Normal table of embryonic development in the Anji salamander Hynobius amjiensis (Hynobiidae).

We established a normal embryonic development table for the Anji salamander Hynobius amjiensis, a critically endangered tailed amphibian of the family Hynobiidae with a very limited distribution in East China, following the standards set by the early developmental table of vertebrates. Put together 32 embryonic stages for the Anji salamander was defined. The total embryonic period from oviposition to hatching is approximately 30 days at 9 °C. Stages 1-16 represent early development from cleavage to neurulation. Stages 17-32 represent organogenesis documenting later developmental events such as tail, gill, and limb formation, and hatching (Stage 32). We provided a detailed description of the external morphology and color changes of the head, trunk, limbs, tail, external gills, and balancers at various stages from egg-laying to hatching. We also described several cases of abnormal embryonic development. The establishment of the embryonic development table in H. amjiensis contributes to better understanding of the ontogeny in tailed amphibians, distinguishing closely related species, and identifying abnormal embryonic amphibians.

Survival of Polyploid hybrid salamander embryos.

BACKGROUND: Animals with polyploid, hybrid nuclei offer a challenge for models of gene expression and regulation during embryogenesis. To understand how such organisms proceed through development, we examined the timing and prevalence of mortality among embryos of unisexual salamanders in the genus Ambystoma. RESULTS: Our regional field surveys suggested that heightened rates of embryo mortality among unisexual salamanders begin in the earliest stages of embryogenesis. Although we expected elevated mortality after zygotic genome activation in the blastula stage, this is not what we found among embryos which we reared in the laboratory. Once embryos entered the first cleavage stage, we found no difference in mortality rates between unisexual salamanders and their bisexual hosts. Our results are consistent with previous studies showing high rates of unisexual mortality, but counter to reports that heightened embryo mortality continues throughout embryo development. CONCLUSIONS: Possible causes of embryonic mortality in early embryogenesis suggested by our results include abnormal maternal loading of RNA during meiosis and barriers to insemination. The surprising survival rates of embryos post-cleavage invites further study of how genes are regulated during development in such polyploid hybrid organisms.

The publications of embryologist Lev V. Beloussov.

A list of papers and books of the late Lev V. Beloussov was compiled and is available in Word and EndNote Supplements. The breadth of his work is briefly described.

Early limb patterning in the direct-developing salamander Plethodon cinereus revealed by sox9 and col2a1.

Direct-developing amphibians form limbs during early embryonic stages, as opposed to the later, often postembryonic limb formation of metamorphosing species. Limb patterning is dramatically altered in direct-developing frogs, but little attention has been given to direct-developing salamanders. We use expression patterns of two genes, sox9 and col2a1, to assess skeletal patterning during embryonic limb development in the direct-developing salamander Plethodon cinereus. Limb patterning in P. cinereus partially resembles that described in other urodele species, with early formation of digit II and a generally anterior-to-posterior formation of preaxial digits. Unlike other salamanders described to date, differentiation of preaxial zeugopodial cartilages (radius/tibia) is not accelerated in relation to the postaxial cartilages, and there is no early differentiation of autopodial elements in relation to more proximal cartilages. Instead, digit II forms in continuity with the ulnar/fibular arch. This amniote-like connectivity to the first digit that forms may be a consequence of the embryonic formation of limbs in this direct-developing species. Additionally, and contrary to recent models of amphibian digit identity, there is no evidence of vestigial digits. This is the first account of gene expression in a plethodontid salamander and only the second published account of embryonic limb patterning in a direct-developing salamander species.

Posterior tail development in the salamander Eurycea cirrigera: exploring cellular dynamics across life stages.

During embryogenesis, the body axis elongates and specializes. In vertebrate groups such as salamanders and lizards, elongation of the posterior body axis (tail) continues throughout life. This phenomenon of post-embryonic tail elongation via addition of vertebrae has remained largely unexplored, and little is known about the underlying developmental mechanisms that promote vertebral addition. Our research investigated tail elongation across life stages in a non-model salamander species, Eurycea cirrigera (Plethodontidae). Post-embryonic addition of segments suggests that the tail tip retains some aspects of embryonic cell/tissue organization and gene expression throughout the life cycle. We describe cell and tissue differentiation and segmentation of the posterior tail using serial histology and expression of the axial tissue markers, MF-20 and Pax6. Embryonic expression patterns of HoxA13 and C13 are shown with in situ hybridization. Tissue sections reveal that the posterior spinal cord forms via cavitation and precedes development of the underlying cartilaginous rod after embryogenesis. Post-embryonic tail elongation occurs in the absence of somites and mesenchymal cells lateral to the midline express MF-20. Pax6 expression was observed only in the spinal cord and some mesenchymal cells of adult Eurycea tails. Distinct temporal and spatial patterns of posterior Hox13 gene expression were observed throughout embryogenesis. Overall, important insights to cell organization, differentiation, and posterior Hox gene expression may be gained from this work. We suggest that further work on gene expression in the elongating adult tail could shed light on mechanisms that link continual axial elongation with regeneration.

