Skeletal gene expression in the temporal region of the reptilian embryos: implications for the evolution of reptilian skull morphology
© Tokita et al.; licensee Springer. 2013
Received: 25 June 2013
Accepted: 8 July 2013
Published: 23 July 2013
Reptiles have achieved highly diverse morphological and physiological traits that allow them to exploit various ecological niches and resources. Morphology of the temporal region of the reptilian skull is highly diverse and historically it has been treated as an important character for classifying reptiles and has helped us understand the ecology and physiology of each species. However, the developmental mechanism that generates diversity of reptilian skull morphology is poorly understood. We reveal a potential developmental basis that generates morphological diversity in the temporal region of the reptilian skull by performing a comparative analysis of gene expression in the embryos of reptile species with different skull morphology. By investigating genes known to regulate early osteoblast development, we find dorsoventrally broadened unique expression of the early osteoblast marker, Runx2, in the temporal region of the head of turtle embryos that do not form temporal fenestrae. We also observe that Msx2 is also uniquely expressed in the mesenchymal cells distributed at the temporal region of the head of turtle embryos. Furthermore, through comparison of gene expression pattern in the embryos of turtle, crocodile, and snake species, we find a possible correlation between the spatial patterns of Runx2 and Msx2 expression in cranial mesenchymal cells and skull morphology of each reptilian lineage. Regulatory modifications of Runx2 and Msx2 expression in osteogenic mesenchymal precursor cells are likely involved in generating morphological diversity in the temporal region of the reptilian skull.
KeywordsReptiles Skull Morphology Development Osteogenesis Heterotopy
Amniotes (Amniota) consist of two large groups of tetrapod vertebrates, Synapsida and Reptilia, that diverged from one another over 300 million years ago (Ma) (Carroll, 1988;Modesto & Anderson,, Modesto & Anderson, Modesto & Anderson,2004, Benton, 2005). The synapsids are represented today by mammals while reptiles by extant turtles, tuatara, lizards, snakes, crocodiles, birds, and their extinct relatives, including dinosaurs and pterosaurs. Over time, reptiles have evolved highly diverse morphological and physiological traits that allow them to exploit various ecological niches and resources on the land, in water, and in the air.
In reptiles, phylogenetic position of turtles is highly controversial. Traditionally, turtles have been regarded as the only surviving clade of stem reptiles based on the pattern of their skull morphology: an anapsid skull whose temporal region is completely roofed with bones (Williston, 1917, Gregory, 1946, Romer, 1968, Gaffney, 1980;Reisz & Laurin,, Reisz & Laurin, Reisz & Laurin,1991, Lee, 1993;Laurin & Reisz,, Laurin & Reisz, Laurin & Reisz,1995, Lee, 1996 1997, Reisz, 1997, Lee, 2001). However, recent comprehensive analysis of morphological traits (Rieppel & deBraga, 1996;deBraga & Rieppel,, deBraga & Rieppel, deBraga & Rieppel,1997, Rieppel, 2000, Hill, 2005, Li et al., 2008, but see Lyson et al., 2010 2013 for opposed conclusion) and molecular phylogenetic studies (Hedges & Poling, 1999;Kumazawa & Nishida,, Kumazawa & Nishida, Kumazawa & Nishida,1999, Iwabe et al., 2005, Hugall et al., 2007, Shedlock et al., 2007, Shen et al., 2011, Tzika et al., 2011, Chiari et al., 2012, Crawford et al., 2012, Fong et al., 2012, Lyson et al., 2012, Wang et al., 2013) suggest that there is a close relationship of turtles to diapsid reptiles, implying that the temporal fenestrae were secondarily closed in turtles. In this study, we employ the hypothesis that turtles are descendent of diapsid reptiles.
