The evolution,development and skeletal identity of the crocodylian pelvis: Revisiting a forgotten scientific debate

Unlike most tetrapods, in extant crocodylians the acetabulum is formed by only two of the three skeletal elements that constitute the pelvis, the ilium, and ischium. This peculiar arrangement is further confused by various observations that suggest the crocodylian pelvis initially develops from four skeletal elements: the ilium, ischium, pubis, and a novel element, the prepubis. According to one popular historical hypothesis, in crocodylians (and many extinct archosaurs), the pubis fuses with the ischium during skeletogenesis, leaving the prepubis as a distinct element, albeit one which is excluded from the acetabulum. Whereas the notion of a distinct prepubic element was once a topic of considerable interest, it has never been properly resolved. Here, we combine data gleaned from a developmental series of Alligator mississippiensis embryos, with a revised interpretation of fossil evidence from numerous outgroups to Crocodylia. We demonstrate that the modern crocodylian pelvis is composed of only three elements: the ilium, ischium, and pubis. The reported fourth pelvic element is an unossified portion of the ischium. Interpretations of pelvic skeletal homology have featured prominently in sauropsid systematics, and the unambiguous identification of the crocodylian pubis provides an important contribution to address larger scale evolutionary questions associated with locomotion and respiration. J. Morphol. , 2012. © 2012 Wiley Periodicals, Inc.     

Genetics of shoulder girdle formation: roles of Tbx15 and aristaless-like genes

The diverse cellular contributions to the skeletal elements of the vertebrate shoulder and pelvic girdles during embryonic development complicate the study of their patterning. Research in avian embryos has recently clarified part of the embryological basis of shoulder formation. Although dermomyotomal cells provide the progenitors of the scapular blade, local signals appear to have an essential guiding role in this process. These signals differ from those that are known to pattern the more distal appendicular skeleton. We have studied the impact of Tbx15, Gli3, Alx4 and related genes on formation of the skeletal elements of the mouse shoulder and pelvic girdles. We observed severe reduction of the scapula in double and triple mutants of these genes. Analyses of a range of complex genotypes revealed aspects of their genetic relationship, as well as functions that had been previously masked due to functional redundancy. Tbx15 and Gli3 appear to have synergistic functions in formation of the scapular blade. Scapular truncation in triple mutants of Tbx15, Alx4 and Cart1 indicates essential functions for Alx4 and Cart1 in the anterior part of the scapula, as opposed to Gli3 function being linked to the posterior part. Especially in Alx4/Cart1 mutants, the expression of markers such as Pax1, Pax3 and Scleraxis is altered prior to stages when anatomical aberrations are visible in the shoulder region. This suggests a disorganization of the proximal limb bud and adjacent flank mesoderm, and is likely to reflect the disruption of a mechanism providing positional cues to guide progenitor cells to their destination in the pectoral girdle.     

Inductive signals from the somatopleure mediated by bone morphogenetic proteins are essential for the formation of the sternal component of avian ribs

The posterior five pairs of avian ribs are composed of vertebral and sternal components, both derived from the somitic mesoderm. For the patterning of the rib cartilage, inductive signals from neighboring tissues on the somitic mesoderm have been suggested to play critical roles. The notochord and surface ectoderm overlying the somitic mesoderm are essentially required for the development of proximal and distal regions of the ribs, respectively. Involvement of the somatopleure in rib development has already been suggested but is less understood than those of the notochord and surface ectoderm. In this study, we reinvestigated the role of the somatopleure during rib development. We first identified the chicken homologue of the mouse Mesenchymal forkhead-1 (cMfh-1) gene based on sequence similarities. cMfh-1 was observed to be expressed in the nonaxial mesoderm, including the somitic mesoderm, and, subsequently, in cartilage forming the ribs, vertebrae, and appendicular skeletal system. In the interlimb region, corresponding to somites 21-25 (or 26), cMfh-1-positive somitic mesoderm was seen penetrating the somatopleure of E4 embryos, and cMfh-1 was used as a molecular marker demarcating prospective rib cartilage. A series of experiments affecting the penetration of the somitic mesoderm into the somatopleure was performed in the present study, resulting in defects in sternal rib formation. The inductive signals emanating from the somatopleure mediated by BMP family proteins were observed to be essentially involved in the ingrowth of the somitic mesoderm. BMP4 alone, however, could not completely replace inductive signals from the somatopleure, suggesting the involvement of additional signals for rib formation.     

