The dinosaurian femoral head experienced a morphogenetic shift from torsion to growth along the avian stem.

Significant evolutionary shifts in locomotor behaviour often involve comparatively subtle anatomical transitions. For dinosaurian and avian evolution, medial overhang of the proximal femur has been central to discussions. However, there is an apparent conflict with regard to the evolutionary origin of the dinosaurian femoral head, with neontological and palaeontological data suggesting seemingly incongruent hypotheses. To reconcile this, we reconstructed the evolutionary history of morphogenesis of the proximal end of the femur from early archosaurs to crown birds. Embryological comparison of living archosaurs (crocodylians and birds) suggests the acquisition of the greater overhang of the femoral head in dinosaurs results from additional growth of the proximal end in the medial-ward direction. On the other hand, the fossil record suggests that this overhang was acquired by torsion of the proximal end, which projected in a more rostral direction ancestrally. We reconcile this apparent conflict by inferring that the medial overhang of the dinosaur femoral head was initially acquired by torsion, which was then superseded by mediad growth. Details of anatomical shifts in fossil forms support this hypothesis, and their biomechanical implications are congruent with the general consensus regarding broader morpho-functional evolution on the avian stem.

The developing bird pelvis passes through ancestral dinosaurian conditions.

Living birds (Aves) have bodies substantially modified from the ancestral reptilian condition. The avian pelvis in particular experienced major changes during the transition from early archosaurs to living birds1,2. This stepwise transformation is well documented by an excellent fossil record2-4; however, the ontogenetic alterations that underly it are less well understood. We used embryological imaging techniques to examine the morphogenesis of avian pelvic tissues in three dimensions, allowing direct comparison with the fossil record. Many ancestral dinosaurian features2 (for example, a forward-facing pubis, short ilium and pubic 'boot') are transiently present in the early morphogenesis of birds and arrive at their typical 'avian' form after transitioning through a prenatal developmental sequence that mirrors the phylogenetic sequence of character acquisition. We demonstrate quantitatively that avian pelvic ontogeny parallels the non-avian dinosaur-to-bird transition and provide evidence for phenotypic covariance within the pelvis that is conserved across Archosauria. The presence of ancestral states in avian embryos may stem from this conserved covariant relationship. In sum, our data provide evidence that the avian pelvis, whose early development has been little studied5-7, evolved through terminal addition-a mechanism8-10 whereby new apomorphic states are added to the end of a developmental sequence, resulting in expression8,11 of ancestral character states earlier in that sequence. The phenotypic integration we detected suggests a previously unrecognized mechanism for terminal addition and hints that retention of ancestral states in development is common during evolutionary transitions.

Embryonic muscle splitting patterns reveal homologies of amniote forelimb muscles.

Limb muscles are remarkably complex and evolutionarily labile. Although their anatomy is of great interest for studies of the evolution of form and function, their homologies among major amniote clades have remained obscure. Studies of adult musculature are inconclusive owing to the highly derived morphology of modern amniote limbs but correspondences become increasingly evident earlier in ontogeny. We followed the embryonic development of forelimb musculature in representatives of six major amniote clades and found, contrary to current consensus, that these early splitting patterns are highly conserved across Amniota. Muscle mass cleavage patterns and topology are highly conserved in reptiles including birds, irrespective of their skeletal modifications: the avian flight apparatus results from slight early topological modifications that are exaggerated during ontogeny. Therian mammals, while conservative in their cleavage patterns, depart drastically from the ancestral amniote musculoskeletal organization in terms of topology. These topological changes occur through extension, translocation and displacement of muscle groups later in development. Overall, the simplicity underlying the apparent complexity of forelimb muscle development allows us to resolve conflicting hypotheses about homology and to trace the history of each individual forelimb muscle throughout the amniote radiations.

Hidden limbs in the "limbless skink" Brachymeles lukbani: Developmental observations.