Although skull morphology has been regarded as an important character in classification of reptiles and in understanding the ecological and physiological aspects of each reptilian species, the developmental mechanism underlying diversification of reptilian skull morphology is poorly understood (Rieppel, 1993a, Evans, 2008). As a consequence, a general genetic and developmental model of reptile skull diversity does not yet exist. In this paper, we test the hypothesis that changes of skeletal gene expression patterns cause diversification of reptilian skull morphology through comparative analyses of gene expression in the embryos of representative reptilian species and reveal a potential developmental basis underlying reptilian skull evolution. First, we describe the pattern of early phases of cranial morphogenesis in a crocodile species with both upper and lower temporal bars surrounding temporal fenestrae, using molecular markers specific for musculoskeletal tissue precursors. Then, we compare these data with cranial morphogenesis in a turtle species. We found a broader expression of the early osteogenic genes, Runx2 and Msx2 in the mesenchymal cells at the temporal region of turtle embryos, compared to that in crocodile embryos. Finally, to obtain a broader picture of reptilian skull morphogenesis, we examined expression patterns of Runx2 and Msx2 in cranial morphogenesis of a snake species without temporal bars on the skull and compared with the patterns in crocodile and turtle embryos. Our findings suggest that there is a possible correlation between the expression patterns of Runx2 and Msx2 and the architectural pattern seen in the temporal region of the reptilian skull.
In previous studies in which cranial osteogenesis of reptilian embryos was described, whole-mount clearing and staining with Alizarin red was used to detect mineralization of intramembranous bones that comprise the dermatocranium (Kamal et al., 1970;Haluska & Alberch,, Haluska & Alberch, Haluska & Alberch,1983, Rieppel, 1993b, Rieppel, 1993c, Kuratani, 1999;Rieppel & Zaher,, Rieppel & Zaher, Rieppel & Zaher,2001, Sheil, 2003, Sheil, 2005, Boughner et al., 2007;Vickaryous & Hall,, Vickaryous & Hall, Vickaryous & Hall,2008;Sánchez-Villagra et al.,, Sánchez-Villagra et al., Sánchez-Villagra et al.,2009, Werneburg et al., 2009). However, this method is unable to identify the distribution of the precursor cells of bones: osteoblasts, as reported by others (Kerney et al., 2010). To overcome this, we conducted section in situ hybridization analysis, which labels tissues located deep inside of the embryonic body and is effective for detecting tissue-specific domains of expression. We used a probe to Runx2, which is a molecular marker for osteogenic mesenchymal precursor cells (Ducy et al., 1997, Bobola et al., 2003, Abzhanov et al., 2007, Han et al., 2007, Kerney et al., 2010) and described its expression pattern in the temporal region of reptilian embryos where mineralization of bones has not been initiated. Furthermore, to describe distribution pattern of "non-osteoblast" cell lineages relative to that of osteoblasts in the cranial tissue of the embryos, we also examined expression of other tissue-specific markers: MyoD for skeletal muscle precursors (Hacker & Guthrie, 1998, Noden et al., 1999), Sox9 for cartilage precursors (Wright et al., 1995, Bell et al., 1997), Scleraxis (Scx) and Six2 for precursor of connective tissues, including ligaments and tendons (Oliver et al., 1995, Schweitzer, 2001, Dreyer et al., 2004;Edom-Vovard & Duprez,, Edom-Vovard & Duprez, Edom-Vovard & Duprez,2004, Schweitzer et al., 2010).
Differential expression of early osteoblast marker, Runx2, in the head of crocodile and turtle embryos
Expression of potential upstream osteogenic regulatory genes in the head of crocodile and turtle embryos
Through comparative analysis of expression patterns of tissue-specific marker genes, we noticed a difference in the spatial pattern of expression of the early osteoblast marker, Runx2 in the head of crocodile and turtle embryos. To reveal potential mechanisms that account for such differential distribution of osteogenic mesenchymal precursor cells between two reptilian lineages with or without temporal fenestrae, we next examined expression patterns of some candidate genes that are known to regulate cranial osteogenesis. In the present study, we focused on Bmp4, Msx1, and Msx2. Bmp4 is a signaling molecule and plays a key role in the Bmp signaling pathway. Because exogenous Bmp4 increases tissue volume in calvarial bone tissue culture, this protein is considered to be involved in calvarial bone growth (Kim et al., 1998, Rice et al., 2003). Both Msx1 and Msx2 are members of the muscle segment homeobox (msh) gene family of transcription factors and both loss-of- and gain-of-function analyses of these genes suggest their essential roles in vertebrate cranial osteogenesis (Satokata & Maas, 1994, Satokata et al., 2000).
