The first report on circulating microRNAs at Pre- and Post-metamorphic stages of axolotls.

MicroRNAs (miRNAs) are endogenously coded small RNAs, implicated in post-transcriptional gene regulation by targeting messenger RNAs (mRNAs). Circulating miRNAs are cell-free molecules, found in body fluids, such as blood and saliva, and emerged recently as potential diagnostic biomarkers. Functions of circulating miRNAs and their roles in target tissues have been extensively investigated in mammals, and the reports on circulating miRNAs in non-mammalian clades are largely missing. Salamanders display remarkable regenerative potential, and the Mexican axolotl (Ambystoma mexicanum), a critically endangered aquatic salamander, has emerged as a powerful model organism in regeneration and developmental studies. This study aimed to explore the circulating miRNA signature in axolotl blood plasma. Small RNA sequencing on plasma samples revealed 16 differentially expressed (DE) circulating miRNAs between neotenic and metamorphic stages out of identified 164 conserved miRNAs. Bioinformatics predictions provided functional annotation of detected miRNAs for both stages and enrichment of DE miRNAs in cancer-related and developmental pathways was notable. Comparison with previous reports on axolotl miRNAs unraveled common and unique members of the axolotl circulating miRNome. Overall, this work provides novel insights into non-mammalian aspects of circulating miRNA biology and expands the multi-omics toolkit for this versatile model organism.

Generation of Chimeric Axolotls with Mutant Haploid Limbs Through Embryonic Grafting.

A growing set of genetic techniques and resources enable researchers to probe the molecular origins of the ability of some species of salamanders, such as axolotls, to regenerate entire limbs as adults. Here, we outline techniques used to generate chimeric axolotls with Cas9-mutagenized haploid forelimbs that can be used for exploring gene function and the fidelity of limb regeneration. We combine several embryological and genetic techniques, including haploid generation via in vitro activation, CRISPR/Cas9 mutagenesis, and tissue grafting into one protocol to produce a unique system for haploid genetic screening in a model organism of regeneration. This strategy reduces the number of animals, space, and time required for the functional analysis of genes in limb regeneration. This also permits the investigation of regeneration-specific functions of genes that may be required for other essential processes, such as organogenesis, tissue morphogenesis, and other essential embryonic processes. The method described here is a unique platform for conducting haploid genetic screening in a vertebrate model system.

Multiplex CRISPR/Cas screen in regenerating haploid limbs of chimeric Axolotls.

Axolotls and other salamanders can regenerate entire limbs after amputation as adults, and much recent effort has sought to identify the molecular programs controlling this process. While targeted mutagenesis approaches like CRISPR/Cas9 now permit gene-level investigation of these mechanisms, genetic screening in the axolotl requires an extensive commitment of time and space. Previously, we quantified CRISPR/Cas9-generated mutations in the limbs of mosaic mutant axolotls before and after regeneration and found that the regenerated limb is a highfidelity replicate of the original limb (Flowers et al. 2017). Here, we circumvent aforementioned genetic screening limitations and present methods for a multiplex CRISPR/Cas9 haploid screen in chimeric axolotls (MuCHaChA), which is a novel platform for haploid genetic screening in animals to identify genes essential for limb regeneration.

Stability and plasticity of positional memory during limb regeneration in Ambystoma mexicanum.

BACKGROUND: Urodele amphibians are capable of regenerating their organs after severe damage. During such regeneration, participating cells are given differentiation instructions by the surrounding cells. Limb regeneration has been investigated as a representative phenomenon of organ regeneration. Cells known as blastema cells are induced after limb amputation. In this process, dermal fibroblasts are dedifferentiated and become undifferentiated similar to limb bud cells. Just like limb bud cells, the induced blastema cells are positioned along the three limb developmental axes: the dorsoventral, the anteroposterior, and the proximodistal. The accurate developmental axes are essential for reforming the structures correctly. Despite the importance of the developmental axes, the relationship between the newly establishing developmental axes and existing limb axes was not well described with molecular markers. RESULTS: In this study, we grafted skin from GFP-transgenic axolotls and traced the cell lineage with position-specific gene expressions in order to investigate the correlation of the newly established axes and cellular origin. Shh- and Lmx1b-expressing cells emerged from the posterior skin and dorsal skin, respectively, even though the skin was transplanted to an inconsistent position. Shox2, a posterior marker gene, could be activated in cells derived from distal skin. CONCLUSIONS: Our results suggest that the location memories on anteroposterior and dorsoventral axes are relatively stable in a regenerating blastema though cellular differentiation is reprogrammed.

