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The vertebrate head as a major novelty is directly linked to the evolutionary success of the vertebrates. Sequential information on the embryonic pattern of cartilaginous head development are scarce, but important for the understanding of its evolution. In this study, we use the oriental fire bellied toad, Bombina orientalis, a basal anuran to investigate the sequence and timing of larval cartilaginous development of the head skeleton from the appearance of mesenchymal Anlagen in post-neurulation stages until the premetamorphic larvae. We use different methodological approaches like classic histology, clearing and staining, and antibody staining to examine the larval skeletal morphology. Our results show that in contrast to other vertebrates, the ceratohyals are the first centers of chondrification. They are followed by the palatoquadrate and the basihyal. The latter later fuses to the ceratohyal and the branchial basket. Anterior elements like Meckel's cartilage and the rostralia are delayed in development and alter the ancestral anterior posterior pattern observed in other vertebrates. The ceratobranchials I-IV, components of the branchial basket, follow this strict anterior-posterior pattern of chondrification as reported in other amphibians. Chondrification of different skeletal elements follows a distinct pattern and the larval skeleton is nearly fully developed at Gosner Stage 28. We provide baseline data on the pattern and timing of early cartilage development in a basal anuran species, which may serve as guidance for further experimental studies in this species as well as an important basis for the understanding of the evolutionary changes in head development among amphibians and vertebrates.

Xenopus laevis is widely used as a model organism in biological research. Morphological descriptions of the larval cartilaginous skeleton are more than half a century old and comprehensive studies of early cartilage differentiation and development are missing. A proper understanding of early cranial skeletal development in X. laevis requires a detailed description that can function as a baseline for experimental studies. This basis makes it possible to evaluate skeletal defects produced by experiments on gene interactions, such as gain- or loss-of function experiments. In this study, we provide a detailed description of the pattern and timing of early cartilage differentiation and development in the larval head of X. laevis. Methods used include antibody staining, confocal laser scanning microscopy and 3D-reconstruction. Results were than compared to earlier studies based on classical histological approaches and clearing-and-staining. The first cartilage to chondrify is, in contrast to other vertebrates investigated so far, the ceratohyal. The components of the branchial basket chondrify in anterior-to-posterior direction as reported for other amphibians. Chondrification of different cartilages begins at different stages and the majority of cartilages are fully developed at Ziermann and Olsson stage 17. Our baseline data on the pattern and timing of early cartilaginous development in X. laevis is useful for evaluation of experiments which alter head skeletal development as well as for identifying heterochronic shifts in head development in other amphibians.

The head is considered the major novelty of the vertebrates and directly linked to their evolutionary success. Its form and development as well as its function, for example in feeding, is of major interest for evolutionary biologists. In this study, we describe the skeletal development of the cranium and pectoral girdle in Siberian (Acipenser baerii) and Russian sturgeon (A. gueldenstaedtii), two species that are commonly farmed in aquaculture and increasingly important in developmental studies. This study comprises the development of the neuro-, viscero- and dermatocranium and the dermal and chondral components of the pectoral girdle, from first condensation of chondrocytes in prehatchlings to the early juvenile stage and reveals a clear pattern in formation. The otic capsules, the parachordal cartilages, and the trabeculae cranii are the first centers of chondrification, at 8.4mm TL. These are followed by the mandibular, then the hyoid, and later the branchial arches. Teeth form early on the dentary, dermopalatine, and palatopterygoid, and then appear later in the buccal cavity as dorsal and ventral toothplates. With ongoing chondrification in the neurocranium a capsule around the brain and a strong rostrum are formed. Dermal ossifications start to form before closure of the dorsal neurocranial fenestrae. Perichondral ossification of cartilage bones occurs much later in ontogeny. Our results contribute data bearing on the homology of elements such as the lateral rostral canal bone that we regard homologous to the antorbital of other actinopterygians based on its sequence of formation, position and form. We further raise doubts on the homology of the posterior ceratobranchial among Actinopteri based on the formation of the hyoid arch elements. We also investigate the basibranchials and the closely associated unidentified gill-arch elements and show that they are not homologous. J. Morphol. 278:418-442, 2017. © 2017 Wiley Periodicals, Inc.