In contrast to Bmp4 and Msx1, we detected differential expression patterns of Msx2 in the head of crocodile and turtle embryos. In crocodile embryos at stage 14 and 15, Msx2 was expressed in a thin layer of mesenchymal cells surrounding the dorsal aspect of the brain (Figures 6C and 6E). In the posterior part of the head, the ventral edge of this Msx2-expressing cell population is located dorsal to the eye. In these crocodilian embryos, Msx2 expression was also observed in a population of the mesenchyme that occupied the domain between the ventrolateral part of quadrate cartilage and surface epidermis (Figures 6C and 6E). These mesenchymal cells expressed Msx1 as well (Figure 6B) and appeared to differentiate into the quadratojugal bone later. In crocodile embryos at stage 17, specific expression of Msx2 was detected at a population of mesenchymal cells in close proximity of Runx2-expressing precursors of postorbital and quadratojugal bones, as well as in a thin layer of the mesenchyme surrounding the brain dorsally where future parietal bones were developed (Figures 7C and 7F). We observed that the space adjacent to Msx2-positive precursors of these dermatocranial elements was filled with Msx2-negative mesenchymal cells. Interestingly, we observed broader expression of Msx2 in turtle embryos, compared to that in stage-matched crocodile embryos. In turtle embryos examined, Msx2 was expressed in mesenchymal cells that populate lateral aspect of the head of embryos (Figures 6I and 6K; Figures 7I and 7J). The ventral edge of the Msx2-expressing mesenchymal layer was terminated ventral to the eye and these cells covered MyoD-expressing external adductor muscle laterally. Showing its dorsoventrally broadened expression pattern, the domain of Msx2 expression largely overlapped with that of Runx2 in turtle embryos (Figures 4B and 4G; Figure 5B).
Expression of Runx2 and Msx2 in the head of snake embryos
Expression domains of the genes in the head of crocodile, turtle, and snake embryos
St.14/15: The domain dorsal to the oral cavity where the ventral part of the braincase and future palatine and pterygoid bones develop; a domain dorsolateral to the orbit where the future dorsal projection of the postorbital bone forms; the domain ventrolateral to the orbit where future jugal and postorbital bones form; the mesenchyme that later differentiates into the main body of the postorbital bone.
St.14/15: A population of cells medial to the precursor of the jaw adductor muscles; the mesenchyme localized at the domain dorsolateral and ventrolateral to the orbit; a thick layer of the mesenchymal cells that completely covers the brain and the precursor of jaw adductor muscle laterally.
St.26: The mesenchyme occupying the space medial to the quadrate cartilage precursor; the mesenchyme ventral to the orbit; a layer of mesenchymal cells surrounding the brain laterally.
St.17: The cell populations localized to the area where the future dermatocranium differentiates (palatine, parietal, postorbital, pterygoid, quadratojugal bones).
St.17: A thick layer of mesenchymal cells surrounding the braincase and jaw adductor muscle laterally; the mesenchyme associated with the quadrate cartilage and the ventral part of the braincase.
St.29: The precursors of palatine and pterygoid bones; the mesenchyme accompanying jaw cartilages; a layer of loose mesenchyme that later forms a precursor of parietal bones; the precursors of the maxilla bones.
St.31: The precursors of the dermatocranial elements, including the parietal.
St.14/15: Precursor cells of each jaw muscle in the first pharyngeal arch; eye muscle precursors.
St.14/15: The primordia of jaw and eye muscles.
St.26: The primordia of jaw and eye muscles.
St.17: Differentiated jaw and eye muscles.
St.17: Differentiated jaw and eye muscles.
St.29-31: Differentiated jaw and eye muscles.
St.14/15: Cartilage precursors that later differentiate into the quadrate, Meckel's cartilage, and the braincase.
St.14/15: Precursor cells of the braincase, quadrate, and Meckel's cartilages.
St.26: The precursors of quadrate and Meckel's cartilages and the braincase; a layer of mesenchyme surrounding the brain laterally.
St.17: Differentiated chondrocranium and splanchnocranium components (the braincase, quadrate, and Meckel's).
St.17: Differentiated chondrocranium and splanchnocranium components (the braincase, quadrate, and Meckel's).
St.29-31: Differentiated chondrocranium and splanchnocranium components (the braincase, quadrate, and Meckel's).
St.14/15: Tendon precursor cells within jaw muscle primordia; connective tissue within eye muscles.
St.14/15: A layer of mesenchymal cells located at the periphery of the jaw adductor and eye muscle precursors.
St.17: Tendinous tissues accompanying jaw muscles; connective tissue associated with eye muscles.
St.17: Tendinous tissues at the periphery of jaw adductor muscles; the precursor of the bodenaponeurosis (central tendon of external adductor) within the jaw adductor muscular tissue.