Fgf-signaling is compartmentalized within the mesenchyme and controls proliferation during salamander limb development.

Although decades of studies have produced a generalized model for tetrapod limb development, urodeles deviate from anurans and amniotes in at least two key respects: their limbs exhibit preaxial skeletal differentiation and do not develop an apical ectodermal ridge (AER). Here, we investigated how Sonic hedgehog (Shh) and Fibroblast growth factor (Fgf) signaling regulate limb development in the axolotl. We found that Shh-expressing cells contributed to the most posterior digit, and that inhibiting Shh-signaling inhibited Fgf8 expression, anteroposterior patterning, and distal cell proliferation. In addition to lack of a morphological AER, we found that salamander limbs also lack a molecular AER. We found that amniote and anuran AER-specific Fgfs and their cognate receptors were expressed entirely in the mesenchyme. Broad inhibition of Fgf-signaling demonstrated that this pathway regulates cell proliferation across all three limb axes, in contrast to anurans and amniotes where Fgf-signaling regulates cell survival and proximodistal patterning.

Salamanders are a group of amphibians that are well-known for their ability to regenerate lost limbs and other body parts. At the turn of the twentieth century, researchers used salamander embryos as models to understand the basic concepts of how limbs develop in other four-limbed animals, including amphibians, mammals and birds, which are collectively known as "tetrapods". However, the salamander's amazing powers of regeneration made it difficult to carry out certain experiments, so researchers switched to using the embryos of other tetrapods ­ namely chickens and mice ­ instead. Studies in chickens, later confirmed in mice and frogs, established that there are two major signaling centers that control how the limbs of tetrapod embryos form and grow: a small group of cells known as the "zone of polarizing activity" within a structure called the "limb bud mesenchyme"; and an overlying, thin ridge of cells called the "apical ectodermal ridge". Both of these centers release potent signaling molecules that act on cells in the limbs. The cells in the zone of polarizing activity produce a molecule often called Sonic hedgehog, or Shh for short. The apical ectodermal ridge produces another group of signals commonly known as fibroblast growth factors, or simply Fgfs. Several older studies reported that salamander embryos do not have an apical ectodermal ridge suggesting that these amphibian's limbs may form differently to other tetrapods. Yet, contemporary models in developmental biology treated salamander limbs like those of chicks and mice. To address this apparent discrepancy, Purushothaman et al. studied how the forelimbs develop in a salamander known as the axolotl. The experiments showed that, along with lacking an apical ectodermal ridge, axolotls did not produce fibroblast growth factors normally found in this tissue. Instead, these factors were only found in the limb bud mesenchyme. Purushothaman et al. also found that fibroblast growth factors played a different role in axolotls than previously reported in chick, frog and mouse embryos. On the other hand, the pattern and function of Shh activity in the axolotl limb bud was similar to that previously observed in chicks and mice. These findings show that not all limbs develop in the same way and open up questions for evolutionary biologists regarding the evolution of limbs. Future studies that examine limb development in other animals that regenerate tissues, such as other amphibians and lungfish, will help answer these questions.

Inhibiting fibroblast aggregation in skin wounds unlocks developmental pathway to regeneration.

Salamanders are capable of full-thickness skin regeneration where removal of epidermis, dermis and hypodermis results in scar-free repair. What remains unclear is whether regeneration of these tissues recapitulates the cellular events of skin development or occurs through a process unique to regenerative healing. Unfortunately, information on the post-embryonic development of salamander skin is severely lacking, having focused on compartments or cell types, but never on the skin as a complete organ. By examining coordinated development of the epidermis and dermis in axolotls we establish six distinct stages of skin development (I-VI): I-V for normally paedomorphic adults and a sixth stage following metamorphosis. Raising animals either in isolation (zero density pressure) or in groups (density pressure) we find that skin development progresses as a function of animal size and that density directly effects developmental rate. Using keratins, p63, and proliferative markers, we show that when the dermis transforms into the stratum spongiosum and stratum compactum, keratinocytes differentiate into at least three distinct phenotypes that reveal a cryptic stratification program uncoupled from metamorphosis. Lastly, comparing skin regeneration to skin development, we find that dermal regeneration occurs through a unique process, relying heavily on remodeling of the wound extracellular matrix, rather than proceeding through direct development of a dermal lamella produced by the epidermis. By preventing fibroblast influx into the wound bed using beryllium nitrate, we show that in the absence of fibroblast generated ECM production skin regeneration occurs through an alternate route that recapitulates development.