As part of an ongoing comparative study of pigment patterns and their formation in embryos and larvae of ambystomatid salamanders, Ambystoma maculatum from two differnt populations, one in the northern (New York) and one in the central (Tennessee) United States, were investigated. Scanning electron microscopy was used to study early neural crest development. Light microscopy in combination with markers for the two pigment cell types (xanthophores and melanophores) made it possible to follow pigment cell migration before the pigment cells were fully differentiated. A bilateral pigment pattern consisting of two horizontal melanophore stripes surrounding an interstripe area populated by xanthophores formed in the larvae. In both populations, some variation was present in the form of a continuum ranging from clear horizontal stripes to extreme cases with a random pattern. Unlike the other ambystomatids that have been investigated, the neural crest cells in A. maculatum do not form aggregates and no vertical bars are formed. Instead, both the pattern and its formation are very similar to what has been reported for salamandrids. If pattern formation mechanisms can act as developmental constraints we would expect the A. maculatum pattern to be the primitive condition in the Ambystomatidae, using the Salamandridae as the outgroup. There is no strong support for this when aggregate formation is used as a character and mapped onto phylogenies for the group. The aggregate formation mechanism, and the pigment pattern that it leads to, have most likely been secondarily lost in A. maculatum. © 1993 Wiley-Liss, Inc.

The pigment pattern formation in embryos and larvae of three ambystomatid salamanders was investigated in an evolutionary context. Early neural crest development was studied with scanning electron microscopy. Pigment cell migration and pattern formation were investigated at the light microscopy level with markers that labelled the two pigment cell types specifically before they were fully differentiated. In all three species, the pigment pattern formation started when xanthophores that had first formed aggregates in the crest migrated ventrally. As previously observed in other species, vertical bars always form by a mechanism involving earlier onset of migration in melanophores than in xanthophores and aggregate formation in the crest. In Ambystoma talpoideum and A. annulatum, a pattern of vertical chromatophore bars formed, which was superimposed on a pattern of horizontal stripes. In Ambystoma barbouri, the tendency to form this pattern was obscured by the high density of melanophores. It is suggested that variation among species may be due to differences in the chromatophore density and in the melanophore/xanthophore ratio. Mapping of the evolution of vertical bars onto existing phylogenies for the group was confounded by controversies about how to interpret the phylogenetic data. On the phylogeny that takes all the available evidence into account, there are two equally parsimonious mappings. Vertical bars have either evolved only once and been lost twice, or evolved twice and been lost once. This rather conservative pattern can be explained both as an effect of stabilizing selection and as a result of developmental constraints. © 1994 Wiley-Liss, Inc.

We have begun a comparative study of pigment patterns and their mechanisms of formation in ambystomatid salamanders in an attempt to elucidate the evolution of these traits in this family. In Ambystoma t. tigrinum, the migration of the prospective pigment cells was followed by using scanning electron microscopy and light microscopy combined with markers (dopa incubation for detecting melanophores, ammonia-induced pterin fluorescence for detecting xanthophores). The pigment pattern resulting from the cell migration shares features both with the alternating vertical xanthophore and melanophore bars of A. mexicanum and the horizontal stripes of certain salamandrids and ambystomatids. The pigment pattern of A. t. tigrinum is interpreted here as an intermediate evolutionary step between a primitive horizontal stripe pattern and a derived vertical bar pattern. The initiation of pigment pattern formation resembles the situation in A. mexicanum, probably reflecting the close phylogenetic relationship between the two taxa.

We assess cranial neural-crest cell migration and contributions to the larval chondrocranium in the phylogenetically basal and morphologically generalized anuran Bombina orientalis (Bombinatoridae). Methods used include microdissection, scanning electron microscopy, and vital dye labeling, in conjunction with confocal and fluorescence microscopy. Cranial neural-crest cells begin migrating before neural-fold closure and soon form three primary streams. These streams contribute to all cranial cartilages except two medial components of the hyobranchial skeleton (basihyal and basibranchial cartilages), the posterior portion of the trabecular plate, and the otic capsule, the embryonic origin of which is unknown. Chondrogenic fate is regionalized within the cranial neural folds, with the anterior regions contributing to anterior cartilages and the posterior regions to posterior cartilages. A neural-crest contribution also was consistently observed in several cranial nerves and the connective tissue component of many cranial muscles. Notwithstanding minor differences among species in the initial configuration of migratory streams, cranial neural-crest migration and chondrogenic potential in metamorphosing anurans seem to be highly stereotyped and evolutionarily conservative. This includes a primary role for the neural crest in the evolutionary origin of the paired suprarostral and infrarostral cartilages, two prominent caenogenetic features of the rostral skull unique to anuran larvae. Our results provide a model of the ancestral pattern of embryonic head development in anuran amphibians. This model can serve as a basis for examining the ontogenetic mechanisms that underlie the diversity of cranial morphology and development displayed by living frogs, as well as the evolutionary consequences of this diversity. © 1996 Wiley-Liss, Inc.