Reduced limbs and limblessness have evolved independently in many lizard clades. Scincidae exhibit a wide range of limb-reduced morphologies, but only some species have been used to study the embryology of limb reduction (e.g., digit reduction in Chalcides and limb reduction in Scelotes). The genus Brachymeles, a Southeast Asian clade of skinks, includes species with a range of limb morphologies, from pentadactyl to functionally and structurally limbless species. Adults of the small, snake-like species Brachymeles lukbani show no sign of external limbs in the adult except for small depressions where they might be expected to occur. Here, we show that embryos of B. lukbani in early stages of development, on the other hand, show a truncated but well-developed limb with a stylopod and a zeugopod, but no signs of an autopod. As development proceeds, the limb's small size persists even while the embryo elongates. These observations are made based on external morphology. We used florescent whole-mount immunofluorescence to visualize the morphology of skeletal elements and muscles within the embryonic limb of B. lukabni. Early stages have a humerus and separated ulna and radius cartilages; associated with these structures are dorsal and ventral muscle masses as those found in the embryos of other limbed species. While the limb remains small, the pectoral girdle grows in proportion to the rest of the body, with well-developed skeletal elements and their associated muscles. In later stages of development, we find the small limb is still present under the skin, but there are few indications of its presence, save for the morphology of the scale covering it. By use of CT scanning, we find that the adult morphology consists of a well-developed pectoral girdle, small humerus, extremely reduced ulna and radius, and well-developed limb musculature connected to the pectoral girdle. These muscles form in association with a developing limb during embryonic stages, a hint that "limbless" lizards that possess these muscles may have or have had at least transient developing limbs, as we find in B. lukbani. Overall, this newly observed pattern of ontogenetic reduction leads to an externally limbless adult in which a limb rudiment is hidden and covered under the trunk skin, a situation called cryptomelia. The results of this work add to our growing understanding of clade-specific patterns of limb reduction and the convergent evolution of limbless phenotypes through different developmental processes.

A developmental staging system and musculoskeletal development sequence of the common musk turtle (Sternotherus odoratus).

BACKGROUND: The extremely derived body plan of turtles has sparked a great interest in studying their developmental biology. Here, we describe the embryonic development of the Stinkpot, or common musk turtle (Sternotherus odoratus), a small aquatic turtle from the family Kinosternidae. RESULTS: We identify 20 distinct developmental stages, some comparable to stages described by previous studies on other turtles and some in between these, improving the resolution of the generalities of turtle development. We provide a detailed account of both the external morphology and skeletal development, as well as a general look at the early stages of muscular development until the attainment of the adult muscular anatomical pattern. CONCLUSIONS: Several potential skeletal and muscular apomorphies of turtles are identified or elaborated. The musk turtle, with its small size and hard-shelled egg, could become an important species for the study of turtle evolution and development, suitable for in ovo experimentation and late stage imaging of well-advanced anatomical features.

How do Crocodylian embryos process yolk? Morphological evidence from the American alligator, Alligator mississippiensis.

Recent studies have demonstrated a mechanism of embryonic yolk processing in lizards, snakes and turtles that differs markedly from that of birds. In the avian pattern, cells that line the inside of the yolk sac take up products of yolk digestion and deliver nutrients into the vitelline circulation. In contrast, in squamates and turtles, proliferating endodermal cells invade and fill the yolk sac cavity, forming elongated strands of yolk-filled cells that surround small blood vessels. This arrangement provides a means by which yolk material becomes cellularized, digested, and transported for embryonic use. Ultrastructural observations on late-stage Alligator mississippiensis eggs reveal elongated, vascular strands of endodermal cells within the yolk sac cavity. The strands of cells are intermixed with free yolk spheres and clumps of yolk-filled endodermal cells, features that reflect early phases in the yolk-processing pattern. These observations indicate that yolk processing in Alligator is more like the pattern of other reptiles than that of birds.

Dinosaur ossification centres in embryonic birds uncover developmental evolution of the skull.