Lineage tracing of sclerotome cells in amphibian reveals that multipotent somitic cells originate from lateral somitic frontier.

The two somite compartments, dorso-lateral dermomyotome and medio-ventral sclerotome are major vertebrate novelties, but little is known about their evolutionary origin. We determined that sclerotome cells in Xenopus come from lateral somitic frontier (LSF) by lineage tracing, ablation experiments and histological analysis. We identified Twist1 as marker of migrating sclerotome progenitors in two amphibians, Xenopus and axolotl. From these results, three conclusions can be drawn. First, LSF is made up of multipotent somitic cells (MSCs) since LSF gives rise to sclerotome but also to dermomytome as already shown in Xenopus. Second, the basic scheme of somite compartmentalization is conserved from cephalochordates to anamniotes since in both cases, lateral cells envelop dorsally and ventrally the ancestral myotome, suggesting that lateral MSCs should already exist in cephalochordates. Third, the transition from anamniote to amniote vertebrates is characterized by extension of the MSCs domain to the entire somite at the expense of ancestral myotome since amniote somite is a naive tissue that subdivides into sclerotome and dermomyotome. Like neural crest pluripotent cells, MSCs are at the origin of major vertebrate novelties, namely hypaxial region of the somite, dermomyotome and sclerotome compartments. Hence, change in MSCs properties and location is involved in somite evolution.

Amputation-induced reactive oxygen species signaling is required for axolotl tail regeneration.

BACKGROUND: Among vertebrates, salamanders are unparalleled in their ability to regenerate appendages throughput life. However, little is known about early signals that initiate regeneration in salamanders. RESULTS: Ambystoma mexicanum embryos were administered tail amputations to investigate the timing of reactive oxygen species (ROS) production and the requirement of ROS for regeneration. ROS detected by dihydroethidium increased within minutes of axolotl tail amputation and levels remained high for 24 hr. Pharmacological inhibition of ROS producing enzymes with diphenyleneiodonium chloride (DPI) and VAS2870 reduced ROS levels. Furthermore, DPI treatment reduced cellular proliferation and inhibited tail outgrowth. CONCLUSIONS: The results show that ROS levels increase in response to injury and are required for tail regeneration. These findings suggest that ROS provide instructive, if not initiating cues, for salamander tail regeneration. Developmental Dynamics 248:189-196, 2019. © 2018 Wiley Periodicals, Inc.

Experimentally induced metamorphosis in highly regenerative axolotl (ambystoma mexicanum) under constant diet restructures microbiota.

Axolotl (Ambystoma mexicanum) is a critically endangered salamander species and a model organism for regenerative and developmental biology. Despite life-long neoteny in nature and in captive-bred colonies, metamorphosis of these animals can be experimentally induced by administering Thyroid hormones (THs). However, microbiological consequences of this experimental procedure, such as host microbiota response, remain largely unknown. Here, we systematically compared host bacterial microbiota associated with skin, stomach, gut tissues and fecal samples, between neotenic and metamorphic axolotls based on 16S rRNA gene sequences. Our results show that distinct bacterial communities inhabit individual organs of axolotl and undergo substantial restructuring through metamorphosis. Skin microbiota among others, shifted sharply, as highlighted by a major transition from Firmicutes-enriched to Proteobacteria-enriched relative abundance and precipitously decreased diversity. Fecal microbiota of neotenic and metamorphic axolotl shared relatively higher similarity, suggesting that diet continues to shape microbiota despite fundamental transformations in the host digestive organs. We also reproduced the previous finding on reduction in regenerative capacity in limbs of axolotl following metamorphosis, highlighting the need to investigate whether shifts in microbiota is causally linked to regenerative capacity of axolotl. The initial results on axolotl microbiota provide novel insights into microbiological aspects of axolotl metamorphosis and will establish a baseline for future in-depth studies.

Embryonic yolk removal affects neither morphology nor escape performance of larval axolotls.

