Hybridization and its impact on the ontogenetic allometry of skulls in macaques.

The role of hybridization in morphological diversification is a fundamental topic in evolutionary biology. However, despite the accumulated knowledge on adult hybrid variation, how hybridization affects ontogenetic allometry is less well understood. Here, we investigated the effects of hybridization on postnatal ontogenetic allometry in the skulls of a putative hybrid population of introduced Taiwanese macaques (Macaca cyclopis) and native Japanese macaques (Macaca fuscata). Genomic analyses indicated that the population consisted of individuals with varying degrees of admixture, formed by male migration from Japanese to Taiwanese macaques. For overall skull shape, ontogenetic trajectories were shifted by hybridization in a nearly additive manner, with moderate transgressive variation observed throughout development. In contrast, for the maxillary sinus (hollow space in the face), hybrids grew as fast as Taiwanese macaques, diverging from Japanese macaques, which showed slow growth. Consequently, adult hybrids showed a mosaic pattern, that is, the maxillary sinus is as large as that of Taiwanese macaques, while the overall skull shape is intermediate. Our findings suggest that the transgressive variation can be caused by prenatal shape modification and nonadditive inheritance on regional growth rates, highlighting the complex genetic and ontogenetic bases underlying hybridization-induced morphological diversification.

Heterochronic development of pelvic fins in zebrafish: possible involvement of temporal regulation of pitx1 expression.

Anterior and posterior paired appendages of vertebrates are notable examples of heterochrony in the relative timing of their development. In teleosts, posterior paired appendages (pelvic fin buds) emerge much later than their anterior paired appendages (pectoral fin buds). Pelvic fin buds of zebrafish (Danio rerio) appear at 3 weeks post-fertilization (wpf) during the larva-to-juvenile transition (metamorphosis), whereas pectoral fin buds arise from the lateral plate mesoderm on the yolk surface at the embryonic stage. Here we explored the mechanism by which presumptive pelvic fin cells maintain their fate, which is determined at the embryonic stage, until the onset of metamorphosis. Expression analysis revealed that transcripts of pitx1, one of the key factors for the development of posterior paired appendages, became briefly detectable in the posterior lateral plate mesoderm at early embryonic stages. Further analysis indicated that the pelvic fin-specific pitx1 enhancer was in the poised state at the larval stage and is activated at the juvenile stage. We discuss the implications of these findings for the heterochronic development of pelvic fin buds.

Air-breathing behavior underlies the cell death in limbs of Rana pirica tadpoles.

Amphibians shape their limbs by differential outgrowth of digits and interdigital regions. In contrast, amniotes employ cell death, an additional developmental system, to determine the final shape of limbs. Previous work has shown that high oxygen availability is correlated with the induction of cell death in developing limbs. Given the diversity of life histories of amphibians, it is conceivable that some amphibians are exposed to a high-oxygen environment during the tadpole phase and exhibit cell death in their limbs. Here, we examined whether air-breathing behavior underlies the cell death in limbs of aquatic tadpoles of the frog species Rana pirica. Our experimental approach revealed that R. pirica tadpoles exhibit cell death in their limbs that is likely to be induced by oxidative stress associated with their frequent air-breathing behavior.

Regulation of the limb shape during the development of the Chinese softshell turtles.

Interdigital cell death is an important mechanism employed by amniotes to shape their limbs; inhibiting this process leads to the formation of webbed fingers, as seen in bats and ducks. The Chinese softshell turtle Pelodiscus sinensis (Reptilia: Testudines: Trionychidae) has a distinctive limb morphology: the anterior side of the limbs has partially webbed fingers with claw-like protrusions, while the posterior fingers are completely enclosed in webbings. Here, P. sinensis embryos were investigated to gain insights on the evolution of limb-shaping mechanisms in amniotes. We found cell death and cell senescence in their interdigital webbings. Spatial or temporal modulation of these processes were correlated with the appearance of indentations in the webbings, but not a complete regression of this tissue. No differences in interdigital cell proliferation were found. In subsequent stages, differential growth of the finger cartilages led to a major difference in limb shape. While no asymmetry in bone morphogenetic protein signaling was evident during interdigital cell death stages, some components of this pathway were expressed exclusively in the clawed digit tips, which also had earlier ossification. In addition, a delay and/or truncation in the chondrogenesis of the posterior digits was found in comparison with the anterior digits of P. sinensis, and also when compared with the previously published pattern of digit skeletogenesis of turtles without posterior webbings. In conclusion, modulation of cell death, as well as a heterochrony in digit chondrogenesis, may contribute to the formation of the unique limbs of the Chinese softshell turtles.