Lungfishes are the extant sister group of tetrapods. As such, they are important for the study of evolutionary processes involved in the water to land transition of vertebrates. The evolution of a true neck, that is, the complete separation of the pectoral girdle from the cranium, is one of the most intriguing morphological transitions known among vertebrates. Other salient changes involve new adaptations for terrestrial feeding, which involves both the cranium and its associated musculature. Historically, the cranium has been extensively investigated, but the development of the cranial muscles much less so. Here, we present a detailed study of cephalic muscle development in the Australian lungfish, Neoceratodus forsteri, which is considered to be the sister taxon to all other extant lungfishes. Neoceratodus shows several developmental patterns previously described in other taxa; the tendency of muscles to develop from anterior to posterior, from their region of origin toward insertion, and from lateral to ventral/medial (outside-in), at least in the branchial arches. The m.protractor pectoralis appears to develop as an extension of the most posterior m.levatores arcuum branchialium, supporting the hypothesis that the m.cucullaris and its derivatives (protractor pectoralis, levatores arcuum branchialium) are branchial muscles. We present a new hypothesis regarding the homology of the ventral branchial arch muscles (subarcualis recti and obliqui, transversi ventrales) in lungfishes and amphibians. Moreover, the morphology and development of the cephalic muscles confirms that extant lungfishes are neotenic and have been strongly influenced via paedomorphosis during their evolutionary history.

The skeleton of the jaws and neurocranium of sturgeons (Acipenseridae) are connected only through the hyoid arch. This arrangement allows considerable protrusion and retraction of the jaws and is highly specialized among ray-finned fishes (Actinopterygii). To better understand the unique morphology and the evolution of the jaw apparatus in Acipenseridae, we investigated the development of the muscles of the mandibular and hyoid arches of the Siberian sturgeon, Acipenser baerii. We used a combination of antibody staining and formalin-induced fluorescence of tissues imaged with confocal microscopy and subsequent three-dimensional reconstruction. These data were analyzed to address the identity of previously controversial and newly discovered muscle portions. Our results indicate that the anlagen of the muscles in A. baerii develop similarly to those of other actinopterygians, although they differ by not differentiating into distinct muscles. This is exemplified by the subpartitioning of the m. adductor mandibulae as well as the massive m. protractor hyomandibulae, for which we found a previously undescribed portion in each. The importance of paedomorphosis for the evolution of Acipenseriformes has been discussed before and our results indicate that the muscles of the mandibular and the hyoid may be another example for heterochronic evolution.

The African Clawed Toad, Xenopus laevis, has been a major vertebrate model organism for developmental studies for half a century. Because most studies have focused on the early stages of development, this has had the effect that many aspects of organogenesis and later development remain relatively poorly known in this species. In particular, little is known about cranial muscle development even at the level of morphology and histological differentiation of muscle anlagen and muscle fibers. In this study, we document the morphogenesis and histological differentiation of cranial muscles in X. laevis. We provide a detailed account of the timing of development for each of the cranial muscles, and also describe a new muscle, the m. transversus anterior. The cranial musculature of X. laevis larvae generally develops in a rostrocaudal sequence. The first muscles to differentiate are the extrinsic eye muscles. Muscles of the mandibular and hyoid arches develop almost simultaneously, and are followed by the muscles of the branchial arches and the larynx, and by the mm. geniohyoideus and rectus cervicis. Despite the fact that differentiation starts at different stages in the different muscles, most are fully developed at Stage 14. These baseline data on the timing of muscle differentiation in the X. laevis can serve as a foundation for comparative studies of heterochronic changes in cranial muscle development in frogs and other lissamphibians.

The development of the vertebrate head is a complex process involving interactions between a multitude of cell types and tissues. This thesis describes the development of the cranial neural crest and of the visceral arch muscles in the head of two species. One, the Mexican axolotl (Ambystoma mexicanum), is a basal tetrapod, whereas the other, the Australian lungfish (Neoceratodus forsteri), belongs to the Dipnoi, the extant sister group of the Tetrapoda. The migration of neural crest cells, which form most of the bones and connective tissues in the head, and the morphogenesis of the jaw, was determined in the Mexican axolotl. It was shown that both the upper and lower jaws form from ventral condensations of neural crest cells in the mandibular arch. The dorsal condensation, earlier considered to give rise to the upper jaw, was shown to form the trabecula cranii.The normal spatio-temporal development of visceral arch muscles was investigated in both the Mexican axolotl and the Australian lungfish. In axolotl, the muscles tended to start forming almost simultaneously in all visceral arches at their future origins and extend towards their future insertions at the onset of muscle fibre formation. In lungfish, fibres formed simultaneously throughout most of each muscle anlage in the first and second visceral arch, but were delayed in the branchial arches. The anlagen were first observed at their future insertion, from which they developed towards future origins. To test the ability of neural crest cells to pattern the visceral arch muscles, migrating crest cells were extirpated from axolotl embryos, which resulted in a wide range of muscle malformations. In most cases, the muscles appeared in the right position but were small and extended in abnormal directions. This shows that neural crest cells are responsible not for the position of the muscles but for their correct anatomical pattern. Fate mapping showed that connective tissue surrounding myofibers is, at least partly, neural crest derived.In conclusion, the work presented in this thesis shows that although early development may map out the patterns of later development, the differences between axolotl and lungfish head development are not seen until during morphogenesis. Further investigation of morphogenesis is needed to explain the great variation of head morphology seen in vertebrates today.

