Amputation level-dependent patterning in urodele limb regeneration

Comparison of mesopodial skeletal patterns found in native and regenerated limbs of the salamander Plethodon cinereus reveals variant patterns unique to each group. Variant patterns in native limbs are based on fusions between laterally adjacent elements (i.e., in the anteroposterior axis). Variant patterns in the mesopodia of regenerated limbs usually exhibit fusions among proximodistally adjacent elements. Analysis of regenerates derived from limb amputation at different levels shows that the axis of fusion between regenerated mesopodial elements remains the same (i.e., proximodistal) independent of amputation level. However, the frequency of specific fusion combinations is unexpectedly sensitive to amputation level. Proximal (stylopodial) amputation results in mesopodial patterns with predominantly preaxial fusion combinations; distal amputation produces mesopodial patterns with predominantly postaxial fusion combinations. This finding is discussed in the context of other recent studies in which amputation level influenced limb regeneration patterning.     

Ontogeny and phytogeny of the mesopodial skeleton in mosasauroid reptiles

Current phylogenics of mosasauroid reptiles are reviewed and a new phylogeny examining aigialosaur interrelationships presented. Patterns of mesopodial ossification and overall limb morphology are described for adult mosasauroids. Ossification sequences are mapped onto a phylogeny in order to assess the distribution of ontogenetic characters. Consistent and ordered distributions are found. Based on the phylogenetic distribution of ossification patterns, an overall mesopodial ossification sequence for mosasaurs is proposed. Carpal sequence: ulnare—distal carpal four (dc4)—intermedium—dc3—radiale or dc2—de1 or pisiform and dc5. Tarsal sequence: astragalus—distal tarsal four or calcaneum. Skeletal paedomorphosis is recognized as a dominant pattern in the evolution of mosasauroid limbs. Apomorphic characters of skeletal paedomorphosis, apparent in most taxa, reach extremes in tylosaurs. Arguments for the presence of a single proximal cartilage in the tarsus of mosasaurs are made. This cartilage is presumed to include ossification centres from which both the astragalus and calcaneum will ossify.     

Patterns of chondrification and ossification in the skull of Graptemys pseudogeographica,the false map turtle (Emydidae)

Patterns of ossification and chondrification are well‐described for several species of turtles, but details of the chondrocranial anatomy are known for only a handful of species. Cleared and double‐stained embryos of Graptemys pseudogeographica were used to examine the fully formed chondrocranium and the formation, chondrification, and ossification of the cranium. The chondrocranium of G. pseudogeographica possesses an unusually large, irregularly shaped foramen epiphaniale that is joined with the fenestra olfactoria. As in other emydids, and many turtles generally, the taenia marginalis is present only as a small projection and the taenia medialis is lacking in mature stages of embryonic development. Ossification data for G. pseudogeographica are consistent with those of other Testudines in that the dentary and maxilla (dermal elements of the upper and lower jaws) ossify early, whereas the articular (an endochondral bone of the lower jaw) ossifies relatively late. Additionally, comparative ossification shows that the vomer is quite variable in its relative timing of ossification across Testudines.     

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.     

Ossification patterns in the tetrapod limb – conservation and divergence from morphogenetic events

Two different patterns of the condensation and chondrification of the limbs of tetrapods are known from extensive studies on their early skeletal development. These are on the one hand postaxial dominance in the sequential formation of skeletal elements in amniotes and anurans, and on the other, preaxial dominance in urodeles. The present study investigates the relative sequence of ossification in the fore‐ and hindlimbs of selected tetrapod taxa based on a literature survey in comparison to the patterns of early skeletal development, i.e. mesenchymal condensation and chondrification, representing essential steps in the late stages of tetrapod limb development. This reveals the degree of conservation and divergence of the ossification sequence from early morphogenetic events in the tetrapod limb skeleton. A step‐by‐step recapitulation of condensation and chondrification during the ossification of limbs can clearly be refuted. However, some of the deeper aspects of early skeletal patterning in the limbs, i.e. the general direction of development and sequence of digit formation are conserved, particularly in anamniotes. Amniotes show a weaker coupling of the ossification sequence in the limb skeleton with earlier condensation and chondrification events. The stronger correlation between the sequence of condensation/chondrification and ossification in the limbs of anamniotes may represent a plesiomorphic trait of tetrapods. The pattern of limb ossification across tetrapods also shows that some trends in the sequence of ossification of their limb skeleton are shared by major clades possibly representing phylogenetic signals. This review furthermore concerns the ossification sequence of the limbs of the Palaeozoic temnospondyl amphibian Apateon sp. For the first time this is described in detail and its patterns are compared with those observed in extant taxa. Apateon sp. shares preaxial dominance in limb development with extant salamanders and the specific order of ossification events in the fore‐ and hindlimb of this fossil dissorophoid is almost identical to that of some modern urodeles.     

