Development of limbs in urodeles and the origin of tetrapod limbs

On the basis of studies on serial sections of larval Ranodon sibiricus limbs and published data, the hypothesis of the origin of tetrapod limbs from the biserial archipterygium is proposed. The mesomeres of the central axis of the biserial fin correspond (in proximodistal direction) to the humerus, ulna, ulnare, all carpalia distalia, metacarpale 1, and phalanges of the first digit in the forelimb of caudate amphibians and to the femur, fibula, fibulare, tarsalia distalia, metatarsale 1, and phalanges of the first digit in the hind limb. The preaxial elements of the zygopodium and autopodium, which are positioned proximal to the digital arch, correspond to the preaxial rays of the biserial fin, and digits 2–5 correspond to its postaxial rays. As the fin transformed into the limb, the central axis curved preaxially, forming the digital arch and resulting in partial reduction and fusion of preaxial rays.     

Autopodial development in the sea turtles Chelonia mydas and Caretta caretta

The manus and pes were studied using whole-mount and histological preparations of ontogenetic series of Chelonia mydas and Caretta caretta. Patterns of connectivity and sequences of chondrification events are similar to those reported for other turtle species, with respect to both the primary axis and the digital arch. There is no evidence of anterior condensations in the region distal to the radius and the tibia, supporting the hypothesis that the radiale and tibiale are absent in turtles. The three middle metacarpals are the first elements to start ossification in the manus of C. mydas, while ossification has not started in the pes. In the hatchling of C. mydas, most carpals have started ossification, whereas tarsals are mostly still cartilaginous. In C. caretta, the first carpals to ossify are the ulnare and intermedium, followed by the pisiform. Among metatarsals, the fifth hooked metatarsal is the last one to start ossification. The fibulare and intermedium fuse early in chondrogenesis, later becoming the astragalocalcaneum. Ossification in the carpals of C. caretta starts while tarsals are still cartilaginous. The derived autopodial proportions in each autopodium of adults are laid out at the condensation stage, and features that were present in basal turtles are absent at all stages examined (developmental penetrance). In contrast to this, conservatism is expressed in the presence of similar patterns of connectivity during early chondrogenesis, and in the development of overall proportions of the manus versus pes. As in adult anatomy, the development of the autopodium of marine turtles is a mosaic of derived and plesiomorphic features.     

Limb development in the gekkonid lizard gonatodes albogularis: A reconsideration of homology in the lizard carpus and tarsus

Despite the attention squamate lizards have received in the study of digit and limb loss, little is known about limb morphogenesis in pentadactyl lizards. Recent developmental studies have provided a basis for understanding lizard autopodial element homology based on developmental and comparative anatomy. In addition, the composition and identity of some carpal and tarsal elements of lizard limbs, and reptiles in general, have been the theme of discussions about their homology compared to non‐squamate Lepidosauromorpha and basal Amniota. The study of additional embryonic material from different lizard families may improve our understanding of squamate limb evolution. Here, we analyze limb morphogenesis in the gekkonid lizard Gonatodes albogularis describing patterns of chondrogenesis and ossification from early stages of embryonic development to hatchlings. Our results are in general agreement with previous developmental studies, but we also show that limb development in squamates probably involves more chondrogenic elements for carpal and tarsal morphogenesis, as previously recognized on the grounds of comparative anatomy. We provide evidence for the transitory presence of distal carpale 1 and intermedium in the carpus and tibiale, intermedium, distal centralia, and distal tarsale 2 in the tarsus. Hence, we demonstrate that some elements that were believed to be lost in squamate evolution are conserved as transitory elements during limb development. However, these elements do not represent just phylogenetic burden but may be important for the morphogenesis of the lizard autopodium. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

Autopodial skeleton evolution in side-necked turtles (Pleurodira)

Carpal and tarsal anatomy was documented based on the observation of dry skeletons of adult specimens representing 25 species in 15 genera and on data taken from the literature. In addition, histological sections and cleared and double‐stained autopodia of recently hatched and juvenile specimens representing seven chelid and pelomedusoid species were studied. There is much more morphological diversity in the manus than in the pes. Variation in autopodial skeletons includes: the astragalus and calcaneum are either separated or fused; fusion of distal carpals 3–4−5 or just 4–5; number of centralia in the carpus; and presence/absence of a pisiform and of an accessory radial element. The widespread and probably basal phalangeal formula for Pleurodira is 2.3.3.3.3. Deviations are Pelomedusa subrufa, exhibiting a reduction to 2.2.2.2.2, Pelusios spp. with one phalanx less in digit I and for one species in digit V as well, and Acanthochelys pallidipectoris with an additional phalanx in the fourth finger. Six discrete characters itemizing some of the morphological variation observed were plotted on a composite pleurodire phylogeny, revealing not only homoplastic patterns but also the utility of some characters in supporting the monophyly of several clades. The pisiform is the last carpal element to ossify in Chelus fimbriatus. We hypothesize that the so‐called fifth hooked metatarsal represents the fusion of distal tarsal 5 with metatarsal V. The accessory radial element that was occasionally present in the turtles examined may represent an atavism of the otherwise lost radiale of turtles.     