Maternal effects, the influences of maternal phenotype on the phenotypes of her offspring, mediate early ontogenetic traits through maternal investment. In amphibians, provisioning eggs with yolk is the main source of maternal investment. While larger eggs generally result in larger, higher-quality offspring, the relationship between egg size and offspring phenotype is complicated because offspring can evolve to be more or less responsive to variation in yolk provisions. Previous studies of several ambystomatid salamanders suggest that the effects of embryonic yolk reserve reduction on hatchling life history traits increase with egg size. In this study, a similar controlled experimental yolk removal technique in Ambystoma mexicanum was used to determine the effects of reduced yolk reserves on phenotypes including hatching time and stage, hatchling and larval size and performance in predation trials with fish. Surprisingly, yolk reduction revealed no effects on any traits. These findings suggest that larval morphology in A. mexicanum is highly canalized and larval phenotypes are decoupled from yolk reserve variation. This surprising lack of yolk removal effects in hatchling and larval axolotls illustrates the evolutionary flexibility of early life history traits. Traits can evolve to increase or decrease their response to resources and can even become completely unresponsive. Since we found no effects in early life history, we hypothesize that domestication of the axolotl may have altered yolk properties or allocation dynamics and that maternal investment in yolk reserves may manifest at later life stages by reducing the time to reproductive maturity or increasing fecundity.

Repeated removal of developing limb buds permanently reduces appendage size in the highly-regenerative axolotl.

Matching appendage size to body size is fundamental to animal function. Generating an appropriately-sized appendage is a robust process executed during development which is also critical for regeneration. When challenged, larger animals are programmed to regenerate larger limbs than smaller animals within a single species. Understanding this process has important implications for regenerative medicine. To approach this complex question, models with altered appendage size:body size ratios are required. We hypothesized that repeatedly challenging axolotls to regrow limb buds would affect their developmental program resulting in altered target morphology. We discovered that after 10 months following this experimental procedure, limbs that developed were permanently miniaturized. This altered target morphology was preserved upon amputation and regeneration. Future experiments using this platform should provide critical information about how target limb size is encoded within limb progenitors.

The posterior neural plate in axolotl gives rise to neural tube or turns anteriorly to form somites of the tail and posterior trunk.

Classical grafting experiments in the Mexican axolotl had shown that the posterior neural plate of the neurula is no specified neuroectoderm but gives rise to somites of the tail and posterior trunk. The bipotentiality of this region with neuromesodermal progenitor cell populations was revealed more recently also in zebrafish, chick, and mouse. We reinvestigated the potency of the posterior plate in axolotl using grafts from transgenic embryos, immunohistochemistry, and in situ hybridization. The posterior plate is brachyury-positive except for its more anterior parts which express sox2. Between anterior and posterior regions of the posterior plate a small domain with sox2+ and bra+ cells exists. Lineage analysis of grafted GFP-labeled posterior plate tissue revealed that posterior GFP+ cells move from dorsal to ventral, form the posterior wall, turn anterior bilaterally, and join the gastrulated paraxial presomitic mesoderm. More anterior sox2+/GFP+ cells, however, are integrated into the developing spinal cord. Tail notochord is formed from axial mesoderm involuted already during gastrulation. Thus the posterior neural plate is a postgastrula source of paraxial mesoderm, which performs an anterior turn, a novel morphogenetic movement. More anterior plate cells, in contrast, do not turn anteriorly but become specified to form tail spinal cord.

Mechanics of neurulation: From classical to current perspectives on the physical mechanics that shape, fold, and form the neural tube.

Neural tube defects arise from mechanical failures in the process of neurulation. At the most fundamental level, formation of the neural tube relies on coordinated, complex tissue movements that mechanically transform the flat neural epithelium into a lumenized epithelial tube (Davidson, 2012). The nature of this mechanical transformation has mystified embryologists, geneticists, and clinicians for more than 100 years. Early embryologists pondered the physical mechanisms that guide this transformation. Detailed observations of cell and tissue movements as well as experimental embryological manipulations allowed researchers to generate and test elementary hypotheses of the intrinsic and extrinsic forces acting on the neural tissue. Current research has turned toward understanding the molecular mechanisms underlying neurulation. Genetic and molecular perturbation have identified a multitude of subcellular components that correlate with cell behaviors and tissue movements during neural tube formation. In this review, we focus on methods and conceptual frameworks that have been applied to the study of amphibian neurulation that can be used to determine how molecular and physical mechanisms are integrated and responsible for neurulation. We will describe how qualitative descriptions and quantitative measurements of strain, force generation, and tissue material properties as well as simulations can be used to understand how embryos use morphogenetic programs to drive neurulation. Birth Defects Research 109:153-168, 2017. © 2016 Wiley Periodicals, Inc.