Appendicular skeleton in Bachia bicolor (Squamata: Gymnophthalmidae): osteology,limb reduction and postnatal skeletal ontogeny

The osteology of the appendicular skeleton and its postnatal development are described in Bachia bicolor, a serpentiform lizard with reduced limbs. The pectoral girdle is well developed and the forelimb consists of a humerus, ulna, radius, five carpal elements (ulnare, radiale, distal carpals 4–3, centrale), four metacarpals (II, III, IV, V) and phalanges (phalangeal formula X‐2‐2‐2‐2). In the hindlimb, the femur is small and slender, and articulates distally with a series of ossified amorphous and extremely reduced elements that correspond to a fibula, tibia and proximal and distal tarsals 4 and 3. The pelvic girdle consists of ischium, pubis and ilium, but its two halves are widely separated; the ilium is the least reduced element. We describe the ossification and development during postnatal skeletal ontogeny, especially of epiphyseal secondary centres, ossifications of carpal elements, apophyseal ossifications and sesamoids. Compared to other squamates, B. bicolor shows an overall reduction in limb size, an absence of skeletal elements, a fusion of carpal elements, an early differentiation of apophyseal centres, and a low number of sesamoids and apophyseal centres. These observations suggest that the reductions are produced by heterochronic changes during postnatal development and probably during embryonic development; therefore the appendicular skeleton exhibits a pattern of paedomorphic features.     

What is bred in the bone: Ecomorphological associations of pelvic girdle form in greater Antillean Anolis lizards

Ecological niche partitioning of Anolis lizards of the Greater Antillean islands has been the focus of many comparative studies, and much is known about external morphological convergence that characterizes anole ecomorphs. Their internal anatomy, however, has rarely been explored in an ecomorphological context, and it remains unknown to what degree skeletal morphology tracks the diversity and ecological adaptation of these lizards. Herein, we employ CT scanning techniques to visualise the skeleton of the pelvic girdle in situ, and 3D geometric morphometrics to compare the form of the ilium, ischium, and pubis within and between ecomorphs. We examine 26 species of anoles representing four ecomorphs (trunk‐ground, trunk‐crown, crown‐giant, twig) from three islands (Jamaica, Hispaniola, and Puerto Rico). The subtle variations in pelvic girdle morphology discovered are directly associable with all three parameters that we set out to focus on: phylogenetic relationship, specimen size, and assigned ecomorph category. Morphometric variation that correlates with size and/or phylogenetic signal varies between species and cannot be eliminated from the data set without markedly reducing its overall variability. The discovered patterns of skeletal variation are consistent with the demands of locomotor mechanics pertinent to the structural configuration of the microhabitat of three of the four ecomorphs, with the fourth having no discernible distinctive features. This manifests itself chiefly in the relative anteroposterior extent and anteroventral inclination of the ilium and pubis, which differ between ecomorphs and are postulated to reflect optimization of the direction of muscle vectors of the femoral protractors and retractors. Our investigation of the form of the pelvic girdle of anoles allows us to generalize our findings to entire ecomorph categories within a broad phylogenetic and biogeographic context. Differences in the form and configuration of the postcranial skeleton are directly related to ecological patterns.     

Dual origin and segmental organisation of the avian scapula

Bones of the postcranial skeleton of higher vertebrates originate from either somitic mesoderm or somatopleural layer of the lateral plate mesoderm. Controversy surrounds the origin of the scapula, a major component of the shoulder girdle, with both somitic and lateral plate origins being proposed. Abnormal scapular development has been described in the naturally occurring undulated series of mouse mutants, which has implicated Pax1 in the formation of this bone. Here we addressed the development of the scapula, firstly, by analysing the relationship between Pax1 expression and chondrogenesis and, secondly, by determining the developmental origin of the scapula using chick quail chimeric analysis. We show the following. (1) The scapula develops in a rostral-to-caudal direction and overt chondrification is preceded by an accumulation of Pax1-expressing cells. (2) The scapular head and neck are of lateral plate mesodermal origin. (3) In contrast, the scapular blade is composed of somitic cells. (4) Unlike the Pax1-positive cells of the vertebral column, which are of sclerotomal origin, the Pax1-positive cells of the scapular blade originate from the dermomyotome. (5) Finally, we show that cells of the scapular blade are organised into spatially restricted domains along its rostrocaudal axis in the same order as the somites from which they originated. Our results imply that the scapular blade is an ossifying muscular insertion rather than an original skeletal element, and that the scapular head and neck are homologous to the 'true coracoid' of higher vertebrates.     