Developmental basis of limb homology in Pleurodiran turtles,and the identity of the hooked element in the chelonian tarsus

Although Pleurodiran turtles represent an important component of extant turtle radiation, our knowledge of the development and homology of limb bones in turtles rests mostly upon observations made on derived members of the Cryptodiran clade. Herein, we describe limb development in three pleurodirans: Podocnemis unifilis Troschel, 1848, Podocnemis sextuberculata Cornalia, 1849 and Phrynops hilarii (Dumeril and Bibron, 1835), in an effort to contribute to filling this anatomical gap. For earlier stages of limb development, we described the Y‐shaped condensation that gave rise to the zeugopodial cartilages, and differentiation of the primary axis/digital arch that reveals the invariant pattern common to tetrapods. There are up to four central cartilaginous foci in the carpus, and the proximal tarsale is formed by the fusion of the fibulare, intermedium, and centrale 4. Digital development is similar for the five digits. Changes in toe V occur predominantly in the distal tarsale 5. Ontogenetic reduction of phalanges is observed in toe V of Podocnemis. Based on these results, we suggest that the hooked element present in the chelonian tarsus, and traditionally recognized as a modified fifth metatarsale, is actually the fifth distal tarsale. Additionally, our data on limb development of pleurodiran turtles supply more taxonomically comprehensive information to interpret limb configuration within the chelonian clade. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 155 , 845–866.     

Heterochronic differences of Hoxa-11 expression in Xenopus fore- and hind limb development: Evidence for lower limb identity of the anuran ankle bones

 The wrist (carpus) and ankle (tarsus) of most tetrapods, as well as the wrist of anurans, contains relatively small nodular skeletal elements. The anuran tarsus, however, comprises a pair of long bones, the proximal tarsals tibiale and fibulare, which resemble the lower leg bones, tibia and fibula (zeugopodium). In this paper we investigate whether the proximal tarsals of Xenopus are of zeugopodial character identity, i.e. whether they develop under the influence of the same genes that pattern the lower limb. We compare Hoxa-11 expression in the forelimb bud with that in the hind limb bud by whole-mount in situ hybridization. Hoxa-11 has been implicated in the development of the lower limb. In Xenopus we note three differences between Hoxa-11 expression in fore- and hind limb buds: (1) Hoxa-11 expression is maintained until the hind limb bud reaches a larger size (2 mm) than that of the forelimb bud (1.5 mm); (2) Hoxa-11 expression is maintained over larger spatial domains than in the forelimb; and (3) Hoxa-11 expression has a pronounced posterior polarity in the hind limb, but not in the forelimb. Hind limb expression of Hoxa-11 can be understood as a heterochronic prolonging of the expression dynamic in the forelimb. Finally we found that the proximal tarsals start to develop within the expression domain of Hoxa-11, while in the forelimb the lower arm elements reach the distal expression limit of Hoxa-11. The gene expression data presented here support the notion of a zeugopodial identity of the proximal tarsal elements in Xenopus. Received: 20 January 1998 / Accepted: 27 March 1998     

