-eared slider turtle (Trachemys scripta). PLoS ONE 2013, 8:e66357. Current Opinion in Genetics & Development 20172017, 4545:124–131
130 Developmental mechanisms, patterning and evolution
24. Cordero GA, Janzen FJ: An enhanced developmental staging table for the painted turtle, Chrysemys picta (Testudines: Emydidae). J Morphol 2014, 275:442-455. 25. Moustakas-Verho JE, Zimm R, Cebra-Thomas J, Lempia¨inen NK, Kallonen A, Mitchell KL, Ha¨ma¨la¨inen K, Salazar-Ciudad I, Jernvall J, Gilbert SF: The origin and loss of periodic patterning in the turtle shell. Development 2014, 141:3033-3039. The molecular and developmental basis of scute evolution in turtles that combine evolutionary, laboratory, and mathematical experiments to describe the diversification of the skin of the turtle shell. 26. Pascual-Anaya J, Hirasawa T, Sato I, Kuraku S, Kuratani S: Comparative analysis of pleurodiran and cryptodiran turtle embryos depicts the molecular ground pattern of the turtle carapacial ridge. Int J Dev Biol 2014, 58:743-750. Consideration of differences reported in the developmental expression of turtle shell development from different species, combined with hypothesis testing using additional species of turtle and newly published genomic resources. 27. Nomura T, Yamashita W, Gotoh H, Ono K: Genetic manipulation of reptilian embryos: toward an understanding of cortical development and evolution. Front Neurosci 2015, 9:1-11. Development of techniques forin ovo electroporation and short-term ex vivo culture of turtle embryos. 28. Chang C, Wu P, Baker RE, Maini PK, Alibardi L, Chuong CM: Reptile scale paradigm: Evo-Devo, pattern formation, and regeneration. Int J Dev Biol 2009, 53:813-826. 29. Dhouailly D: A new scenario for the evolutionary origin of hair, feather, and avian scales. J Anat 2009, 214:587-606. 30. Thesleff I, Mikkola ML: Development of ectodermal organs. Semin Cell Dev Biol 2014, 25–26:1-2. 31. Moustakas-Verho JE, Cherepanov GO: The integumental appendages of the turtle shell: an evo-devo perspective. J Exp Zool (Mol Dev Evol) 2015, 324B:221-229. A perspective on the evolution of the organs of the skin of the turtle shell. 32. Ruckes H: Studies in chelonian osteology. Part II. The morphological relationships between the girdles, ribs and carapace. Ann N Y Acad Sci 1929, 31:81-120. 33. Burke AC: Development of the turtle carapace: implications for the evolution of a novel bauplan. J Morphol 1989, 199:363-378. 34. Harris MP, Fallon JF, Prum RO: Shh-Bmp2 signaling module and the evolutionary origin and diversification of feathers. J Exp Zool 2002, 294:160-176. 35. Bardot B, Lecoin L, Fliniaux I, Huillard E, Marx M, Viallet JP: Drm/ Gremlin, a BMP antagonist, defines the interbud region during feather development. Int J Dev Biol 2004, 48:149-156. 36. Di-Poı¨ N, Milinkovitch MC: The anatomical placode in reptile scale morphogenesis indicates shared ancestry among skin appendages in amniotes. Sci Adv 2016, 2:e1600708. This work establishes that the skin appendages of reptiles share not only molecular signals in their developmental program, but also the anatomical placode with other amniotes. 37. Turing AM: The chemical basis of morphogenesis. Philos Trans R Soc Lond B Biol Sci 1952, 237:37-72. 38. Gierer A, Meinhardt H: A theory of biological pattern formation. Kybernetik 1972, 12:30-39. 39. Shearman RM, Burke AC: Ontogeny and phylogeny of the lateral somatic frontier. J Exp Zool (Mol Dev Evol) 2009, 312B:603-612. 40. Rice R, Gilbert SF, Riccio P, Cebra-Thomas J: Emerging from the rib: resolving turtle controversies. J Exp Zool (Mol Dev Evol) 2015, 324B:208-220. Consideration of differences in the developmental expression of turtle shell development in hard-shelled versus soft-shelled species. 41. Kawashima-Ohya Y, Narita Y, Nagashima H, Usuda R, Kuratani S: Hepatocyte growth factor is crucial for development of the carapace in turtles. Evol Dev 2011, 13:260-268. 42. Ohya KY, Kuraku S, Kuratani S: Hox code in embryos of Chinese soft-shelled turtle Pelodiscus sinensis correlates with the evolutionary innovation in the turtle. J Exp Zool (Mol Dev Evol) 2005, 304B:107-118. Current Opinion in Genetics & Development 2017, 45:124–131
43. Rice R, Kallonen A, Cebra-Thomas J, Gilbert SF: Development of the turtle plastron, the order-defining skeletal structure. PNAS 2016, 113:5317-5322. This work describes the molecular and histological development of the mesenchymal condensation in turtle plastron development, and proposes a developmental program for the induction of osteogenesis and comcomitant suppression of chondrogenesis in the evolution of the plastron. 44. Burke AC: Turtles . . .
. . . again. Evol Dev 2009, 11:622-624.