The formation of the avian scapula blade takes place in the hypaxial domain of the somites and requires somatopleure-derived BMP signals

The avian scapula is a long bone located dorsally on the thorax. The cranial part that articulates with the upper limb is derived from the somatopleure of the forelimb field, while the caudal part, the scapula blade, originates from the dermomyotomes of brachial and thoracic somites. In previous studies, we have shown that scapula blade formation is intrinsically controlled by segment-specific information as well as extrinsically by ectoderm-derived signals. Here, we addressed the role of signals derived from the lateral plate mesoderm on scapula development. Chick-quail chimera experiments revealed that scapula precursor cells are located within the hypaxial domain of the dermomyotome adjacent to somatopleural cells. Barrier implantation between these two cell populations inhibited scapula blade formation. Furthermore, we identified BMPs as scapula-inducing signals from the somatopleure using injection of Noggin-producing cells into the hypaxial domain of scapula-forming dermomyotomes. We found that inhibition of BMP activity interfered with scapula-specific Pax1 expression and scapula blade formation. Taken together, we demonstrate that the scapula-forming cells located within the hypaxial somitic domain require BMP signals derived from the somatopleure for their specification and differentiation.     

A somitic contribution to the pectoral girdle in the axolotl revealed by long-term fate mapping

The pectoral girdle is a unique skeletal element that underwent drastic morphological changes during its evolution, especially in association with the fin-to-limb transition. Comparative studies of its development are needed to gain a deeper understanding of its evolution. Transplantation experiments using the quail-chick chimeric system have revealed that not only lateral plate mesoderm but also somites contribute to the pectoral girdle in birds. Studies in mice and turtles also document somitic contributions to the pectoral girdle, but extirpation experiments in a salamander did not affect shoulder girdle development. Somitic contributions to the pectoral girdle therefore have been interpreted as a feature unique to amniotes. Here, we present a long-term fate map of single somites in the Mexican axolotl, based on transplantations of somites two to six from GFP-transgenic donors into wild-type hosts, as well as injections of fluorescein dextran into single somites. The results show a somitic derivation of the dorsal region of the suprascapula, demonstrating that somitic contributions to the pectoral girdle are not restricted to amniotes. Comparison with the few other species studied so far leads us to suggest a position-dependent origin of the pectoral girdle. We propose that embryonic origin is determined by the proximity of the developing pectoral girdle to the somites or to the lateral plate mesoderm, respectively. This position-dependent origin and the diversity of the anatomy of the pectoral girdle among vertebrates implies that the embryonic origin of the pectoral girdle is too variable to be useful for defining homologies or for phylogenetic analysis.     

Heterochronic shifts in the ossification sequences of surface- and subsurface-dwelling skinks are correlated with the degree of limb reduction

Scincid lizards exhibit a variety of limb anatomies which reflect the functional requirements of different modes of life. Besides surface dwellers which show neither body elongation nor limb reduction, there are numerous examples that can be arranged as increasingly serpentiform taxa moving in sand, humus or leaf litter. We explored the question of whether limb reduction and body elongation in skinks are linked to heterochronic shifts in the ossification sequences. The study material comprises skinks showing four different morphotypes: Liopholis whitii, Lerista bougainvillii, Hemiergis peronii and Saiphos equalis. Results showed that (i) scincid lizards with limb reductions exhibit an earlier onset of ossification in the cervical vertebrae, and (ii) ossification starts earlier in the pectoral girdle (scapula and coracoid) and pelvic girdle (ilium, ischium and pubis) relative to the timing of the onset in elements of the forelimbs and hind limbs. Furthermore, they show (iii) an earlier strengthening of the premaxilla, which first completes the anterior part of the dorsal cranial roof, and (iv) an earlier onset of ossification in the forelimb elements than in the equivalent elements of the hind limbs. The species showing the least limb reduction (L. bougainvillii) had the greatest developmental similarity to the normally proportioned surface-dwelling species (L. whitii). S. equalis, as the morphotype with the greatest deviation from the normally proportioned, pentadactyle form, varies the most from L. whitii. The heterochronic shifts in the ossification sequences are linked to a shift in the emphasis from limbed locomotion to trunk locomotion in the species with body elongation and/or limb reduction.     