St.14/15: The mesenchyme surrounding the eyes and cartilaginous precursors of the braincase, quadrate, and Meckel's; the mesenchyme between jaw muscle precursors and the skeletal tissues to which the muscles attach; the mesenchyme that dorsally surrounds the brain.
St.14/15: The mesenchyme surrounding the eye; the mesenchyme associated with the braincase and jaw cartilages; the mesenchyme within the jaw muscle precursors.
St.17: The mesenchyme localized around the jaw articulation between quadrate and Meckel's; the mesenchyme associated with the braincase, postorbital bone, and jaw muscles.
St.17: The mesenchyme surrounding jaw adductor muscles, braincase, and jaw cartilages.
St.14-17: The epithelium of cochlear
canal; the mesenchyme surrounding the eye; the mesenchyme distributed in the medial part of jaw primordia; the precursors of the palatine bones; a population of mesenchymal cells covering the brain dorsally.
St.14-17: The epithelium if cochlear canal; The mesenchyme dorsolateral and ventrolateral to the eye; a limited population of the mesenchyme in close proximity of the jaw articulation.
St.14-17: The epithelium of the cochlear canal; the mesenchyme adjacent to the jaw articulation; the mesenchyme lateral to the quadrate and Meckel's cartilages; a thin layer of mesenchymal cells covering the brain dorsally.
St.14-17: The epithelium of the cochlear canal; the mesenchyme adjacent to the jaw articulation; the mesenchyme lateral to quadrate and Meckel's cartilages; the mesenchyme that populates the domain dorsal to the eye.
St.14/15: A thin layer of mesenchymal cells surrounding the dorsal aspect of the brain; the mesenchyme located dorsal to the eye; the mesenchyme occupying the domain between the ventrolateral part of quadrate cartilage and surface epidermis.
St.14-17: Mesenchymal cells populating lateral aspect of the head (lateral to external adductor muscle).
St.26: The mesenchyme medial to the quadrate precursor; a mesenchymal layer surrounding the brain dorsally.
St.17: A population of mesenchymal cells in close proximity of postorbital and quadratojugal bone precursors; a thin layer of the mesenchyme surrounding the brain dorsally where future parietal bones form.
St.29: The precursors of palatine and pterygoid bones; the mesenchyme accompanying jaw cartilages; a layer of loose mesenchyme that later forms a precursor of parietal bones.
St.31: The precursors of the dermatocranial elements, including the parietal.
Potential developmental basis of anapsid skull in turtles
Skull morphology, especially the osteological configuration of the temporal region, has historically been treated as the most important character in the classification of major lineages of reptiles. Based on their anapsid skull, turtles have been regarded as a sole descendent of stem reptiles (Williston, 1917, Gregory, 1946, Romer, 1968, Gaffney, 1980;Reisz & Laurin,, Reisz & Laurin, Reisz & Laurin,1991, Lee, 1993;Laurin & Reisz, 1995, Lee, 1996 1997, Reisz, 1997, Lee, 2001) despite the contrary argument that turtles were derived from an ancestor with a diapsid skull (Lakjer, 1926, Goodrich, 1930). Recent phylogenetic studies where the interrelationships of both extant and extinct reptiles were surveyed through comprehensive analysis of multiple osteological traits concluded that turtles were closely related to lepidosaurian diapsids (Rieppel & deBraga, 1996;deBraga & Rieppel , deBraga & Rieppel deBraga & Rieppel 1997Rieppel 2000, Hill, 2005, Li et al., 2008). Furthermore, results of molecular phylogenetic studies have strongly suggested diapsid affinity of turtles (Hedges & Poling, 1999;Kumazawa & Nishida,, Kumazawa & Nishida, Kumazawa & Nishida,1999, Iwabe et al., 2005, Hugall et al., 2007, Shedlock et al., 2007Shen et al. 2011, Tzika et al., 2011, Chiari et al., 2012, Crawford et al., 2012, Fong et al., 2012, Lyson et al., 2012, Wang et al., 2013). If turtles were derived from a diapsid ancestor, then the anapsid skull of turtles evolved independently from that of ancestral lineages of reptiles by secondary closure of the temporal fenestrae. However, although the phylogenetic position of turtles within amniotes still remains inconclusive (Lyson et al., 2010 2013, Kuratani et al., 2011), there has been no study in which the process of development of their anapsid skull is described with molecular markers for labeling precursor cells of the dermatocranium. In the present study, we examined early cranial morphogenesis of representative reptilian species through comparative analysis of gene expression patterns and found unique expression patterns of Runx2 and Msx2 in turtle embryos that are not observed in crocodile and snake embryos.