Environmental Oxygen is a Key Modulator of Development and Evolution: From Molecules to Ecology: Oxygen-sensitive pathways pattern the developing organism, linking genetic and environmental components during the evolution of new traits.

Oxygen is a key regulator of both development and homeostasis and a promising candidate to bridge the influence of the environment and the evolution of new traits. To clarify the various ways in which oxygen may modulate embryogenesis, its effects are reviewed at distinct organizational levels. First, the role of pathways that sense dioxygen levels and reactive oxygen species are reviewed. Then, the effects of microenvironmental oxygen on metabolism, stemness, and differentiation throughout embryogenesis are discussed. Last, the interplay between ecology and development are reexamined with a focus on the evolution of tetrapods, including during the emergence of a novel mechanism that shapes amniote limbs-interdigital cell death. Both genetic and environmental components work together during the formation of organisms, highlighting the importance of a multidisciplinary approach for understanding the evolution of new traits.

Phenotypic plasticity in the mandibular morphology of Japanese macaques: captive-wild comparison.

Despite the accumulating evidence suggesting the importance of phenotypic plasticity in diversification and adaptation, little is known about plastic variation in primate skulls. The present study evaluated the plastic variation of the mandible in Japanese macaques by comparing wild and captive specimens. The results showed that captive individuals are square-jawed with relatively longer tooth rows than wild individuals. We also found that this shape change resembles the sexual dimorphism, indicating that the mandibles of captive individuals are to some extent masculinized. By contrast, the mandible morphology was not clearly explained by ecogeographical factors. These findings suggest the possibility that perturbations in the social environment in captivity and resulting changes of androgenic hormones may have influenced the development of mandible shape. As the high plasticity of social properties is well known in wild primates, social environment may cause the inter- and intra-population diversity of skull morphology, even in the wild. The captive-wild morphological difference detected in this study, however, can also be possibly formed by other untested sources of variation (e.g. inter-population genetic variation), and therefore this hypothesis should be validated further.

Environmental Oxygen Exposure Allows for the Evolution of Interdigital Cell Death in Limb Patterning.

Amphibians form fingers without webbing by differential growth between digital and interdigital regions. Amniotes, however, employ interdigital cell death (ICD), an additional mechanism that contributes to a greater variation of limb shapes. Here, we investigate the role of environmental oxygen in the evolution of ICD in tetrapods. While cell death is restricted to the limb margin in amphibians with aquatic tadpoles, Eleutherodactylus coqui, a frog with terrestrial-direct-developing eggs, has cell death in the interdigital region. Chicken requires sufficient oxygen and reactive oxygen species to induce cell death, with the oxygen tension profile itself being distinct between the limbs of chicken and Xenopus laevis frogs. Notably, increasing blood vessel density in X. laevis limbs, as well as incubating tadpoles under high oxygen levels, induces ICD. We propose that the oxygen available to terrestrial eggs was an ecological feature crucial for the evolution of ICD, made possible by conserved autopod-patterning mechanisms.

Evolution of the avian digital pattern.