Anatomy and relationships of Pachyrhachis problematicus,a primitive snake with hindlimbs

The anatomy of Pachyrhachis problematicus, an elongate, limb-reduced squamate from the Upper Cretaceous of Israel, is described and evaluated in detail. Previously considered a snake-like ''lizard'' of uncertain affinities, it is here shown to be the most primitive snake, and the sister-group to all other snakes. Pachyrhachis exhibits numerous derived characters uniting it with modern snakes (scolecophidians and alethinophidians): e.g. mobile premaxilla-maxilla articulation, braincase enclosed by frontals and parietals, sagittal parietal crest, absence of tympanic recess, single postdentary bone, over 140 presacral vertebrae, and complete loss of shoulder girdle and forelimb. However, it is more primitive than all modern snakes in retaining some strikingly primitive (lizard-like) features: presence of a jugal, squamosal, normal sacral attachment, and well-developed hindlimb composed of femur, tibia, fibula, and tarsals. Pachyrhachis provides additional support for the hypothesis that snakes are most closely related to Cretaceous marine lizards (mosasauroids). Almost all of the derived characters proposed to unite snakes and mosasauroids are highly developed in Pachyrhachis: the mobile mandibular symphysis, intramandibular joint, long and recurved pterygoid teeth, quadrate suspended by the supratemporal, loosely united pelvic elements (ilium, ischium, and pubis), and separate astragalus and calcaneum.     

Ontogeny and phylogeny of the pelvis in Gorilla, Pongo, Pan, Australopithecus and Homo

To examine the evolutionary differences between hominoid locomotor systems, a number of observations concerning the growth of the pelvis among the great apes as compared to modern and fossil hominids are reported. We are interested in the size and shape of the coxal bones at different developmental stages across species that may elucidate the relationship between ontogeny and phylogeny (i.e., heterochrony) in the hominoid pelvis. Our hypotheses are: (1) do rates of absolute growth differ?, (2) do rates of relative growth differ?, and (3) does heterochrony explain these differences? Bivariate and multivariate analyses of pelvic dimensions demonstrate both the diversity of species-specific ontogenetic patterns among hominoids, and an unequivocal separation of hominids and the great apes. Heterochrony alone fails to account for the ontogenetic differences between hominids and the great apes. Compared to recent Homo,Australopithecus can be described as 'hyper-human' from the relative size of the ischium, and short but broad ilium. Australopithecus afarensis differs from Australopithecus africanus by its relatively long pubis. In multivariate analyses of ilium shape, the most complete coxal bone attributed to Homo erectus, KNM-ER 3228, falls within the range of juvenile and adult Australopithecus, whereas Broken Hill falls within the range of modern Homo, suggesting that the modern human ilium shape arose rather recently. Among the great apes, patterns of pelvic ontogeny do not exclusively separate the African apes from Pongo.     

Identification of distinct genes with restricted expression in the somitic mesoderm in Xenopus embryo

We have used whole-mount in situ hybridisation to identify genes expressed in the somitic mesoderm during Xenopus early development. We report here the analysis of eight genes whose expression pattern has not been described previously. They include the Xenopus homologues of eukaryotic initiation factor 2beta, methionine adenosyltransferase II, serine dehydratase, alpha-adducin, oxoglutarate dehydrogenase, fragile X mental retardation syndrome related protein 1, monocarboxylate transporter and voltage-dependent anion channel 1. Interestingly, these genes exhibit very dynamic expression pattern during early development. At early gastrula stages several genes do not show localised expression pattern, while other genes are expressed in the marginal mesoderm or in ectoderm. As development proceeds, the expression of these genes is gradually restricted to different compartments of somite. This study thus reveals an unexpected dynamic expression pattern for various genes with distinct function in vertebrates.     

An evaluation of the sexual dimorphism of the human innominate bone

The author analyses the sexual dimorphism of the innominate bone of Homo sapiens by seven measurements of angles and 15 measurements of distances. It appears that the dimorphism is very significant for the ischium, less for the ilium and is not significant for the pubis. The results are discussed and compared with data found in the literature.     