In this study, we focused on several candidate molecules that potentially regulate Runx2 expression and examined their expression patterns in reptilian embryos. Bmp4 is known to be involved in osteogenesis of vertebrates where it regulates expression of other osteogenic regulatory genes, including Msx1, Msx2, and Runx2 (Marazzi et al., 1997, Kim et al., 1998, Hollnagel et al., 1999, Tribulo et al., 2003, Zhang et al., 2003, Brugger et al., 2004). Msx1 is a transcription factor known to regulate growth and patterning of calvarial bones in mouse embryos (Satokata & Maas, 1994, Han et al., 2007, Roybal et al., 2010). Although, as previously reported in mouse embryos (Rice et al., 2003, Han et al., 2007), both Bmp4 and Msx1 are expressed in limited populations of cranial mesenchyme in embryos of crocodiles and turtles, we could not detect any substantial differences in their expression domains between the two species. On the other hand, we observed spatially different expression patterns of Msx2 in the head of embryos of all reptilian species we examined. Expression of Msx2 was detected in cranial mesenchyme and dermal bone precursors as reported in mouse embryos (Jabs et al., 1993, Ishii et al., 2003, Rice et al., 2003, Han et al., 2007, Roybal et al., 2010). Furthermore, its expression spatially overlapped with that of Runx2 in reptilian embryos, as in mouse embryos (Ishii et al., 2003, Rice et al., 2003, Han et al., 2007). In turtle embryos, expression domain of Msx2 in the mesenchyme distributed in the temporal region of the head was broad in a dorsal-ventral direction, showing similar pattern with Runx2 in the mesenchyme. A mutation in the homeobox of Msx2 gene causes craniosynostosis in human and mouse (Jabs et al., 1993, Liu et al., 1999). Similarly, overexpression of Msx2 promotes osteogenesis (Cheng et al., 2003, Ichida et al., 2004) and causes overgrowth of dermal bones of the skull by increasing the number of proliferative osteoblasts (Dodig et al., 1999, Liu et al., 1999). In contrast, loss-of-function of Msx2 results in defects of skull ossification in mammals (Satokata et al., 2000, Wilkie et al., 2000, Ishii et al., 2003, Antonopoulou et al., 2004, Han et al., 2007). Furthermore, Msx2 is known to positively regulate downstream Runx2 expression (Ishii et al., 2003, Han et al., 2007, Watanabe et al., 2008). Considering the evidence provided by previous studies, regulatory changes in Msx2 expression in turtle embryos may influence expression patterns of downstream Runx2, which regulate osteoblast differentiation. Dorsoventrally broadened distribution of osteogenic mesenchymal precursor cells in the temporal region of the head owing to the regulatory alteration of these osteogenic genes may allow this reptilian lineage to reacquire the anapsid skull. Although the precise mechanism underlying regulatory change of Msx2 expression in the head of turtle embryos has not been identified, recent findings that early stage arrest of Msx2 expression in neural crest-derived odontoblasts may account for the absence of teeth in turtles (Tokita et al., 2012) supports the hypothesis that this transcription factor may play a pivotal role in the development of their unique cranial morphology.
The development of the dermatocranium occurs in multiple steps (Ishii et al., 2003). The first phase includes the genesis, migration, and initial specification of osteogenic mesenchymal precursor cells. The second phase consists of the differentiation of the mesenchyme into osteoblasts. And the last phase includes deposition of osteogenic extracellular matrix around the osteoblasts and mineralization of the matrix. The dermatocranium of vertebrates is formed from cranial mesenchyme derived from two distinct embryonic sources: neural crest and mesoderm (Jiang et al., 2002;Gross & Hanken , Gross & Hanken Gross & Hanken 2005;Noden & Trainor,, Noden & Trainor, Noden & Trainor,2005). Unfortunately, fate mapping studies of each dermatocranial element as performed in avian and mammalian embryos (Le Lièvre, 1978, Noden, 1978 1983, Couly et al., 1993;Köntges & Lumsden,, Köntges & Lumsden, Köntges & Lumsden,1996, Jiang et al., 2002) have not been done in non-avian reptiles. Interestingly, the pattern of migration and distribution of cranial neural crest cells from which some cranial dermal bones should form is almost identical in early stage embryos of crocodiles and turtles (Meier & Packard, 1984;Hou & Takeuchi,, Hou & Takeuchi, Hou & Takeuchi,1994;Kundrát,, Kundrát, Kundrát,2008). Such data may support that differentiation or maturation processes of osteogenic mesenchyme are more responsible for producing diversity of reptilian skull morphology. We speculate that the developmental program, which determines cranial mesenchymal populations where early-phase osteogenic transcription factors Msx2 and Runx2 are activated, may be important in the patterning of reptilian skull morphology.