CAENOGENESIS,DEVELOPMENTAL VARIABILITY,AND EVOLUTION IN THE CARPUS AND TARSUS OF THE MARBLED NEWT TRITURUS MARMORATUS

In this paper, after a comparative analysis of the development of Triturus marmoratus, we explore the existence of caenogenetic events and their ontogenetic and phylogenetic consequences. The adult morphology of the Triturus marmoratus limb, in terms of number and spatial arrangement of skeletal elements, agrees with the general pattern of urodeles. The congruence in the typical pattern of adult morphology does not hint at the striking differences in embryonic development. These differences can be summarized as follows: 1) Presence of a “central axis” that develops in a distal-to-proximal direction. It originates in the basale commune giving rise to the centrale and the intermedium. Thus, there is no postaxial branching as found in Ambystoma mexicanum. 2) Again, unlike in Ambystoma mexicanum, we find a postaxial structure composed of the ulnare (fibulare)-distal carpal (tarsal) 4-metacarpal (metatarsal) 4 which is independent of the “digital arch.” 3) The (forelimb) digits, in particular, digits 1, 2, and 3, undergo disproportionate elongation. For example, the second digit, composed of a thin continuous, cartilaginous rod, becomes longer than the rest of the limb. Our study of the patterns of embryonic connectivity suggests the coexistence of three directions of growth and morphogenesis in the development of the Triturus marmoratus limb. 1) A proximo-distal one that gives rise to the preaxial axis, “primary axis,” and individual digits. 2) An anterio-posterior axis of development that gives rise to the “digital arch” and determines the number of digits. 3) A disto-proximal central axis that originates in the basale commune and sequentially generates the centrale and the intermedium. We speculate that heterochronic interspecific variation in the time of onset of limb bud formation is related to the degree of precocious digital elongation. Selection for long extremities in early larval stages, associated with functional demands for locomotion and balancing, may be the cause for the above listed changes in developmental pattern. Thus, the reported system is an example of how selection during ontogeny can result in the evolution of the developmental process.     

Patterns of chondrogenesis and calcification in the developing limb of the lizard,Calotes versicolor

The first skeletal condensation appears deep at the base of the limb bud near the somites, when the apical ectodermal ridge (AER) is maximally developed. Thereafter the skeletal elements generally appear in a proximodistal sequence but most of the mesopodial cartilages appear well after the metapodial ones and one of them, tarsalia-1, even after the phalangeal ones. The skeletal elements that fuse or “disappear” during the development are the cartilaginous condensation of fibulare, and the precartilaginous condensation of the distal centrale in the tarsus, and possibly the mesenchymatous condensation of the intermedium in the carpus. The calcification of all the long cartilages is perichondral and osseous while that of all the mesopodial and other cartilages, like epiphyses and sesamoids, is endochondral and nonosseous except the partly osseous astragalus and fibulare. The limbs of the mature adult have many sesamoids and metaplastic calcifications. The AER starts regressing after the appearance of the first skeletal condensation but is retained on the digital tips, though in a moderately regressed condition, almost till the time of the appearance of all the phalangeal condensations. These studies on the mesopodium differ with most studies on reptilian and avian mesopodia in favoring the view that very few skeletal condensations fuse or disappear during the development. They thus raise important issues concerning the ontogeny and phylogeny of the pentadactyl limb. While the AER has a substantial role in the limb morphogenesis, it most probably is not responsible for the information to mesoderm regarding the number, size, shape and relative position of the skeletal elements in the limb.     

The mechanism of forearm rotation in Varanus exanthematicus

In the sprawling gait of Varanus exanthematicus, the bicondylar distal humerus requires both the radius and ulna to rotate in the same direction. The joints between the radius and radiale and between the ulna and ulnare and pisiform accomodate these specific rotations. A ligament system between radius, ulna, radiale, and ulnare causes the radius and ulna to approximate one another during external rotation of the forearm. This approximation is conveyed distally resulting in a narrowing of the hand during external rotation of radius and ulna or during pronation of the free hand. The significance of these and related linkages is discussed.     

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.     

The chick oligozeugodactyly (ozd) mutant lacks sonic hedgehog function in the limb

We have analyzed a new limb mutant in the chicken that we name oligozeugodactyly (ozd). The limbs of this mutant have a longitudinal postaxial defect, lacking the posterior element in the zeugopod (ulna/fibula) and all digits except digit 1 in the leg. Classical recombination experiments show that the limb mesoderm is the defective tissue layer in ozd limb buds. Molecular analysis revealed that the ozd limbs develop in the absence of Shh expression, while all other organs express Shh and develop normally. Neither Ptc1 nor Gli1 are detectable in mutant limb buds. However, Bmp2 and dHAND are expressed in the posterior wing and leg bud mesoderm, although at lower levels than in normal embryos. Activation of Hoxd11-13 occurs normally in ozd limbs but progressively declines with time. Phase III of expression is more affected than phase II, and expression is more severely affected in the more 5' genes. Interestingly, re-expression of Hoxd13 occurs at late stages in the distal mesoderm of ozd leg buds, correlating with formation of digit 1. Fgf8 and Fgf4 expression are initiated normally in the mutant AER but their expression is progressively downregulated in the anterior AER. Recombinant Shh protein or ZPA grafts restore normal pattern to ozd limbs; however, retinoic acid fails to induce Shh in ozd limb mesoderm. We conclude that Shh function is required for limb development distal to the elbow/knee joints, similar to the Shh(-/-) mouse. Accordingly we classify the limb skeletal elements as Shh dependent or independent, with the ulna/fibula and digits other than digit 1 in the leg being Shh dependent. Finally we propose that the ozd mutation is most likely a defect in a regulatory element that controls limb-specific expression of Shh.     

