Journal of Experimental Zoology Part B: Molecular and Developmental Evolution

The aim of this study is to profile the transcriptome of teeth and the surrounding tissues of an adult lizard dentition (Eublepharis macularius) that is actively replacing teeth throughout life. Bulk RNAseq was used to compare teeth that are in function versus unerupted, developing teeth and single cell RNA-seq was carried out on jaw segments containing the dental forming tissues. In bulk RNAseq data, we found that functional teeth expressed genes involved in bone and tooth resorption. Indeed, multinucleated odontoclasts were abundant in tissue sections of functional teeth undergoing resorption. Unexpectedly, chemotaxis gene SEMA3A was expressed within odontoblasts and in adjacent mesenchyme, confirmed using RNAscope. Semaphorins may be involved in regulating odontoclasts during tooth resorption. The scRNA-seq experiment successfully isolated dental mesenchyme and epithelial cells. We confirmed that some of these genes are expressed in the earliest tooth buds within the tooth forming field. In addition, we found evidence of convergent evolution in the tooth eruption trait. Geckos evolved a means for second generation teeth to communicate with the functional teeth. Instead of a dental follicle inducing an eruption pathway as in the mammal, the gecko and other squamate reptiles use the enamel organ of the successional teeth to trigger tooth resorption of the functional teeth, thus creating an eruption pathway. New molecules such as SEMA3A and SFRP2 may also participate in this process. Future studies on the gecko will uncover the molecular basis of convergent evolution in the dentition.

Recent molecular phylogenetic studies have raised two questions about the evolutionary history of the calcified exoskeleton of mollusks. The first question concerns the homology of the two types of skeleton; whether spicules and shell plates share an evolutionary origin. The second question is the homology of the shell plates between chitons and other mollusks, including gastropods and bivalves. To gain insight into these questions, we examined the early development of shell plates and spicules in chitons. We identified several developmental genes that are involved in both shell plates and spicules, suggesting that spicules and shell plates share a common evolutionary origin. We also found that subpopulations of the dorsal shell field (the ridge and the plate field) have specific gene expression profiles. The differential gene expression of the ridge and plate field is not identical to the profiles of the zones of the gastropod shell field. This observation may suggest an independent evolutionary origin of the shell plates in chitons and gastropods.

Developmental plasticity, the ability of a genotype to produce different phenotypes in response to environmental conditions, has been subject to intense studies in the last four decades. The self-fertilizing nematode Pristionchus pacificus has been developed as a genetic model system for studying developmental plasticity due to its mouth-form polyphenism that results in alternative feeding strategies with a facultative predatory and a non-predatory mouth form. Many studies linked molecular aspects of the regulation of mouth-form polyphenism with investigations of its evolutionary and ecological significance. Also, several environmental factors influencing P. pacificus feeding structure expression were identified including temperature, culture condition and population density. However, the nutritional plasticity of the mouth form has never been properly investigated although polyphenisms are known to be influenced by changes in nutritional conditions. For instance, studies in eusocial insects and scarab beetles have provided significant mechanistic insights into the nutritional regulation of polyphenisms but also other forms of plasticity. Here, we study the influence of nutrition on mouth-form polyphenism in P. pacificus through experiments with monosaccharide and fatty acid supplementation. We show that in particular glucose supplementation renders worms non-predatory. Subsequent transcriptomic and mutant analyses indicate that de novo fatty acid synthesis and peroxisomal beta-oxidation pathways play an important role in the mediation of this plastic response. Finally, the analysis of fitness consequences through fecundity counts suggests that non-predatory animals have an advantage over predatory animals grown in the glucose-supplemented condition. Research highlightsThis study represents the first systematic attempt to investigate the influence of nutrition on mouth-form polyphenism in the genetic model organism Pristionchus pacificus. Through glucose and oleic acid supplementation we show that high nutritional conditions influence feeding plasticity and render worms non-predatory. Mutant analysis indicates a role of de novo fatty acid synthesis and peroxisomal beta-oxidation pathways for these responses.

Evolutionary novelties entail the origin of morphologies that enable new functions. These features can arise through changes to gene function and regulation. One important novelty is the fused rod at the end of the vertebral column in anurans, the urostyle. This feature is composed of a coccyx and an ossifying hypochord, and both structures ossify during metamorphosis. We used Laser Capture Micro-dissection of these identified tissues and subjected them to RNA-seq and ATAC-seq analyses at three developmental stages in tadpoles of Xenopus tropicalis. These experiments reveal that the coccyx and hypochord have two different molecular signatures. ATAC-seq data reveals potential regulatory regions that are observed in proximity to candidate genes identified from RNA-seq. Neuronal (TUBB3) and muscle markers (MYH3) are upregulated in coccygeal tissues, whereas T-box genes (TBXT, TBXT.2), corticosteroid stress hormones (CRCH.1), and matrix metallopeptidases (MMP1, MMP8, MMP13) are upregulated in the hypochord. Even though an ossifying hypochord is only present in anurans, this ossification between the vertebral column and the notochord appears to resemble a congenital vertebral anomaly seen prenatally in humans, caused by an ectopic expression of the TBXT/TBXT.2 gene. This work opens the way to functional studies that help us better elucidate anuran bauplan evolution.