Conserved and novel functions of programmed cellular senescence during vertebrate development.

Cellular senescence, a form of stable cell cycle arrest that is traditionally associated with tumour suppression, has been recently found to occur during mammalian development. Here, we show that cell senescence is an intrinsic part of the developmental programme in amphibians. Programmed senescence occurs in specific structures during defined time windows during amphibian development. It contributes to the physiological degeneration of the amphibian pronephros and to the development of the cement gland and oral cavity. In both contexts, senescence depends on TGFß but is independent of ERK/MAPK activation. Furthermore, elimination of senescent cells through temporary TGFß inhibition leads to developmental defects. Our findings uncover conserved and new roles of senescence in vertebrate organogenesis and support the view that cellular senescence may have arisen in evolution as a developmental mechanism.

Analysis of embryonic development in the unsequenced axolotl: Waves of transcriptomic upheaval and stability.

The axolotl (Ambystoma mexicanum) has long been the subject of biological research, primarily owing to its outstanding regenerative capabilities. However, the gene expression programs governing its embryonic development are particularly underexplored, especially when compared to other amphibian model species. Therefore, we performed whole transcriptome polyA+ RNA sequencing experiments on 17 stages of embryonic development. As the axolotl genome is unsequenced and its gene annotation is incomplete, we built de novo transcriptome assemblies for each stage and garnered functional annotation by comparing expressed contigs with known genes in other organisms. In evaluating the number of differentially expressed genes over time, we identify three waves of substantial transcriptome upheaval each followed by a period of relative transcriptome stability. The first wave of upheaval is between the one and two cell stage. We show that the number of differentially expressed genes per unit time is higher between the one and two cell stage than it is across the mid-blastula transition (MBT), the period of zygotic genome activation. We use total RNA sequencing to demonstrate that the vast majority of genes with increasing polyA+ signal between the one and two cell stage result from polyadenylation rather than de novo transcription. The first stable phase begins after the two cell stage and continues until the mid-blastula transition, corresponding with the pre-MBT phase of transcriptional quiescence in amphibian development. Following this is a peak of differential gene expression corresponding with the activation of the zygotic genome and a phase of transcriptomic stability from stages 9-11. We observe a third wave of transcriptomic change between stages 11 and 14, followed by a final stable period. The last two stable phases have not been documented in amphibians previously and correspond to times of major morphogenic change in the axolotl embryo: gastrulation and neurulation. These results yield new insights into global gene expression during early stages of amphibian embryogenesis and will help to further develop the axolotl as a model species for developmental and regenerative biology.

Multicellular Mathematical Modelling of Mesendoderm Formation in Amphibians.

The earliest cell fate decisions in a developing embryo are those associated with establishing the germ layers. The specification of the mesoderm and endoderm is of particular interest as the mesoderm is induced from the endoderm, potentially from an underlying bipotential group of cells, the mesendoderm. Mesendoderm formation has been well studied in an amphibian model frog, Xenopus laevis, and its formation is driven by a gene regulatory network (GRN) induced by maternal factors deposited in the egg. We have recently demonstrated that the axolotl, a urodele amphibian, utilises a different topology in its GRN to specify the mesendoderm. In this paper, we develop spatially structured mathematical models of the GRNs governing mesendoderm formation in a line of cells. We explore several versions of the model of mesendoderm formation in both Xenopus and the axolotl, incorporating the key differences between these two systems. Model simulations are able to reproduce known experimental data, such as Nodal expression domains in Xenopus, and also make predictions about how the positional information derived from maternal factors may be interpreted to drive cell fate decisions. We find that whilst cell-cell signalling plays a minor role in Xenopus, it is crucial for correct patterning domains in axolotl.

Cellular dynamics underlying regeneration of appropriate segment number during axolotl tail regeneration.