Variation in digit number has occurred multiple times in the history of archosaur evolution. The five digits of dinosaur limbs were reduced to three in bird forelimbs, and were further reduced in the vestigial forelimbs of the emu. Regulation of digit number has been investigated previously by examining genes involved in anterior-posterior patterning in forelimb buds among emu (Dromaius novaehollandiae), chicken (Gallus gallus) and zebra finch (Taeniopygia guttata). It was described that the expression of posterior genes are conserved among these three birds, whereas expression of anterior genes Gli3 and Alx4 varied significantly. Here we re-examined the expression pattern of Gli3 and Alx4 in the forelimb of emu, chicken and zebra finch. We found that Gli3 is expressed in the anterior region, although its range varied among species, and that the expression pattern of Alx4 in forelimb buds is broadly conserved in a stage-specific manner. We also found that the dynamic expression pattern of the BMP antagonist Gremlin1 (Grem1) in limb buds, which is critical for autopodial expansion, was consistent with the digital pattern of emu, chicken and zebra finch. Furthermore, in emu, variation among individuals was observed in the width of Grem1 expression in forelimb buds, as well as in the adult skeletal pattern. Our results support the view that the signalling system that regulates the dynamic expression of Grem1 in the limb bud contributes substantially to variations in avian digital patterns.

Cux2 refines the forelimb field by controlling expression of Raldh2 and Hox genes.

In vertebrates, two pairs of buds that give rise to the fore- and hindlimbs form at discrete positions along the rostral-caudal axis of the body. The mechanism responsible for the positioning of the limb buds is still largely unknown. Here we show a novel function for Cut homeobox transcription factor 2 (Cux2), the ortholog of Drosophila cut, in refining the forelimb field during chick development. Cux2 is expressed in the forelimb field before the emergence of the limb buds. Knocking down the expression of Cux2 using small interfering RNA (siRNA) resulted in a caudal shift of the forelimb bud, whereas misexpression of Cux2 or the constitutively active Cux2-VP16 caused a rostral shift of the forelimb bud or reduction of the forelimb field along the anterior-posterior axis. Further functional analyses revealed that expression of Hoxb genes and retinaldehyde dehydrogenase 2 (Raldh2), which are involved in limb positioning, are directly activated by Cux2 in the lateral plate mesoderm. Our data suggest that Cux2 in the lateral plate mesoderm refines the forelimb field via regulation of Raldh2 and Hoxb genes in chicken embryos.

Involvement of HGF/MET signaling in appendicular muscle development in cartilaginous fish.

In amniotes, limb muscle precursors de-epithelialize from the ventral dermomyotome and individually migrate into limb buds. In catsharks, Scyliorhinus, fin muscle precursors are also derived from the ventral dermomyotome, but shortly after de-epithelialization, they reaggregate within the pectoral fin bud and differentiate into fin muscles. Delamination of muscle precursors has been suggested to be controlled by hepatocyte growth factor (HGF) and its tyrosine kinase receptor (MET) in amniotes. Here, we explore the possibility that HGF/MET signaling regulates the delamination of appendicular muscle precursors in embryos of the catshark Scyliorhinus canicula. Our analysis reveals that Hgf is expressed in pectoral fin buds, whereas c-Met expression in fin muscle precursors is rapidly downregulated. We propose that alteration of the duration of c-Met expression in appendicular muscle precursors might underlie the evolution of individually migrating muscle precursors, which leads to the emergence of complex appendicular muscular systems in amniotes.

Localization of ß-Catenin and Islet in the Pelvic Fin Field in Zebrafish.

In zebrafish, pelvic fin buds appear at 3 weeks post fertilization (wpf) during the larval to juvenile transition (metamorphosis), but their fate is already determined during embryogenesis. Thus, presumptive pelvic fin cells appear to memorize their positional information for three weeks, but no factors expressed in the pelvic fin field from the embryonic to the metamorphic stages have been identified. In mice, Islet1 is proposed to promote nuclear accumulation of ß-catenin in the hindlimb field, which leads to the initiation of hindlimb bud outgrowth through activation of the Wnt/ßcatenin pathway. Here, we examined the distribution of ß-catenin and islet proteins in the pelvic fin field of zebrafish from the embryonic to the metamorphic stages. We found that transcripts of islet2a, but not islet1, are detected in the posterior lateral plate mesoderm, including the presumptive pelvic fin field, at the embryonic stage as well as in the pelvic fin bud at the metamorphic stage. Immunolocalization revealed that ß-catenin and islet proteins, which are synthesized during the embryonic stage, remain in the cytoplasm of the presumptive pelvic fin cells during the larval stage, and are then translocated into the nuclei of the pelvic fin bud at the metamorphic stage. We propose that cytoplasmic localization of these proteins in the presumptive pelvic fin cells that remained during the larval stage may underlie the mechanism by which pelvic fin cells memorize their positional information from the embryonic stage to the metamorphic stage.