The first vertebrate mineralized skeleton was an external bony armor coated with dental structures. The subsequent emergence of a mineralized endoskeleton and of teeth are considered key innovations in the diversification of vertebrates. Although time clouds our understanding of the initial evolution of these mineralized structures, recent re-emergences may shed light on the underlying processes. Loricarioid catfishes are a lineage that, much like the ancestral vertebrates, bear denticle-clad bony armor from head to tail. Loricarioid denticles (LDs) and oral teeth are very similar in superstructure. We show here that other extra-oral dental structures are found as ancestral characters only in lineages that are distantly related to loricarioids such as sharks or coelacanth, indicating that LDs have independently re-emerged in loricarioid catfishes. We investigate whether the similarities between LDs and teeth extend to their developmental and genetic context, and how their development compares to that of other vertebrate integument structures. Our detailed study of the development of LDs, and gene expression analyses through in situ hybridization confirm that all 12 genes from the tooth-forming gene regulatory network (oGRN) are expressed in developing LDs in a similar way as they are expressed in developing teeth. We then compare the developmental, structural, and genetic aspects of LD and teeth with that of other integument appendages such as fish scales, shark dermal denticles, feathers and hairs. We find that LDs share all developmental cues with teeth and, to a lesser extent, with the other vertebrate integument structures. Taken together, our results indicate that denticles have re-emerged on the trunk of loricarioid catfishes through the ectopic co-option of the oGRN rather than the resurrection of an ancestral trunk-specific denticle genetic pathway.

A widely accepted model for the evolution of cave animals posits colonization by surface ancestors followed by the acquisition of adaptations over many generations. However, the speed of cave adaptation in some species suggests mechanisms operating over shorter timescales. To address these mechanisms, we used Astyanax mexicanus, a teleost with ancestral surface morphs (surface fish, SF) and derived cave morphs (cavefish, CF). We exposed SF to completely dark conditions and identified numerous altered traits at both the gene expression and phenotypic levels. Remarkably, most of these alterations mimicked CF phenotypes. Our results indicate that cave-related traits can appear within a single generation by phenotypic plasticity. In the next generation, plasticity can be further refined. The initial plastic responses are random in adaptive outcome but may determine the subsequent course of evolution. Our study suggests that phenotypic plasticity contributes to the rapid evolution of cave-related traits in A. mexicanus.

The loss of pigmentation is a hallmark adaptation of cave-dwelling animals but the underlying mechanisms are poorly understood. This study investigates the cellular and biochemical basis of albinism, the loss of melanin pigment, which convergently evolved across a wide range of cave animals. Is albinism caused by the interruption of pigment synthesis or elimination of the pigment cell lineage? The results suggest that albinism evolved by a common mechanism, a block in the first step of the melanin biosynthesis pathway, the conversion of L-tyrosine to L-DOPA, in diverse albino cave animals ranging from annelids and mollusks to vertebrates. Pigment cells are conserved in all tested albino cave species and distributed in patterns resembling pigmentation in their close surface relatives. The cells capable of melanin synthesis when provided with L-DOPA substrate were detected at the sites of injuries in a cave annelid, cave mollusk, and cave teleost, suggesting roles in innate immunity during tissue repair, which may explain why pigment cells are conserved despite the loss of pigment production. Next, we focused on the longstanding issue of the evolutionary forces involved in the regression of pigmentation in cave animals and explored hypotheses of why losing pigmentation might be adaptive in cave environments. First, we tested the hypothesis that pigment regression may conserve energy, which is important for survival in the food-limited cave environment. Our results indicate that melanin synthesis has a marked energetic cost in the cavefish, Astyanax mexicanus. Lastly, we show that the disruption of melanin synthesis at its first biosynthetic step is correlated with increased dopamine levels in multiple depigmented cavefish populations, including populations that evolved albinism independently. This result supports a widespread tradeoff between the melanin and catecholamine synthesis pathways in A. mexicanus. We conclude that the interruption of the conversion of L-tyrosine to L-DOPA in the synthesis of melanin confers multiple advantages that could serve as targets of natural selection, supporting an adaptive hypothesis for the evolution of albinism in cave animals.