BACKGROUND: Salamanders regenerate their tails after amputation anywhere along their length. How the system faithfully reconstitutes the original number of segments and length is not yet known. METHODS: To gain quantitative insight into how the system regenerates the appropriate length, we amputated tails at 4 or 16 myotomes post-cloaca and measured blastema size, cell cycle kinetics via cumulative Bromodeoxyuridine (BrdU) incorporation and the method of Nowakowski, and myotome differentiation rate. RESULTS: In early stages until day 15, blastema cells were all proliferative and divided at the same rate at both amputation levels. A larger blastema was formed in 4th versus 16th myotome amputations indicating a larger founding population. Myotome differentiation started at the same timepoint in the 4th and 16 th level blastemas. The rate of myotome formation was more rapid in 4th myotome blastemas so that by day 21 the residual blastema from the two amputation levels achieved equivalent size. At that time point, only a fraction of blastema cells remain in cycle, with the 4th myotome blastema harboring double the number of cycling cells as the 16th myotome blastema allowing it to grow faster and further reconstitute the larger number of missing myotomes. CONCLUSIONS: These data suggest that there are two separable phases of blastema growth. The first is level-independent, with cells displaying unrestrained proliferation. In the second phase, the level-specific growth is revealed, where differing fractions of cells remain in the cell cycle over time.

Positional plasticity in regenerating Amybstoma mexicanum limbs is associated with cell proliferation and pathways of cellular differentiation.

BACKGROUND: The endogenous ability to dedifferentiate, re-pattern, and re-differentiate adult cells to repair or replace damaged or missing structures is exclusive to only a few tetrapod species. The Mexican axolotl is one example of these species, having the capacity to regenerate multiple adult structures including their limbs by generating a group of progenitor cells, known as the blastema, which acquire pattern and differentiate into the missing tissues. The formation of a limb regenerate is dependent on cells in the connective tissues that retain memory of their original position in the limb, and use this information to generate the pattern of the missing structure. Observations from recent and historic studies suggest that blastema cells vary in their potential to pattern distal structures during the regeneration process; some cells are plastic and can be reprogrammed to obtain new positional information while others are stable. Our previous studies showed that positional information has temporal and spatial components of variation; early bud (EB) and apical late bud (LB) blastema cells are plastic while basal-LB cells are stable. To identify the potential cellular and molecular basis of this variation, we compared these three cell populations using histological and transcriptional approaches. RESULTS: Histologically, the basal-LB sample showed greater tissue organization than the EB and apical-LB samples. We also observed that cell proliferation was more abundant in EB and apical-LB tissue when compared to basal-LB and mature stump tissue. Lastly, we found that genes associated with cellular differentiation were expressed more highly in the basal-LB samples. CONCLUSIONS: Our results characterize histological and transcriptional differences between EB and apical-LB tissue compared to basal-LB tissue. Combined with our results from a previous study, we hypothesize that the stability of positional information is associated with tissue organization, cell proliferation, and pathways of cellular differentiation.

Dual embryonic origin and patterning of the pharyngeal skeleton in the axolotl (Ambystoma mexicanum).

The impressive morphological diversification of vertebrates was achieved in part by innovation and modification of the pharyngeal skeleton. Extensive fate mapping in amniote models has revealed a primarily cranial neural crest derivation of the pharyngeal skeleton. Although comparable fate maps of amphibians produced over several decades have failed to document a neural crest derivation of ventromedial elements in these vertebrates, a recent report provides evidence of a mesodermal origin of one of these elements, basibranchial 2, in the axolotl. We used a transgenic labeling protocol and grafts of labeled cells between GFP+ and white embryos to derive a fate map that describes contributions of both cranial neural crest and mesoderm to the axolotl pharyngeal skeleton, and we conducted additional experiments that probe the mechanisms that underlie mesodermal patterning. Our fate map confirms a dual embryonic origin of the pharyngeal skeleton in urodeles, including derivation of basibranchial 2 from mesoderm closely associated with the second heart field. Additionally, heterotopic transplantation experiments reveal lineage restriction of mesodermal cells that contribute to pharyngeal cartilage. The mesoderm-derived component of the pharyngeal skeleton appears to be particularly sensitive to retinoic acid (RA): administration of exogenous RA leads to loss of the second basibranchial, but not the first. Neural crest was undoubtedly critical in the evolution of the vertebrate pharyngeal skeleton, but mesoderm may have played a central role in forming ventromedial elements, in particular. When and how many times during vertebrate phylogeny a mesodermal contribution to the pharyngeal skeleton evolved remain to be resolved.