There exists substantial diversity in the skull morphology within turtles and most living turtle species do not have fully anapsid skulls and instead possess varying degrees of dorsal and/or ventral emargination on their skull (Jones et al., 2012, Werneburg, 2012a). In the present study, we could not sample and analyze the embryos of turtle species with fully anapsid skull, such as marine turtles (Kuratani, 1999, Jones et al., 2012), alligator snapping turtle () (Sheil, Macrochelys temminckii 2005), and big-headed turtle (Platysternon megacephalum), owing to difficulty in the access to the materials. Instead, we analyzed the embryos of a soft-shelled turtle species with highly emarginated skull. In fact, soft-shelled turtles have only a narrow bar of bone across the temporal region lateral to the external adductor muscles due to large scale emargination from the dorsal and ventral margins of the cheek (Ogushi, 1911, Sheil, 2003). In normal development of soft-shelled turtles, the postorbital bone does not grow in a posterior direction significantly, keeping its relatively small size within the dermatocranium (Sheil, 2003;Sánchez-Villagra et al.,, Sánchez-Villagra et al., Sánchez-Villagra et al.,2009). Therefore, the small postorbital bone of soft-shelled turtles does not largely contribute to the formation of a bony roof at the temporal region of the skull.
It is interesting that we observed dorsoventrally broadened distribution of the mesenchymal cells that express Runx2 at the temporal region of the embryos of a soft-shelled turtle species with highly emarginated skull. Dermal bone development occurs through a multi-step molecular pathway regulated by different transcription factors (Zhang, 2010). As an initial step, Runx2 is required for the differentiation of mesenchymal cells into preosteoblasts. In subsequent stage where these preosteoblasts differentiate into mature osteoblasts, Osx, a downstream gene of Runx2, is necessary (Nakashima et al., 2002, Nishio et al., 2006). Furthermore, in the later stages where the osteoblasts produce osteogenic extracellular matrix and the mineralization of these extracellular matrix is occurred, many additional molecules such as bone sialoprotein, osteopontin, and osteocalcin are involved (Zhang, 2010). We speculate that in soft-shelled turtles only a limited population of cells within Runx2-positive preosteoblasts distributed in the temporal region of the head is allowed to differentiate into mature osteoblasts and eventually osteocytes through regulation of expression of down stream genes (e.g. Osx), to form a pair of relatively small postorbital bones. Although the regulatory mechanism of Osx expression in osteogenic mesenchyme is not fully understood, both Runx2-dependent and -independent pathways have been suggested (Lee et al., 2003;Celil & Campbell,, Celil & Campbell, Celil & Campbell,2005, Maehata et al., 2006, Xing et al., 2007, Zhang, 2010). Histological analysis reveals that late stage soft-shelled turtle embryos have a layer of (non-muscular) fibrous connective tissue lateral to the external adductor muscles (Additional file 3). Judging from its position, the connective tissue layer appears to be derived from Runx2-positive preosteoblasts and have a potential to ossify themselves as other connective tissues represented by tendons and ligaments (Okawa et al., 1998;Tokita et al., 2007). Interestingly, similar type of connective tissue layer is absent in the temporal region of crocodile and snake embryos (Additional file 3). Those histological observations support the above hypothesis that later processes of cranial osteogenesis may largely contribute to the construction of the main body of each dermatocranial element from the osteogenic mesenchymal progenitor pool.