Tongue and taste organ development in the ontogeny of direct-developing salamander Plethodon cinereus (Lissamphibia: Plethodontidae).

The latest research on direct developing caecilian and anuran species indicate presence of only one generation of taste organs during their ontogeny. This is distinct from indirect developing batrachians studied thus far, which possess taste buds in larvae and anatomically distinct taste discs in metamorphs. This study is a description of the tongue and taste organ morphology and development in direct developing salamander Plethodon cinereus (Plethodontidae) using histology and electron microscopy techniques. The results reveal two distinct stages tongue morphology (primary and secondary), similar to metamorphic urodeles, although only one stage of taste organ morphology. Taste disc sensory zones emerge on the surface of the oropharyngeal epithelium by the end of embryonic development, which coincides with maturation of the soft tongue. Taste organs occur in the epithelium of the tongue pad (where they are situated on the dermal papillae), the palate and the inner surface of the mandible and the maxilla. Plethodon cinereus embryos only possess taste disc type taste organs. Similar to the direct developing anuran Eleutherodactylus coqui (Eleutherodactylidae), these salamanders do not recapitulate larval taste bud morphology as an embryo. The lack of taste bud formation is probably a broadly distributed feature characteristic to direct developing batrachians. J. Morphol. 277:906-915, 2016. © 2016 Wiley Periodicals, Inc.

Maternal investment mediates offspring life history variation with context-dependent fitness consequences.

Maternal effects, such as per capita maternal investment, often interact with environmental conditions to strongly affect traits expressed early in ontogeny. However, their impact on adult life history traits and fitness components is relatively unknown. Theory predicts that lower per capita maternal investment will have strong fitness costs when the offspring develop in unfavorable conditions, yet few studies have experimentally manipulated per capita maternal investment and followed offspring through adulthood. We used a surgical embryonic yolk removal technique to investigate how per capita maternal investment interacted with an important ecological factor, larval density, to mediate offspring life history traits through reproductive maturity in an amphibian, Ambystoma talpoideum. We predicted that increased larval density would reinforce the life history variation induced by differences in per capita investment (i.e., Controls vs. Reduced Yolk), with Reduced larvae ultimately expressing traits associated with lower fitness than Controls when raised at high densities. We found that Reduced individuals were initially smaller and more developed, caught up in size to Controls within the first month of the larval stage, but were smaller at the end of the larval stage in low densities. Reduced individuals also were more likely to undergo metamorphosis at high densities and mature 'females invested in more eggs for their body sizes than Controls. Together, our results do not support our hypothesis, but instead indicate that Reduced individuals express traits associated with higher fitness when they develop in high-density environments, but lower fitness in low-density environments. The observed life history and fitness patterns are consistent with the "maternal match" hypothesis, which predicts that when the maternal environment (e.g., high density) results in phenotypic variation that is transmitted to the offspring (e.g., reduced per capita yolk investment), and offspring face that same environment (e.g., high larval density), the fitness of both mother and offspring is maximized.

Deep-time evolution of regeneration and preaxial polarity in tetrapod limb development.

Among extant tetrapods, salamanders are unique in showing a reversed preaxial polarity in patterning of the skeletal elements of the limbs, and in displaying the highest capacity for regeneration, including full limb and tail regeneration. These features are particularly striking as tetrapod limb development has otherwise been shown to be a highly conserved process. It remains elusive whether the capacity to regenerate limbs in salamanders is mechanistically and evolutionarily linked to the aberrant pattern of limb development; both are features classically regarded as unique to urodeles. New molecular data suggest that salamander-specific orphan genes play a central role in limb regeneration and may also be involved in the preaxial patterning during limb development. Here we show that preaxial polarity in limb development was present in various groups of temnospondyl amphibians of the Carboniferous and Permian periods, including the dissorophoids Apateon and Micromelerpeton, as well as the stereospondylomorph Sclerocephalus. Limb regeneration has also been reported in Micromelerpeton, demonstrating that both features were already present together in antecedents of modern salamanders 290 million years ago. Furthermore, data from lepospondyl 'microsaurs' on the amniote stem indicate that these taxa may have shown some capacity for limb regeneration and were capable of tail regeneration, including re-patterning of the caudal vertebral column that is otherwise only seen in salamander tail regeneration. The data from fossils suggest that salamander-like regeneration is an ancient feature of tetrapods that was subsequently lost at least once in the lineage leading to amniotes. Salamanders are the only modern tetrapods that retained regenerative capacities as well as preaxial polarity in limb development.