Modification of pectoral fins occurs during the larva-to-juvenile transition in the mudskipper (Periophthalmus modestus).

BACKGROUND: Mudskippers are amphibious fishes that use their pectoral fins to move on land. Their pectoral fins are specifically modified for terrestrial locomotion. Studies of the anatomy and kinematics of adult mudskippers suggest that modifications of the pectoral fins, such as their protrusion and elongation of the proximal radials, may provide greater control and flexibility in pectoral fin-based locomotion. However, it is unknown when and how the unique features of these pectoral fins form during the development of mudskippers, which begin life as a planktonic organism. RESULTS: Here we examined the developmental process of the pectoral fins of the mudskipper Periophthalmus modestus to address these questions. We also observed other developmental characteristics to provide clarified descriptions, including indicative morphological changes that occur during metamorphosis. CONCLUSION: Our results show that the localized cell division of the proximal part of the endoskeletal disc-the primordium of the proximal radials-and subsequent cell division along the proximal-distal axis, which is restricted to the distal part of the disc during the larva-to-juvenile transition (metamorphosis), lead to the elongation of the proximal radials.

Phylogenetic relationship of a fossil macaque (Macaca cf. robusta) from the Korean Peninsula to extant species of macaques based on zygomaxillary morphology.

Little is known about the biogeographical and evolutionary histories of macaques (Macaca spp.) in East Asia because the phylogenetic positions of fossil species remain unclear. Here we examined the zygomaxillary remains of a fossil macaque (M. cf. robusta) from the Durubong Cave Complex, South Korea, that dates back to the late Middle to Late Pleistocene, to infer its phylogenetic relationship to extant species. We took 195 fixed- and semi-landmarks from the zygomaxillary regions of the fossil specimen and from 147 specimens belonging to 14 extant species. We then conducted a generalized Procrustes analysis followed by a multivariate statistical analysis to evaluate the phenetic affinities of the fossil to the extant species and reconstructed the most parsimonious phylogenetic tree using a phylogenetic morphometric approach. We found that the fossil was most similar to Macaca fuscata (Japanese macaque) in the zygomaxillary morphospace although it was at the limit of the range of variation for this species. The second closest in the morphospace was the continental Macaca mulatta (rhesus macaque). Parsimonious reconstruction confirmed that the fossil was most closely related to M. fuscata, even after controlling for the effects of allometry. These findings suggest that in the late Middle to Late Pleistocene, close relatives of M. fuscata that looked like the extant species were distributed on the Korean Peninsula, where no species of macaques are found today. Thus, some morphological characteristics of M. fuscata may have developed before its ancestor dispersed into the Japanese archipelago.

Alterations in anterior-posterior patterning and its accompanying changes along the proximal-distal axis during the fin-to-limb transition.

The evolution from fins to limbs was one of the most successful innovations for vertebrates, allowing them to vastly expand their behaviors and habitats. Fossil records suggest that morphological changes occurred not only along the proximal-distal axis included appearance of the autopod, but also occurred along the anterior-posterior axis included reductions in the size and number of basal bones and digits. This review focuses on recent progress in developmental and genetic studies aimed at elucidating the mechanisms underlying alteration of anterior-posterior patterning and its accompanying changes along the proximal-distal axis during the fin-to-limb transition.

Migratory appendicular muscles precursor cells in the common ancestor to all vertebrates.