Appendicular skeletal morphology in minute salamanders,genus Thorius (amphibia: Plethodontidae): Growth regulation,adult size determination,and natural variation

Patterns of growth and variation of the appendicular skeleton were examined in Thorius, a speciose genus of minute terrestrial plethodontid salamanders from southern Mexico. Observations were based primarily on ontogenetic series of each of five species that collectively span the range of adult body size in the genus; samples of adults of each of seven additional species provided supplemental estimates of the full range of variation of limb skeletal morphology. Limbs are generally reduced, i.e., pedomorphic, in both overall size and development, and they are characterized by a pattern of extreme variation in the composition of the limb skeleton, especially mesopodial elements, both within and between species. Fifteen different combinations of fused carpal or tarsal elements are variably present in the genus, producing at least 18 different overall carpal or tarsal arrangements, many of which occur in no other plethodontid genus. As many as four carpal or tarsal arrangements were observed in single population samples of each of several; five tarsal arrangements were observed in one population of T. minutissimus. Left-right asymmetry of mesopodial arrangement in a given specimen is also common. In contrast, several unique, nonpedomorphic features of the limb skeleton, including ossification of the typically cartilaginous adult mesopodial elements and ontogenetic increase in the degree of ossification of long bones, are characteristic of all species and distinguish Thorius from most related genera. They form part of a mechanism of determinate skeletal growth that restricts skeletal growth after sexual maturity. Interspecific differences in the timing of the processes of appendicular skeletal maturation relative to body size are well correlated with interspecific differences in mean adult size and size at sexual maturity, suggesting that shifts in the timing of skeletal maturation provide a mechanism of achieving adult size differentiation among species. Processes of skeletal maturation that confer determinate skeletal growth in Thorius are analogous to those typical of most amniotes – both groups exhibit ontogenetic reduction and eventual disappearance of the complex of stratified layers of proliferating and maturing cartilage in long bone epiphyses – but, unlike most amniotes, Thorius lacks secondary ossification centers. Thus, the presence of secondary ossification centers cannot be used as a criterion for establishing determinate skeletal growth in all vertebrates.     

Limb ossification patterns of the ichthyosaur Stenopterygius, and a discussion of the proximal tarsal row of ichthyosaurs and other neodiapsid reptiles

Limb ossification patterns for the Lower Jurassic (Toarcian) ichthyosaur, Stenopterygius , are described. It is found that limb ossification follows a continuous proximal to distal sequence from the propodial elements through to the terminal elements of 1st to 4th digit in the manus and the 1st to 3rd digit in the pes. The 5th manal and 4th pedal digit begin ossification later than more preaxial digits and also show evidence of proximal addition of elements near the distal mesopodial row in a manner consistent with delayed ossification of the 5th distal mesopodial in other diapsids. Ossification of manal elements in the Supernumerary 3–4 (S3-4) digit and the 5th digit appear interdependent; if one or the other is highly ossified, ossification of the other is retarded. The 1st pedal digit is considered to be lost in Stenopterygius and the 4th pedal digit is identified as the 5th digit. Delayed ossification of the mesopodium is not observed. The most preaxial proximal tarsal is identified as the centralc; the remaining proximal tarsals are the astragalus and calcaneum, and it is inferred that the astragalus and calcaneum ossified from within a single proximal cartilage.     

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.     

Studies on skeleton formation in reptiles. I. The postembryonic development of the skeleton in Cyrtodactylus pubisulcus (Reptilia: Gekkonidae)

The study of ossification during postembryonic development of the lizard Cyrtodactylus pubisulcus reveals consistent patterns in the skeleton of the body axis and of the limbs. The vertebral column shows a distinct antero-posterior gradient in ossification; the serial homology of sacral ribs and caudal transverse processes with dorsal ribs requires further scrutiny. The sequence of ossification of carpal and tarsal elements is constant, yet different from the pattern of chondrification as described in the literature. The homology of a separate 'intermedium' in the ossified lizard carpus requires further discussion. The development of the lizard astragalus is discussed in detail, as is the ossification of epiphyses in the limbs.     

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.     

Comparative ossification sequence and skeletal development of the postcranium of palaeognathous birds (Aves: Palaeognathae)

Palaeognaths constitute one of the most basal lineages of extant birds, and are also one of the most morphologically diverse avian orders. Their skeletal development is relatively unknown, in spite of their important phylogenetic position. Here, we compare the development of the postcranial skeleton in the emu (Dromaius novaehollandiae), ostrich (Struthio camelus), greater rhea (Rhea americana) and elegant crested‐tinamou (Eudromia elegans), focusing on ossification. All of these taxa are characterized by element loss in the appendicular skeleton, but there are several developmental mechanisms through which this loss occurs, including failure to chondrify, failure to ossify and fusion of cartilages prior to ossification. Further evidence is presented here to support a reduction in size of skeletal elements resulting in a delay in the timing of ossification. This study provides an important first look at the timing and sequence of postcranial ossification in palaeognathous birds, and discusses the influence of changes in the pattern of skeletal development on morphological evolution.     

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.     