The expression of the homeobox gene Msx1 reveals two populations of dermal progenitor cells originating from the somites

Experimental manipulation in birds has shown that trunk dermis has a double origin: dorsally, it derives from the somite dermomyotome, while ventrally, it is formed by the somatopleure. Taking advantage of an nlacZ reporter gene integrated into the mouse Msx1 locus (Msx1(nlacZ) allele), we detected segmental expression of the Msx1 gene in cells of the dorsal mesenchyme of the trunk between embryonic days 11 and 14. Replacing somites from a chick host embryo by murine Msx1(nlacZ )somites allowed us to demonstrate that these Msx1-(beta)-galactosidase positive cells are of somitic origin. We propose that these cells are dermal progenitor cells that migrate from the somites and subsequently contribute to the dorsalmost dermis. By analysing Msx1(nlacZ) expression in a Splotch mutant, we observed that migration of these cells does not depend on Pax3, in contrast to other migratory populations such as limb muscle progenitor cells and neural crest cells. Msx1 expression was never detected in cells overlying the dermomyotome, although these cells are also of somitic origin. Therefore, we propose that two somite-derived populations of dermis progenitor cells can be distinguished. Cells expressing the Msx1 gene would migrate from the somite and contribute to the dermis of the dorsalmost trunk region. A second population of cells would disaggregate from the somite and contribute to the dermis overlying the dermomyotome. This population never expresses Msx1. Msx1 expression was investigated in the context of the onset of dermis formation monitored by the Dermo1 gene expression. The gene is downregulated prior to the onset of dermis differentiation, suggesting a role for Msx1 in the control of this process.     

Gombe chimpanzee sex differences in the pelvis and observations of pubic and preauricular areas

The skeletons of Gombe chimpanzees provide an opportunity for analysis of bony tissue with reference to known sex and parity in combination with observations on other life history variables and behavior. Measurements of the pelvic bones show a mosaic of sex differences. Well-defined resorption areas on the dorsomedial aspect of the pubis and the preauricular area of the ilium have been associated with sex and parity in humans and other species. However, these are not present in either females or males in this chimpanzee skeletal series.     

Origin and segregation of cranial placodes in Xenopus laevis

Cranial placodes are local thickenings of the vertebrate head ectoderm that contribute to the paired sense organs (olfactory epithelium, lens, inner ear, lateral line), cranial ganglia and the adenohypophysis. Here we use tissue grafting and dye injections to generated fate maps of the dorsal cranial part of the non-neural ectoderm for Xenopus embryos between neural plate and early tailbud stages. We show that all placodes arise from a crescent-shaped area located around the anterior neural plate, the pre-placodal ectoderm. In agreement with proposed roles of Six1 and Pax genes in the specification of a panplacodal primordium and different placodal areas, respectively, we show that Six1 is expressed uniformly throughout most of the pre-placodal ectoderm, while Pax6, Pax3, Pax8 and Pax2 each are confined to specific subregions encompassing the precursors of different subsets of placodes. However, the precursors of the vagal epibranchial and posterior lateral line placodes, which arise from the posteriormost pre-placodal ectoderm, upregulate Six1 and Pax8/Pax2 only at tailbud stages. Whereas our fate map suggests that regions of origin for different placodes overlap extensively with each other and with other ectodermal fates at neural plate stages, analysis of co-labeled placodes reveals that the actual degree of overlap is much smaller. Time lapse imaging of the pre-placodal ectoderm at single cell resolution demonstrates that no directed, large-scale cell rearrangements occur, when the pre-placodal region segregates into distinct placodes at subsequent stages. Our results indicate that individuation of placodes from the pre-placodal ectoderm does not involve large-scale cell sorting in Xenopus.     

Intrinsic,Hox-dependent cues determine the fate of skeletal muscle precursors

It is generally held that vertebrate muscle precursors depend totally on environmental cues for their development. We show that instead, somites are predisposed toward a particular myogenic program. This predisposition depends on the somite's axial identity: when flank somites are transformed into limb-level somites, either by shifting somitic boundaries with FGF8 or by overexpressing posterior Hox genes, they readily activate the program typical for migratory limb muscle precursors. The intrinsic control over myogenic programs can only be overridden by FGF4 signals provided by the apical ectodermal ridge of a developing limb.