Radical transformation of the skull characterizes bird evolution. An increase in the relative size of the brain and eyes was presumably related to the loss of two bones surrounding the eye, the prefrontal and postorbital. We report that ossification centres of the prefrontal and postorbital are still formed in bird embryos, which then fuse seamlessly to the developing nasal and frontal bones, respectively, becoming undetectable in the adult. The presence of a dinosaur-like ossification pattern in bird embryos is more than a trace of their evolutionary past: we show how persistent modularity of ossification centres has allowed for evolutionary re-organization of skull architecture in evolution. Our findings also demonstrate that enigmatic mesodermal cells forming the posterior region of the avian frontal correspond to the ossification centre of the postorbital, not the parietal, and link its failure to develop into an adult bone to its incorporation into the expanded braincase of birds.

Greater Growth of Proximal Metatarsals in Bird Embryos and the Evolution of Hallux Position in the Grasping Foot.

In early theropod dinosaurs-the ancestors of birds-the hallux (digit 1) had an elevated position within the foot and had lost the proximal portion of its metatarsal. It no longer articulated with the ankle, but was attached at about mid-length of metatarsal 2 (mt2). In adult birds, the hallux is articulated closer to the distal end of mt2 at ground level with the other digits. However, on chick embryonic day 7, its position is as in early theropods at half-length of mt2. The adult distal location is acquired during embryonic days 8-10. To assess how the adult phenotype is acquired, we produced fate maps of the metatarsals of day 6 chicken embryos injecting the lipophilic tracer DiI. The fates of these marks indicate a larger expansion of the metatarsals at their proximal end, which creates the illusory effect that d1 moves distally. This larger proximal expansion occurs concomitantly with growth and early differentiation of cartilage. Histological analysis of metatarsals shows that the domains of flattened and prehypertrophic chondrocytes are larger toward the proximal end. The results suggest that the distal position of the hallux in the avian foot evolved as a consequence of an embryological period of expansion of the metatarsus toward the proximal end. It also brings attention to the developmental mechanisms leading to differential growth between epiphyses and their evolutionary consequences.

Molecular development of fibular reduction in birds and its evolution from dinosaurs.

Birds have a distally reduced, splinter-like fibula that is shorter than the tibia. In embryonic development, both skeletal elements start out with similar lengths. We examined molecular markers of cartilage differentiation in chicken embryos. We found that the distal end of the fibula expresses Indian hedgehog (IHH), undergoing terminal cartilage differentiation, and almost no Parathyroid-related protein (PTHrP), which is required to develop a proliferative growth plate (epiphysis). Reduction of the distal fibula may be influenced earlier by its close contact with the nearby fibulare, which strongly expresses PTHrP. The epiphysis-like fibulare however then separates from the fibula, which fails to maintain a distal growth plate, and fibular reduction ensues. Experimental downregulation of IHH signaling at a postmorphogenetic stage led to a tibia and fibula of equal length: The fibula is longer than in controls and fused to the fibulare, whereas the tibia is shorter and bent. We propose that the presence of a distal fibular epiphysis may constrain greater growth in the tibia. Accordingly, many Mesozoic birds show a fibula that has lost its distal epiphysis, but remains almost as long as the tibia, suggesting that loss of the fibulare preceded and allowed subsequent evolution of great fibulo-tibial disparity.

Efficient Detection of Indian Hedgehog During Endochondral Ossification by Whole-Mount Immunofluorescence.

Endochondral ossification is a process essential for the formation of the vertebrate skeleton. Indian Hedgehog (IHH) is a key regulator of this process. So far, monitoring IHH expression in whole-mount developing skeletal structures has been hampered by the permeability and the opacity of the tissue. Whole-mount preparations require advanced techniques of fixation, clearing, and staining. We describe a reliable method for fixing, immunostaining, and clearing whole-mount developing cartilages that allows for the detection of IHH in the developing skeleton of avian embryos. The fixation process ensures a proper preservation of cellular structures and, especially, the antigenicity of the tissue, allowing the antibody labelling of IHH. This protocol reveals specific cell staining in localized regions of the developing cartilage, facilitating the study of IHH function during key periods of skeletogenesis.