Generation of aneurogenic larvae by parabiosis of salamander embryos.

Limb regeneration of salamanders is nerve dependent, and the removal of the nerves in early stages of limb regeneration severely curtails the proliferation of the blastemal cells and growth of the regenerate. The removal of the neural tube from a developing salamander embryo results in an aneurogenic larva and the aneurogenic limb (ANL) develops independently without innervation. Paradoxically, the limb in an ANL is capable of regeneration in a nerve-independent manner. Here, we describe a detailed method for the generation of ANL in the spotted salamander, Ambystoma maculatum, for regeneration studies.

Normal table of embryonic development in the four-toed salamander, Hemidactylium scutatum.

We present a complete staging table of normal development for the lungless salamander, Hemidactylium scutatum (Caudata: Plethodontidae). Terrestrial egg clutches from naturally ovipositing females were collected and maintained at 15 °C in the laboratory. Observations, photographs, and time-lapse movies of embryos were taken throughout the 45-day embryonic period. The complete normal table of development for H. scutatum is divided into 28 stages and extends previous analyses of H. scutatum embryonic development (Bishop, 1920; Humphrey, 1928). Early embryonic stage classifications through neurulation reflect criteria described for Xenopus laevis, Ambystoma maculatum and other salamanders. Later embryonic stage assignments are based on unique features of H. scutatum embryos. Additionally, we provide morphological analysis of gastrulation and neurulation, as well as details on external aspects of eye, gill, limb, pigmentation, and tail development to support future research related to phylogeny, comparative embryology, and molecular mechanisms of development.

Assessment of modularity in the urodele skull: an exploratory analysis using ossification sequence data.

The potential presence of developmental modules is studied in the urodele skull using several classical statistical methods that have not previously been used in this context. Principal component analysis (PCA) of ossification sequence data on 21 bones in 21 extant urodele species suggests the presence of up to four developmental modules, but examination of statistically significant correlations using phylogenetic independent contrasts (PIC) and correcting for multiple tests using the false discovery rate suggests the presence of only two modules of uneven size and of two bones that may not be part of these modules. Thus, PCA does not appear to be a reliable method to investigate modularity; direct investigation of statistically significant correlations using PIC or other phylogeny-informed methods is recommended. A binomial test of the distribution of significant correlations between characters shows significant heterogeneity, which suggests that modularity is indeed present in the data. A cluster analysis gives inconsistent results that apparently do not reflect developmental modules. The data include a phylogenetic signal, as shown by a permutation-based test with squared change parsimony, but this is detectable only when the whole matrix is analyzed, and a plot of the tree onto developmental space through Evolutionary PCA shows that homoplasy is pervasive. Evolutionary rates between characters vary about 90-fold. Canonical variates analyses suggest that obligatorily neotenic urodeles may be discriminated from other urodeles on the basis of cranial ossification sequence data.

Analyzing developmental sequences with Parsimov--a case study of cranial muscle development in anuran larvae.

Parsimov is a parsimony-based method for identifying the minimum number of heterochronic event-shifts on all branches of a given phylogenetic framework to explain the developmental sequences seen in the species investigated, and has been used to investigate the evolution of developmental sequences in various animal groups. However, the biological interpretation of the results is difficult not least because Parsimov uses non-independent data resulting from event-pairing as the basis for its analyses. To test the applicability of Parsimov to a large data set, larval cranial muscle development was studied in 15 anurans, three caudates and the Australian lungfish. We analyzed the developmental sequences with Parsimov to investigate: if there are (1) heterochronies on deep branches of a cladogram indicating changes in the ancestral sequences, (2) heterochronies that can be related to larval life-history, and (3) the sensitivity of the analysis to different underlying cladograms. We discovered general patterns of cranial muscle development, such as an anterior-to-posterior gradient, an outside-in pattern and a tendency for cranial muscles to develop from their region of origin toward their insertion. We found most heterochronies on terminal branches and only a few shifts on deep branches in the cladograms indicating changes in the ancestral sequences. No changes could be related to larval life-history. The underlying cladogram clearly influenced the outcome of the analysis. We propose that Parsimov has the potential, combined with other methods, to find evolutionary important changes and to aid the biological interpretation of these changes.