The dorsoventrally broadened distribution of preosteoblasts observed in turtle embryos might be a developmental synapomorphy re-acquired by the common ancestor of turtles. In the course of chelonian evolution, each chelonian lineage may develop the temporal dermal bones (e.g. postorbital, parietal, jugal) with various sizes and shapes, through regulatory changes of the osteogenic down stream molecules. Future studies should investigate expression pattern of Runx2 and Msx2 in the head of embryos of turtle species with fully anapsid skull, as well as expression pattern of downstream genes that regulate differentiation of mature osteoblasts and osteocytes in turtle embryos, to verify a correlation between the gene expression pattern and their skull morphology.
Heterotopy in distribution of osteogenic mesenchymal precursor cells and diversification of reptilian skull morphology
The frame-like skulls possessed by diapsid reptiles evolved in response to functional forces (Rieppel, 1993a, Moazen et al., 2009, Herrel et al., 2007, Curtis et al., 2011) and several studies have suggested heterochrony as a driving force for producing this morphological diversity (Rieppel, 1993a, Whiteside, 1986, Irish, 1989). The ancestral lineage of diapsid reptiles possessed upper and lower temporal bars that encircle temporal fenestrae (Müller, 2003, Moazen et al., 2009). The lower temporal bar that encloses lower temporal fenestra ventrally was probably lost once in the common ancestor of lepidosaurs and archosaurs, possibly as the outcome of paedomorphosis: incomplete ossification of a quadrato-maxillary ligament between jugal and quadratojugal bones (Rieppel, 1993a;Müller,, Müller, Müller,2003). If this is true, the lower temporal bar that possibly results from peramorphosis (hypermorphosis): complete ossification of a quadrato-maxillary ligament was independently re-acquired in the lineages of tuatara and crocodiles, as well as in several extinct reptilian lineages (Rieppel, 1993a;Müller,, Müller, Müller,2003). Furthermore, disappearance of upper temporal bar, which is regarded as an extreme condition of reduction of the dermatocranium in reptiles, may have independently evolved in the skull of geckos (Gekkonidae), miniaturized fossorial lizards (e.g., Typhlosaurus, Dibamus), amphisbaenian, and snakes, as the outcome of paedomorphosis represented by the retardation of ossification (Rieppel, 1993a, Irish, 1989;Cundall & Irish,, Cundall & Irish, Cundall & Irish,2008). In the present study, we revealed a possible correlation between distribution pattern of Runx2 and/or Msx2-expressing osteogenic mesenchymal precursor cells and the skull morphology of each reptilian lineage (Figure 9). In early stage crocodile embryos, we observed focal distribution of osteogenic mesenchyme around the domain where future temporal bars are formed. In early stage snake embryos, osteogenic mesenchymal cells were primarily found adjacent to the primordium of the braincase and the spatial pattern presaged the absence of bony temporal bars in the temporal region of adult animal.
Regulatory modifications of Runx2 and Msx2 expression in osteogenic mesenchymal precursor cells are likely involved in generating morphological diversity in the temporal region of the reptilian skull, including secondary closure of the temporal fenestrae in turtles. Our findings demonstrate that not only heterochrony in ossification of the dermatocranium that has been traditionally regarded as the major factor producing diversity of reptilian cranial morphology but also heterotopy in distribution of the osteogenic precursor cells may play a fundamental role in this process and it should be further investigated in future studies of reptilian cranial development and evolution.
Materials and methods
Sample collection and staging of embryos
Fertilized eggs of Chinese soft-shelled turtle, Pelodiscus sinensis, were purchased commercially from a local breeder in Japan. Fertilized eggs of Siamese crocodile, Crocodylus siamensis, were provided by a local breeder in Thailand. Fertilized eggs of corn snake, Pantherophis guttatus, were obtained by the first author after mating several pairs of the reproductively mature adults in the laboratory. Staging of P. sinensis embryos was performed after Tokita and Kuratani (2001). Because there is no embryonic staging system for C. siamensis at present, we used the system for Alligator mississippiensis embryos (Ferguson, 1985) where each stage was determined based on external morphology of the embryos, for staging of this species. Staging of P. guttatus (Zehr, embryos was performed on the basis of staging table of Thamnophis sirtalis 1962). Interspecific comparisons of gene expression pattern were performed in the embryos that are comparable to each other in terms of overall external morphology. Because snake embryos are limbless, we mainly employed external features of the head of the embryos as primary criteria for determining the stages for comparison. All animal experiments were approved by the University of Tsukuba Committee for Animal Care (No.10-034).
Total RNA was extracted from embryos using ISOGEN reagent (NIPPON GENE CO., LTD).