Nicotinamide adenine dinucleotide levels in cells of developing chick limbs: possible control of muscle and cartilage development

The studies reported here show that NAD⁺ levels are low in chick limbs which have not yet attained the stage of cellular commitment, that these low levels persist during a time period when major chondrogenic commitment and expression occur, that beyond this stage the NAD⁺ levels in chick limbs rise dramatically and continuously, corresponding to the period of major myogenic development, and that developing cultures of stage 24 mesodermal cells seem to mimic these in vivo events in that myogenic cells are observed when NAD⁺ levels are high and chondrogenic cells are observed when NAD⁺ levels are low. These observations are consistent with the hypothesis that pyridine nucleotides may play some role in the control of muscle and cartilage development in embryonic chick limbs.     

Description of the adult skeleton and developmental osteology of the hyperossified horned frog,Ceratophrys cornuta (Anura: Leptodactylidae)

The adult skeleton and tadpole chondrocranium of the leptodcatylid frog, Ceratophrys cornuta (Ceratophryinae), are described in detail, including the ontogenetic development of the chondrocanium and the ossification sequence of the skeleton. The chondrocranium of the carnivorous larvae is unique in lacking a frontoparietal fontanelle and possessing a complete dorsal roof of cartilage. Furthermore, the chondrocranium is extremely robust, particularly those elements involved in the feeding mechanism; these include large palatoquadrate cartilages, stout Meckel's, supra- and infrarostral cartilages, and short, wide, cornua trabeculae. The chondrocranium of C. cornuta resembles that described for Ceratophrys cranwelli, but differs from the chondrocrania reported for the species of Lepidobatrachus. The large adult skull is hyperossified; most elements are fused into a single unit, and nearly all dermal elements are ornamented, casqued, and co-ossified. Calcification is present in nearly every cartilaginous element of the skeleton in larger (older) adults. Several osteological characters previously used in ceratophryine systematics, such as the otic ramus of the squamosal and the columella, are reassessed. Contrary to previous reports, the ossified, dorsal dermal shield above the vertebral column in many ceratophryine anurans is absent in C. cornuta. With few exceptions, the ossification sequence relative to metamorphosis is consistent with those that are known for other anurans. The squamosal arises from three distinct centers of ossification, including an otic element. The frontoparietal arises from two centers of ossification that fuse early in development. A robust postorbital arch is formed primarily by the otic flange of the frontoparietal, which articulates laterally with the medial border of the otic ramus of the squamosal. Changes in the timing of development, or heterochrony, are involved with the evolution of the unusual skull and skeleton of ceratophryine frogs. J Morphol 232:169–206, 1997. © 1997 Wiley-Liss, Inc.     

Comparative ossification and development of the skull in palaeognathous birds (Aves: Palaeognathae)

Ratites and tinamous are a morphologically diverse group of flightless and weakly flighted birds. As one of the most basal clades of extant birds, they are frequently used as an outgroup for studies discussing character evolution within other avian orders. Their skeletal development is not well known in spite of their important phylogenetic position, and studies have historically been plagued with small sample sizes and limited anatomical and temporal scope. Here, I describe the ossification of the skull in the emu (Dromaius novaehollandiae), ostrich (Struthio camelus), greater rhea (Rhea americana), and elegant crested‐tinamou (Eudromia elegans). Skeletal development is remarkably consistent within palaeognaths, in spite of large differences in absolute size and incubation period. Adult morphology appears to play a role in interordinal differences in the sequence and timing of ossification of certain bones. Neither the timing of cranial ossification events relative to stage nor the sequence of ossification events provides any evidence in support of a paedomorphic origin of the palaeognathous palate. This study provides an important first look at the timing and sequence of skull development in palaeognathous birds, providing data that can be compared to better‐studied avian systems in order to polarize ontogenetic characters. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 156 , 184–200.     

An integrative approach to examining a homology question: shell structures in soft‐shell turtles

Understanding the patterns of shell reduction in turtles is relevant when examining both fossils and living forms. The soft‐shelled turtles (Trionychidae) are characterized by the general reduction of the peripheral bony elements of the carapace, and some species possess structures of contested homology. By examining Remane's ‘principal criteria’, we addressed the primary homology of the prenuchal and the posterior peripheral ossicles (= PPOs) of the Asian flapshell turtles, Lissemys spp., thus evaluating their topological equivalence, their structural quality, and the presence of intermediate forms in ontogeny and phylogeny. We conducted an analysis of gross morphology, bone histology, and ontogeny of these elements in a large sample of living and fossil trionychids and their sister‐group, the carettochelyids. We conclude that the prenuchal comprises a neomorphic structure that does not fulfil any of the homology criteria examined. The assessment of the homology of PPOs is less straightforward because of the presence of partly conflicting evidence. Nevertheless, PPOs and standard peripherals share an antero‐posterior polarity of the ossification pattern, which we interpret as a significant shared underlying developmental pattern. Depending on the phylogenetic position of Lissemys in trionychid phylogeny, the hypothesis of PPOs homology with standard peripherals is a straightforward one or, alternatively, one involving homologous developmental processes at other levels of the hierarchy, resulting in similar microstructural characteristics of these bony shell features. In this respect, we consider the antero‐posterior polarity of the ossification pattern of both PPOs and standard peripherals as providing potential evidence for the homology of the genetic control regulating the expression of both these structures, and therefore we interpret these structures as homologues on the basis of a deeply homologous underlying developmental process. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99 , 462–476.