The evolutionary and developmental basis of parallel reduction in mammalian zeugopod elements

Understanding the mechanisms by which parallel evolution occurs has the potential to clarify the complex relationship between evolution and development. In this study, we examine the role of development in the repeated reduction of zeugopod elements during mammalian evolution, a functionally important phenomenon enabling locomotor specialization. By completing a morphometric study (incorporating both analyses of variation and phylogenetics) of mammalian limbs, we are able to demonstrate an evolutionary trend toward width reduction in posterior zeugopod elements of the forelimbs and hindlimbs, the ulna and fibula, respectively. We also examine the developmental basis of limb reduction in three test cases, the bat Carollia perspicillata ulna and fibula and the mouse Mus musculus fibula. The most common pattern of reduction, that of reduced element width, was achieved via the same developmental process in both bat and mouse limbs (i.e., by a slower growth rate relative to other skeletal elements), suggesting that the parallel reduction of the posterior zeugopod element within mammals could have occurred primarily by the repeated evolution of the same developmental mechanism. However, our findings also suggest that the developmental mechanisms behind the parallel evolution of other, more taxon-specific characteristics of limb reduction (i.e., element fusion) are not conserved.     

Skeletal development in the Chinese soft‐shelled turtle Pelodiscus sinensis (Testudines: Trionychidae)

We investigated the development of the whole skeleton of the soft‐shelled turtle Pelodiscus sinensis, with particular emphasis on the pattern and sequence of ossification. Ossification starts at late Tokita‐Kuratani stage (TK) 18 with the maxilla, followed by the dentary and prefrontal. The quadrate is the first endoskeletal ossification and appears at TK stage 22. All adult skull elements have started ossification by TK stage 25. Plastral bones are the first postcranial bones to ossify, whereas the nuchal is the first carapacial bone to ossify, appearing as two unstained anlagen. Extensive examination of ossification sequences among autopodial elements reveals much intraspecific variation. Patterns of ossification of cranial dermal elements are more variable than those of endochondral elements, and dermal elements ossify before endochondral ones. Differences in ossification sequences with Apalone spinifera include: in Pelodiscus sinensis the jugal develops relatively early and before the frontal, whereas it appears later in A. spinifera; the frontal appears shortly before the parietal in A. spinifera whereas in P. sinensis the parietal appears several stages before the frontal. Chelydrids exhibit an early development of the postorbital bone and the palatal elements as compared to trionychids. Integration of the onset of ossification data into an analysis of the sequence of skeletal ossification in cryptodirans using the event‐pairing and Parsimov methods reveals heterochronies, some of which reflect the hypothesized phylogeny considered taxa. A functional interpretation of heterochronies is speculative. In the chondrocranium there is no contact between the nasal capsules and planum supraseptale via the sphenethmoid commissurae. The pattern of chondrification of forelimb and hind limb elements is consistent with a primary axis and digital arch. There is no evidence of anterior condensations distal to the radius and tibia. A pattern of quasi‐ simultaneity is seen in the chondrogenesis of the forelimb and the hind limb. J. Morphol. 2009. © 2009 Wiley‐Liss, Inc.     

The asymmetry of the carpal joint and the evolution of wing folding in maniraptoran theropod dinosaurs

In extant birds, the hand is permanently abducted towards the ulna, and the wrist joint can bend extensively in this direction to fold the wing when not in use. Anatomically, this asymmetric mobility of the wrist results from the wedge-like shape of one carpal bone, the radiale, and from the well-developed convexity of the trochlea at the proximal end of the carpometacarpus. Among the theropod precursors of birds, a strongly convex trochlea is characteristic of Coelurosauria, a clade including the highly derived Maniraptora in addition to tyrannosaurs and compsognathids. The shape of the radiale can be quantified using a ‘radiale angle’ between the proximal and distal articular surfaces. Measurement of the radiale angle and reconstruction of ancestral states using squared-change parsimony shows that the angle was small (15°) in primitive coelurosaurs but considerably larger (25°) in primitive maniraptorans, indicating that the radiale was more wedge-shaped and the carpal joint more asymmetric. The radiale angle progressively increased still further within Maniraptora, with concurrent elongation of the forelimb feathers and the forelimb itself. Carpal asymmetry would have permitted avian-like folding of the forelimb in order to protect the plumage, an early advantage of the flexible, asymmetric wrist inherited by birds.     