45. Li C, Wu X-C, Rieppel O, Wang L-T, Zhao L-J: An ancestral turtle from the Late Triassic of southwestern China. Nature 2008, 456:497-501. 46. Reisz RR, Head JJ: Palaeontology: turtle origins out to sea. Nature 2008, 456:450-451. 47. Cebra-Thomas J, Tan F, Sistla S, Estes E, Bender G, Kim C, Riccio P, Gilbert SF: How the turtle forms its shell: a paracrine hypothesis of carapace formation. J Exp Zool (Mol Dev Evol) 2005, 304B:558-569. 48. Lee MSY: Correlated progression and the origin of turtles. Nature 1993, 379:812-815. 49. Hirasawa T, Nagashima H, Kuratani S: The endoskeletal origin of the turtle carapace. Nat Commun 2013, 4:2107 http://dx.doi.org/ 10.1038/ncomms3107. 50. Cherepanov GO: The origin of the bony shell of turtles as a unique evolutionary model in reptiles. Russ J Herpetol 1997, 4:155-156. 51. Scheyer TM, Bru¨llmann B, Sanchez-Villagra MR: The ontogeny of the shell in side-necked turtles, with emphasis on the homologies of costal and neural bones. J Morphol 2008, 269:1008-1021. 52. Vickaryous MK, Hall BK: Development of the dermal skeleton in Alligator mississippiensis (Archosauria, Crocodylia) with comments on the homology of osteoderms. J Morphol 2008, 269:398-422. 53. Kuraku S, Usuda R, Kuratani S: Comprehensive survey of carapacial ridge-specific genes in turtle implies co-option of some regulatory genes in carapace evolution. Evol Dev 2005, 7:3-17. 54. Wang Z, Pascual-Anaya J, Zadissa A, Li W, Niimura Y, Huang Z, Li C, White S, Xiong Z, Fang D et al.: The draft genomes of softshell turtle and green sea turtle yield insights into the development and evolution of the turtle-specific body plan. Nat Genet 2013, 45:701-706. 55. Schaffer HB, Minx P, Warren DW, Shedlock AM, Thomson RC, Valenzuela N, Abramyan J, Amemiya CT, Badenhorst D, Biggar KK et al.: The western painted turtle genome, a model for the evolution of extreme physiological adaptations in a slowly evolving lineage. Genome Biol 2013, 14:R28. 56. Lyson TR, Bever GS, Scheyer TM, Hsiang AY, Gauthier JA: Evolutionary origin of the turtle shell. Curr Biol 2013, 23: 1113-1119. 57. Schoch RR, Sues H-D: A Middle Triassic stem-turtle and the evolution of the turtle body plan. Nature 2015, 523:584-587. A stem-turtle assignment to a fossil that shows similarities in the morphology of the ribs with other stem turtles, paired gastralia proposed to be homologous with the plastron of turtles, and a diapsid condition of the skull, which supports a diapsid origin for turtles. 58. Lainoff AJ, Moustakas-Verho JE, Hu D, Kallonen A, Marcucio RS, Hlusko LJ: A comparative examination of odontogenic gene expression in both toothed and toothless amniotes. J Exp Zool (Mol Dev Evol) 2015, 324B:255-269. A developmental genetic investigation of the evolutionary convergence of edentulism in amniotes. 59. Rieppel O, deBraga M: Turtles as diapsid reptiles. Nature 1996, 384:453-455. 60. Bever GS, Lyson TR, Field DJ, Bhullar BAS: Evolutionary origin of the turtle skull. Nature 2015, 525:239-242. www.sciencedirect.com
Patterning of the turtle shell Moustakas-Verho, Cebra-Thomas and Gilbert 131
61. Bever GS, Lyson TR, Field DJ, Bhullar BAS: The amniote temporal roof and the diapsid origin of the turtle skull. Zoology 2016, 119:471-473. A discussion of the developmental stage of fossil taxa proposed to be stem turtles, and the possible convergence of the anapsid condition from diapsid skulls.
65. Lyson TR, Schachner ER, Botha-Brink J, Scheyer TM, Lambertz M, Bever GS, Rubidge BS, de Queiroz K: Origin of the unique ventilatory apparatus of turtles. Nat Commun 2014, 5:5211 http://dx.doi.org/10.1038/ncomms6211. This work considers the origin of the ventilation mechanism in turtles, and re-diagnoses the presence of scutes in the stem turtleOdontochelys.
62. Schoch RR, Sues H-D: The diapsid origin of turtles. Zoology 2016, 119:159-161.
66. Gaffney ES: The comparative osteology of the Triassic turtle Proganochelys. Bull Am Mus Nat Hist 1990, 194:1-263.
63. Felsenstein J: Cases in which parsimony or compatibility methods will be positively misleading. Syst Biol 1978, 27:401-410.
67. Joyce WG, Lucas SG, Scheyer TM, Heckert AB, Hunt AP: A thinshelled reptile from the Late Triassic of North America and the origin of the turtle shell. Proc R Soc B 2009, 276:507-513.
64. Crawford NG, Parham JF, Sellas AB, Faircloth BC, Glenn TC, Papenfuss TJ, Henderson JB, Hansen MH, Simison WB: A phylogenomic analysis of turtles. Mol Phylogenet Evol 2015, 83:250-257. A genome-scale phylogeny of turtles produced using ultra-conserved elements and several algorithms, with discussion of the temporal biogeographic origin of turtle clades.
www.sciencedirect.com
68. Paradis E, Claude J, Strimmer K: APE: analyses of phylogenetics and evolution in R language. Bioinformatics 2004, 20:289-290.
Current Opinion in Genetics & Development 20172017, 4545:124–131