Embryonic yolk removal affects a suite of larval salamander life history traits.

Egg size is a key life history trait affecting fitness, and it varies abundantly. The value of egg size to a mother and her offspring is often determined by a trade-off between investing more yolk in a few large eggs or less yolk into many more, smaller eggs. Smaller eggs are generally expected to be phenotypically inferior or females could increase their fitness by making more smaller eggs. However, many females produce a mix of egg sizes and natural yolk variation induces normal developmental responses which may persist into subsequent stages of a complex life history. Since sources of phenotypic variation are easily confounded, I surgically removed yolk from embryonic spotted salamanders (Ambystoma maculatum) using a sham surgery as a control and a split-clutch design to isolate the effects of yolk reserve variation from genetic sources of variation. Yolk removal induced early hatching, reduced developmental stage and hatchling body size. Small hatchlings stayed relatively small through the early larval period, but 17 weeks later the correlation with early larval body size was lost. When the experiment ended, larger individuals were further along in metamorphic development but mortality was independent of early larval body size. Variation in spotted salamander yolk reserves affects a suite of hatchling life history traits that persists into the larval period. Outside the laboratory, egg size effects may cascade throughout complex amphibian life histories. Applied experimentally and comparatively, this simple yolk removal technique may help identify how traits increase or decrease their response to maternal yolk investment.

The importance of cartilage to amphibian development and evolution.

The duality of amphibians is epitomized by their pharyngeal arch skeletons, the larval and adult morphologies of which enable very different feeding and breathing behaviors in aquatic and terrestrial life. To accomplish this duality, amphibian pharyngeal arch skeletons undergo two periods of patterning: embryogenesis and metamorphosis, and two periods of growth: larval and postmetamorphic. Their extreme ontogenetic variation, however, is coupled with relatively limited phylogenetic variation. I propose that amphibians face an evolutionary tradeoff between their ontogenetic and phylogenetic diversification that stems from the need to grow and transform the pharyngeal arch skeleton in cartilage rather than bone. Cartilage differs fundamentally from bone in its histology, function, development and growth. Cartilage is also the first skeletal tissue to form embryonically and provides more cellular pathways for shape change than bone. This article combines morphological, histological and experimental perspectives to explore how pharyngeal arch cartilage shape is controlled in amphibian embryogenesis, growth and metamorphosis, and how amphibian skeletal ontogenies are impacted by using cartilage to evolve a complex life cycle and in evolving away from a complex life cycle.

Gastrulation and pre-gastrulation morphogenesis, inductions, and gene expression: similarities and dissimilarities between urodelean and anuran embryos.

Studies of meso-endoderm and neural induction and subsequent body plan formation have been analyzed using mainly amphibians as the experimental model. Xenopus is currently the predominant model, because it best enables molecular analysis of these induction processes. However, much of the embryological information on these inductions (e.g., those of the Spemann-Mangold organizer), and on the morphogenetic movements of inductively interacting tissues, derives from research on non-model amphibians, especially urodeles. Although the final body pattern is strongly conserved in vertebrates, and although many of the same developmental genes are expressed, it has become evident that there are individually diverse modes of morphogenesis and timing of developmental events. Whether or not this diversity represents essential differences in the early induction processes remains unclear. The aim of this review is to compare the gastrulation process, induction processes, and gene expressions between a urodele, mainly Cynops pyrrhogaster, and an anura, Xenopus laevis, thereby to clarify conserved and diversified aspects. Cynops gastrulation differs significantly from that of Xenopus in that specification of the regions of the Xenopus dorsal marginal zone (DMZ) are specified before the onset of gastrulation, as marked by blastopore formation, whereas the equivalent state of specification does not occur in Cynops until the middle of gastrulation. Detailed comparison of the germ layer structure and morphogenetic movements during the pre-gastrula and gastrula stages shows that the entire gastrulation process should be divided into two phases of notochord induction and neural induction. Cynops undergoes these processes sequentially after the onset of gastrulation, whereas Xenopus undergoes notochord induction during a series of pre-gastrulation movements, and its traditionally defined period of gastrulation only includes the neural induction phase. Comparing the structure, fate, function and state of commitment of each domain of the DMZ of Xenopus and Cynops has revealed that the true form of the Spemann-Mangold organizer is suprablastoporal gsc-expressing endoderm that has notochord-inducing activity. Gsc-expressing deep endoderm and/or superficial endoderm in Xenopus is involved in inducing notochord during pre-gastrulation morphogenesis, rather than both gsc- and bra-expressing tissues being induced at the same time.