Anatomical features associated with preaxial duplication (pd): a recessive mutation in the rat

Preaxial polydactyly of the fore- and hindlimbs was found in Wistar-derived rats in 1978. Genetic analysis indicated that the polydactyly was due to the effects of an autosomal recessive gene (gene symbol; pd). Polydactylous homozygous rats had two or three pollices (six or seven digits) in the forelimbs and one to three preaxial extra digits (six to eight digits) in the hindlimbs. Skeletal examination revealed the presence of the extra carpal, metacarpal, and phalangeal bones that seemed to be complete or incomplete duplication of the navicular, greater multangular, first metacarpal, and phalanges of digit I in the forelimbs. In the hindlimbs, extra tarsal, metatarsal, and phalangeal bones were also observed preaxially. These extra elements seemed to be mirror-image duplications of the talus, navicular, second cuneiform, third cuneiform, cuboid, and metatarsals and phalanges of digits II-V with the absence of the first cuneiform, tibiale, first metatarsal, and phalanges of digit I. In addition, morphological changes were observed in the humerus, radius, and ulna in the forelimbs and femur, tibia, and fibula in the hindlimbs. Especially in the radius and tibia, thickening and bifurcation were found, indicating incomplete duplication of these bones. Based on these findings, the limb anomaly was classified as preaxial carpometacarpal/tarsometatarsal-type polydactyly with incomplete duplication of the radius and tibia. The mutant rats had other associated anomalies such as accessory spleens and cryptorchism. The males are sterile, whereas the females breed normally.     

The tarsus of rhynchocephalian reptiles

A new reconstruction of the tarsus of Stenaulorhynchus stockleyi differs from that previously offered by von Huene (1938) and Schaeffer (1941), and leads to a revaluation of the tarsi of other rhynchosaurs and rhynchocephalians. The tarsi of the rhynchosaurs Stenaulorhynchus, Scaphonyx, Cephalonia, Rhynchosaurus, Hyperodapedon, Howesia and Mesosuchus appear to comprise a proximal row of three bones (tibiale, intermedium, and fibulare) and a distal row of three bones associated with metatarsals–4. The metatarsals increase in length but decrease in thickness from the first to fourth, and the fifth is "hooked". The first metatarsal of Stenaulorhynchus, Scaphonyx , and Rhynchosaurus is unusually short. Among the remaining rhynchocephalians, the tarsi of claraziids and pleurosaurids are simplified in accord with aquatic habits involving the use of the limbs as paddles, and those of sphenodontids and Sapheosaurus are alike in the following respects. An astragalus (intermedium+tibiale+ centrale) and calcaneum (fibulare), or a single conjoined bone, occupy the proximal row of the tarsus and the distal row comprises four (first to fourth), three (second to fourth), or but two (third and fourth) bones; the fourth is the largest. The metatarsals increase in length from first to fourth but do not decrease in thickness. In both rhynchosaurian and spheno-dontid types tarsal movement is largely mesotarsal, a condition derived from their eosuchian ancestors and not independently developed as Schaeffer (1941) has thought. The specialization of the sphenodontid tarsus parallels that seen in lizards which have the same eosuchian ancestry.     

The effect of breed-type and castration on bone growth and distribution in cattle.

Data from carcasses of 210 cattle (119 bulls and 91 steers) from 4 breed types, serially slaughtered from approximately 200-800 kg kg liveweight were used to test the hypothesis of similar gender dimorphism among breeds in relation to carcass bone growth and distribution. Relative to total bone weight, breed types tended to have similar growth rates for all bones other than the cervical vertebrae, ribs, tibia and fibula, and tarsus. Adjusted to the same total bone weight there were significant differences among breed types in bone weight distribution, but the differences were very small and probably of little economic importance. Castration stimulated growth of the lumbar vertebrae, hindlimb bones, patella and hindquarter bones but inhibited growth of the ribs, scapula, carpus, forelimb bone, and forequarter bone. At the same total bone weight, steers as compared to bulls showed a shift in bone weight distribution towards the hindquarter, pistol and long bones. There were small but significant breed x gender interactions in the distribution of some bones.