In amniote embryos, skeletal muscles in the trunk are derived from epithelial dermomyotomes, the ventral margin of which extends ventrally to form body wall muscles. At limb levels, ventral dermomyotomes also generate limb-muscle precursors, an Lbx1-positive cell population that originates from the dermomyotome and migrates distally into the limb bud. In elasmobranchs, however, muscles in the paired fins were believed to be formed by direct somitic extension, a developmental pattern used by the amniote body wall muscles. Here we re-examined the development of pectoral fin muscles in catsharks, Scyliorhinus, and found that chondrichthyan fin muscles are indeed formed from Lbx-positive muscle precursors. Furthermore, these precursors originate from the ventral edge of the dermomyotome, the rest of which extends towards the ventral midline to form body wall muscles. Therefore, the Lbx1-positive, de-epithelialized appendicular muscle precursors appear to have been established in the body plan before the divergence of Chondrichthyes and Osteichthyes.

Publisher Correction: Migratory appendicular muscles precursor cells in the common ancestor to all vertebrates.

In Fig. 2 of this Article originally published, some erroneous lines appeared on the left side of the images in panels c, e and g. The figure should have appeared as shown below. These errors have now been corrected in all versions of the Article.

Transgene Introduction into the Chick Limb Bud by Electroporation.

Electroporation enables delivering bionanomolecules, such as DNAs, RNAs, siRNAs, and morpholinos, into chick embryos in a spatially and temporally restricted fashion. Recent advances in electroporation techniques allowed us to deliver transgenes into the restricted area of the limb bud and to analyze the function of the enhancers in the limb field. Here, we describe the introduction of transgenes by electroporation in the limb field and its application on enhancer analysis.

Expression patterns of Sema3A in developing amniote limbs: With reference to the diversification of peripheral nerve innervation.

Paired limbs were acquired in the ancestor of tetrapods and their morphology has been highly diversified in amniotes in relation to the adaptive radiation to the terrestrial environment. These morphological changes may have been induced by modification of the developmental program of the skeletal or muscular system. To complete limb modification, it is also important to change the neuronal framework, because the functions of the limbs rely on neural circuits that involve coordinated movement. Previous studies have shown that class 3 semaphorins (Sema3 semaphorins), which act as repulsive axonal guidance cues, play a crucial role in the formation of the peripheral nerves in mice. Here, we studied the expression pattern of Sema3A orthologues in embryos of developing amniotes, including mouse, chick, soft-shelled turtle, and ocelot gecko. Sema3A transcripts were expressed in restricted mesenchymal parts of the developing limb primordium in all animals studied, and developing spinal nerves appeared to extend through Sema3A-negative regions. These results suggest that a Sema3A-dependent guidance system plays a key role in neuronal circuit formation in amniote limbs. We also found that Sema3A partially overlapped with the distribution of cartilage precursor cells. Based on these results, we propose a model in which axon guidance and skeletogenesis are linked by Sema3A; such mechanisms may underlie functional neuron rearrangement during limb diversification.

The fin-to-limb transition as the re-organization of a Turing pattern.

A Turing mechanism implemented by BMP, SOX9 and WNT has been proposed to control mouse digit patterning. However, its generality and contribution to the morphological diversity of fins and limbs has not been explored. Here we provide evidence that the skeletal patterning of the catshark Scyliorhinus canicula pectoral fin is likely driven by a deeply conserved Bmp-Sox9-Wnt Turing network. In catshark fins, the distal nodular elements arise from a periodic spot pattern of Sox9 expression, in contrast to the stripe pattern in mouse digit patterning. However, our computer model shows that the Bmp-Sox9-Wnt network with altered spatial modulation can explain the Sox9 expression in catshark fins. Finally, experimental perturbation of Bmp or Wnt signalling in catshark embryos produces skeletal alterations which match in silico predictions. Together, our results suggest that the broad morphological diversity of the distal fin and limb elements arose from the spatial re-organization of a deeply conserved Turing mechanism.