RT–PCR was performed to amplify fragments of P. sinensis Runx2, Six2 and C. siamensis Bmp4, Msx2, MyoD, Runx2, Scleraxis (Scx), Six2, Sox9 and P. guttatus Msx2, Runx2, Sox9 messenger RNA. Primer sequences used for isolation of the fragments of these genes are available upon request. Because Bmp4, Msx1, Msx2, MyoD, Scx, Sox9 of Pelodiscus and MyoD of Pantherophis were already sequenced and sequence data were deposited in the database by other researchers, we isolated the orthologous fragments by RT–PCR with primers constructed by referring to the reported sequence data. The fragments were isolated using the pGEM T-easy vector systems (Promega) or TOPO® TA cloning kit (Invitrogen) and sequenced using an ABI 3130 sequencer (Applied Biosystems). To identify the orthologous genes of the isolated fragments, comparable sequence data were surveyed using a BLAST search, and phylogenetic trees with neighbor joining method were constructed after sequence alignment using the CLUSTALX software. All new DNA sequence data were deposited in the DDBJ database (AB811933-AB811944).
Gene expression analysis
Embryos were fixed in 4% PFA, dehydrated using an methanol series, placed in xylene, embedded in paraffin, and sliced with a microtome. Serial sections were hybridized with digoxigenin-labeled RNA riboprobes as described in Neubüser et al. (1995) with slight modifications. To identify the expression domain of Msx1 in crocodile tissues, chicken Msx1 antisense riboprobe was hybridized. Generally, hetero-specific RNA probes easily hybridize among reptilian lineages (Harris et al., 2006, Tokita et al., 2012). In this study, we only analyzed reptilian embryos at the ontogenetic stages where early cranial osteogenesis occurs. To confirm the expression pattern of each gene in the cranial tissues, two to five individuals representing each embryonic stage were sampled for analysis. The consistency of the gene expression patterns among all individual embryos at the same stage was confirmed. Multiple sections representing several longitudinal (anterior-posterior) planes prepared from the same individual were hybridized with the probes and the sections prepared at corresponding longitudinal planes were compared between different individuals. Corresponding longitudinal planes between different reptilian species were determined based on overall histological configuration of the head of the embryos. For visualization of each cranial tissue and interspecific comparison of general histology of the head, Miligan's Trichrome staining was performed following standard protocols. To identify each anatomical structure in cranial musculoskeletal tissues of the embryos, we took the results of other's researches into account: (Schumacher, 1973, Rieppel, 1993b;Vickaryous & Hall,, Vickaryous & Hall, Vickaryous & Hall,2008;Bona & Desojo,, Bona & Desojo, Bona & Desojo,2011) for crocodile, (Schumacher, 1973, Rieppel, 1990, Rieppel, 1993c;Sánchez-Villagra et al.,, Sánchez-Villagra et al., Sánchez-Villagra et al.,2009, Werneburg, 2012a 2012b) for turtle, and (Kamal et al., 1970, Haas, 1973, Zaher, 1994;Buchtová et al.,, Buchtová et al., Buchtová et al.,2007) for snake.
Jaw adductor muscle or the anlage of jaw adductor muscle
External adductor muscle
Basicranial plate cartilage
precursor of jaw cartilages (quadrate and Meckel's)
Eye muscles or the anlagen of eye muscles
Anlage of jaw muscle complex
Primordium of maxilla bone
Parietal bone or precursor of parietal bone
Primordium of pterygoid bone
Primordium of palatine bone
Postorbital bone or primordium of postorbital bone
Quadrate cartilage or precursor of quadrate cartilage
Quadratojugal bone or primordium of quadratojugal bone
Ganglion of trigeminal nerve
First pharyngeal arch
Second pharyngeal arch
We appreciate Sriracha Crocodile Farm & Product Co., LTD. and the company staffs, especially Nussara Thongprasert in collection of fertilized eggs of Crocodiles. We also thank Manasaree Klomtun, Punnapa Pinweha, and Ekawit Threenet for their kind help in collection of Crocodile eggs. MT thanks Hiroshi Wada who allowed the use of facilities for experiments and analyses, Hiroki Ono who kindly gifted chick Msx1 probe, Matthew Brandley and Richard Schneider for critical reading and editing of a draft of the manuscript, and Johannes Müller for helpful comments to early version of the manuscript. This study was partially supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan to M.T. (22770077).
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