The segmental series of somites in the vertebrate embryo gives rise to the axial skeleton. In amniote models, single vertebrae are derived from the sclerotome of two adjacent somites. This process, known as resegmentation, is well-studied using the quail-chick chimeric system, but the presumed generality of resegmentation across vertebrates remains poorly evaluated. Resegmentation has been questioned in anamniotes, given that the sclerotome is much smaller and lacks obvious differentiation between cranial and caudal portions. Here, we provide the first experimental evidence that resegmentation does occur in a species of amphibian. Fate mapping of individual somites in the Mexican axolotl (Ambystoma mexicanum) revealed that individual vertebrae receive cells from two adjacent somites as in the chicken. These findings suggest that large size and segmentation of the sclerotome into distinct cranial and caudal portions are not requirements for resegmentation. Our results, in addition to those for zebrafish, indicate that resegmentation is a general process in building the vertebral column in vertebrates, although it may be achieved in different ways in different groups.

The segmental series of somites in the vertebrate embryo gives rise to the axial skeleton. In amniote models, single vertebrae are derived from the sclerotome of two adjacent somites. This process, known as resegmentation, is well-studied using the quail-chick chimeric system, but the presumed generality of resegmentation across vertebrates remains poorly evaluated. Resegmentation has been questioned in anamniotes, given that the sclerotome is much smaller and lacks obvious differentiation between cranial and caudal portions. Here, we provide the first experimental evidence that resegmentation does occur in a species of amphibian. Fate mapping of individual somites in the Mexican axolotl (Ambystoma mexicanum) revealed that individual vertebrae receive cells from two adjacent somites as in the chicken. These findings suggest that large size and segmentation of the sclerotome into distinct cranial and caudal portions are not requirements for resegmentation. Our results, in addition to those for zebrafish, indicate that resegmentation is a general process in building the vertebral column in vertebrates, although it may be achieved in different ways in different groups.

We assess cranial neural-crest cell migration and contributions to the larval chondrocranium in the phylogenetically basal and morphologically generalized anuran Bombina orientalis (Bombinatoridae). Methods used include microdissection, scanning electron microscopy, and vital dye labeling, in conjunction with confocal and fluorescence microscopy. Cranial neural-crest cells begin migrating before neural-fold closure and soon form three primary streams. These streams contribute to all cranial cartilages except two medial components of the hyobranchial skeleton (basihyal and basibranchial cartilages), the posterior portion of the trabecular plate, and the otic capsule, the embryonic origin of which is unknown. Chondrogenic fate is regionalized within the cranial neural folds, with the anterior regions contributing to anterior cartilages and the posterior regions to posterior cartilages. A neural-crest contribution also was consistently observed in several cranial nerves and the connective tissue component of many cranial muscles. Notwithstanding minor differences among species in the initial configuration of migratory streams, cranial neural-crest migration and chondrogenic potential in metamorphosing anurans seem to be highly stereotyped and evolutionarily conservative. This includes a primary role for the neural crest in the evolutionary origin of the paired suprarostral and infrarostral cartilages, two prominent caenogenetic features of the rostral skull unique to anuran larvae. Our results provide a model of the ancestral pattern of embryonic head development in anuran amphibians. This model can serve as a basis for examining the ontogenetic mechanisms that underlie the diversity of cranial morphology and development displayed by living frogs, as well as the evolutionary consequences of this diversity. © 1996 Wiley-Liss, Inc.

We have begun a comparative study of pigment patterns and their mechanisms of formation in ambystomatid salamanders in an attempt to elucidate the evolution of these traits in this family. In Ambystoma t. tigrinum, the migration of the prospective pigment cells was followed by using scanning electron microscopy and light microscopy combined with markers (dopa incubation for detecting melanophores, ammonia-induced pterin fluorescence for detecting xanthophores). The pigment pattern resulting from the cell migration shares features both with the alternating vertical xanthophore and melanophore bars of A. mexicanum and the horizontal stripes of certain salamandrids and ambystomatids. The pigment pattern of A. t. tigrinum is interpreted here as an intermediate evolutionary step between a primitive horizontal stripe pattern and a derived vertical bar pattern. The initiation of pigment pattern formation resembles the situation in A. mexicanum, probably reflecting the close phylogenetic relationship between the two taxa.