Skeletal plasticity in response to embryonic muscular activity underlies the development and evolution of the perching digit of birds.

Most birds have an opposable digit 1 (hallux) allowing the foot to grasp, which evolved from the non-opposable hallux of early theropod dinosaurs. An important morphological difference with early theropods is the twisting of the long axis of its metatarsal. Here, we show how embryonic musculature and the onset of its activity are required for twisting of metatarsal 1 (Mt1) and retroversion of the hallux. Pharmacologically paralyzed embryos do not fully retrovert the hallux and have a straight Mt1 shaft, phenocopying the morphology of early tetanuran dinosaurs. Molecular markers of cartilage maturation and ossification show that differentiation of Mt1 is significantly delayed compared to Mt2-4. We hypothesize on how delayed maturation may have increased plasticity, facilitating muscular twisting. Our experimental results emphasize the importance of embryonic muscular activity in the evolutionary origin of a crucial adaptation.

New developmental evidence clarifies the evolution of wrist bones in the dinosaur-bird transition.

From early dinosaurs with as many as nine wrist bones, modern birds evolved to develop only four ossifications. Their identity is uncertain, with different labels used in palaeontology and developmental biology. We examined embryos of several species and studied chicken embryos in detail through a new technique allowing whole-mount immunofluorescence of the embryonic cartilaginous skeleton. Beyond previous controversy, we establish that the proximal-anterior ossification develops from a composite radiale+intermedium cartilage, consistent with fusion of radiale and intermedium observed in some theropod dinosaurs. Despite previous claims that the development of the distal-anterior ossification does not support the dinosaur-bird link, we found its embryonic precursor shows two distinct regions of both collagen type II and collagen type IX expression, resembling the composite semilunate bone of bird-like dinosaurs (distal carpal 1+distal carpal 2). The distal-posterior ossification develops from a cartilage referred to as "element x," but its position corresponds to distal carpal 3. The proximal-posterior ossification is perhaps most controversial: It is labelled as the ulnare in palaeontology, but we confirm the embryonic ulnare is lost during development. Re-examination of the fossil evidence reveals the ulnare was actually absent in bird-like dinosaurs. We confirm the proximal-posterior bone is a pisiform in terms of embryonic position and its development as a sesamoid associated to a tendon. However, the pisiform is absent in bird-like dinosaurs, which are known from several articulated specimens. The combined data provide compelling evidence of a remarkable evolutionary reversal: A large, ossified pisiform re-evolved in the lineage leading to birds, after a period in which it was either absent, nonossified, or very small, consistently escaping fossil preservation. The bird wrist provides a modern example of how developmental and paleontological data illuminate each other. Based on all available data, we introduce a new nomenclature for bird wrist ossifications.

The developmental origin of zygodactyl feet and its possible loss in the evolution of Passeriformes.

The zygodactyl orientation of toes (digits II and III pointing forwards, digits I and IV pointing backwards) evolved independently in different extant bird taxa. To understand the origin of this trait in modern birds, we investigated the development of the zygodactyl foot of the budgerigar (Psittaciformes). We compared its muscular development with that of the anisodactyl quail (Galliformes) and show that while the musculus abductor digiti IV (ABDIV) becomes strongly developed at HH36 in both species, the musculus extensor brevis digiti IV (EBDIV) degenerates and almost disappears only in the budgerigar. The asymmetric action of those muscles early in the development of the budgerigar foot causes retroversion of digit IV (dIV). Paralysed budgerigar embryos do not revert dIV and are anisodactyl. Both molecular phylogenetic analysis and palaeontological information suggest that the ancestor of passerines could have been zygodactyl. We followed the development of the zebra finch (Passeriformes) foot muscles and found that in this species, both the primordia of the ABDIV and of the EBDIV fail to develop. These data suggest that loss of asymmetric forces of muscular activity exerted on dIV, caused by the absence of the ABDIV, could have resulted in secondary anisodactyly in Passeriformes.