Do larval traits re-evolve? Evidence from the embryogenesis of a direct-developing salamander, Plethodon cinereus.

Recent molecular phylogenies suggest the surprising reacquisition of posthatching metamorphosis within an otherwise direct-developing clade of lungless salamanders (family Plethodontidae). Metamorphosis was long regarded as plesiomorphic for plethodontids, yet the genus Desmognathus, which primarily includes metamorphosing species, is now nested within a much larger clade of direct-developing species. The extent to which the putative reacquisition of metamorphosis in Desmognathus represents a true evolutionary reversal is contingent upon the extent to which both larva-specific features and metamorphosis were actually lost during the evolution of direct development. In this study we analyze development of the hyobranchial skeleton, which is dramatically remodeled during salamander metamorphosis, in the direct-developing red-backed salamander, Plethodon cinereus. We find dramatic remodeling of the hyobranchial skeleton during embryogenesis in P. cinereus and the transient appearance of larva-specific cartilages. Hyobranchial development in this direct-developing plethodontid is highly similar to that in metamorphosing plethodontids (e.g., Desmognathus). The proposed reacquisition of hyobranchial metamorphosis within Desmognathus does not represent the "re-evolution" of a lost phenotype, but instead the elaboration of an existing developmental sequence.

Embryonic staging table for a direct-developing salamander, Plethodon cinereus (Plethodontidae).

This work presents a refined staging table for the direct-developing red-backed salamander Plethodon cinereus, which is based on the incomplete staging system of James Norman Dent (J Morphol 1942; 71:577-601). This common species from eastern North America is a member of the species-rich lungless salamander family Plethodontidae. The staging table presented here covers several stages omitted by Dent and reveals novel developmental features of P. cinereus embryos. These include putative Leydig cells and open gill clefts, which are found in larvae of metamorphosing species but were previously reported as absent in direct-developing Plethodon. Other features found in larvae of metamorphosing salamander species, such as the palatopterygoid bone and lateral line neuromasts, were not observed in this material. The occurrence of larval and metamorphic features in these embryos has direct bearing on the patterns of life history evolution within the plethodontidae family. This study emphasizes the degree to which typically larval structures are retained in this direct-developing species and provides a staging table for further investigations into the development and evolution of plethodontid salamanders.

[On the diversity of the primary steps of embryonic development in the caudate amphibians].

Literary data on the peculiarities of the egg cleavage process in various representatives of the order of caudate amphibians consisting of 10 families have been considered. It has been recognized that in considerable number of species of Plethodontidae, Cryptobranchidae, and some other families, the synchrony of divisions is lost already after the 8-celled stage of the cleavage in large, yolk-rich and unpigmented eggs. A"standard" cleavage of early embryos of caudate amphibians, which had been described in the text-books on developmental biology and consists approximately of 10 synchronous divisions of comparatively small eggs, is characteristic only of the families Ambystomatidae and Salamandridae including 19.3% of species within the order Caudata. However, within each of these families there seems to be a number of species with a "nonstandard" type of early cleavage. The evolutionary relationships between two main types of early embryogenesis within the order Caudata are discussed.

The aneurogenic limb identifies developmental cell interactions underlying vertebrate limb regeneration.

The removal of the neural tube in salamander embryos allows the development of nerve-free aneurogenic limbs. Limb regeneration is normally nerve-dependent, but the aneurogenic limb regenerates without nerves and becomes nerve-dependent after innervation. The molecular basis for these tissue interactions is unclear. Anterior Gradient (AG) protein, previously shown to rescue regeneration of denervated limbs and to act as a growth factor for cultured limb blastemal cells, is expressed throughout the larval limb epidermis and is down-regulated by innervation. In an aneurogenic limb, the level of AG protein remains high in the epidermis throughout development and regeneration, but decreases after innervation following transplantation to a normal host. Aneurogenic epidermis also shows a fivefold difference in secretory gland cells, which express AG protein. The persistently high expression of AG in the epithelial cells of an aneurogenic limb ensures that regeneration is independent of the nerve. These findings provide an explanation for this classical problem, and identify regulation of the epidermal niche by innervation as a distinctive developmental mechanism that initiates the nerve dependence of limb regeneration. The absence of this regulation during anuran limb development might suggest that it evolved in relation to limb regeneration.