Molluscs are one of the most morphologically diverse clades of metazoans, exhibiting an immense diversification of calcium carbonate structures, such as the shell. Biomineralization of the calcified shell is dependent on shell matrix proteins (SMPs). While SMP diversity is hypothesized to drive molluscan shell diversity, we are just starting to unravel SMP evolutionary history and biology. Here we leveraged two complementary model mollusc systems, Crepidula fornicata and Crepidula atrasolea, to determine the lineage-specificity of 185 Crepidula SMPs. We found that 95% of the adult C. fornicata shell proteome belongs to conserved metazoan and molluscan orthogroups, with molluscan-restricted orthogroups containing half of all SMPs in the shell proteome. The low number of C. fornicata-restricted SMPs contradicts the generally-held notion that an animals biomineralization toolkit is dominated by mostly novel genes. Next, we selected a subset of lineage-restricted SMPs for spatial-temporal analysis using in situ hybridization chain reaction (HCR) during larval stages in C. atrasolea. We found that 12 out of 18 SMPs analyzed are expressed in the shell field. Notably, these genes are present in 5 expression patterns, which define at least three distinct cell populations within the shell field. These results represent the most comprehensive analysis of gastropod SMP evolutionary age and shell field expression patterns to date. Collectively, these data lay the foundation for future work to interrogate the molecular mechanisms and cell fate decisions underlying molluscan mantle specification and diversification.

Body shape diversity in vertebrates reflects a complex interplay between functional demands, environmental constraints, and internal developmental mechanisms. Various environments have promoted diverse morphological adaptations not only under natural but also domesticated conditions. One of the most remarkable examples of artificially induced morphology is found in the domesticated ornamental goldfish (Carassius auratus), which has diversified into numerous strains with strikingly different body shapes through prolonged human selection. In this study, we compared the body shapes of representative goldfish strains: the single-tail common goldfish (wild-type), Ryukin, Oranda, Pearl scale, and Ranchu. Our analysis revealed that the Ryukin and Pearl scale strains exhibit significantly greater body circularity in dorsal view compared to the other strains. Further anatomical and histological analyses showed that Pearl scale goldfish possess a thicker lateral body wall along with increased adipose tissue accumulation and reduced muscle fiber density, unlike Ryukin goldfish. These findings suggest that similar globular body shapes in different goldfish strains have arisen through distinct developmental pathways, exemplifying morphological convergence accompanied by histological divergence. We further discuss adipose accumulation in Pearl scale goldfish in relation to natural examples, providing insight into how function, morphology, and tissue organization may be interlinked in the evolution of globular body shapes.

Comparing gene regulatory patterns between seemingly similar phenotypic traits can provide important insights on the molecular mechanisms underlying the evolution of those traits. In this study, we investigate the molecular basis of the formation of a spade-shaped caudal fin, which is a rare phenotype among teleost fish characterized by an elongated medial region of the fin. We examined the expression patterns of candidate fin-shape genes in the spade-shaped caudal fin of the related species Lamprologus tigripictilis, an East African cichlid in the tribe Lamprologini. The candidate gene set consisted of a previously identified gene regulatory network (GRN) associated with the elongation of fin regions in another Lamprologini cichlid species and further genes selected on the basis of co-expression data and transcription factor prediction. Unexpectedly, the anatomical features of elongated fin rays differed and gene expression patterns associated with fin elongation were only weakly conserved between the two related species. We report 20 genes and transcription factors (including angptl5, cd63, csrp1a, cx43, esco2, gbf1 and rbpj), whose expression levels differed between the elongated and the short caudal fin regions of L. tigripictilis, and which are therefore candidates for the regulation of the spade-like fin shape.

Eisenia fetida, the common vermicomposting earthworm, shows robust regeneration of posterior segments removed by amputation. During the period of regeneration, the newly formed tissue initially contains only undifferentiated cells but subsequently differentiates into a variety of cell types including muscle, nerve and vasculature. Transcriptomics analysis, reported previously, provided a number of candidate non-coding RNAs that were induced during regeneration. We found that one such long non-coding RNA (lncRNA) is expressed in the skin, only at the base of newly formed chaetae. The spatial organization and precise arrangement of the regenerating chaetae and the cells expressing the lncRNA on the ventral side clearly support a model wherein the regenerating tissue contains a zone of growth and cell division at the tip and a zone of differentiation at the site of amputation. The temporal expression pattern of the lncRNA, christened Neev, closely resembled the pattern of chitin synthase genes, implicated in chaetae formation. We found that the lncRNA harbours 49 sites for binding a set of four miRNAs while the Chitin Synthase 8 mRNA comprises 478 sites. The over-representation of shared miRNA sites suggests that lncRNA Neev may act as a miRNA sponge to transiently de-repress chitin synthase 8 during formation of new chaetae in the regenerating segments of Eisenia fetida. Summary statementThe earthworm, Eisenia fetida, regenerates posterior segments following amputation. The transcriptome of the regenerating worm revealed a novel lncRNA, expressed only at the base of regenerating chaetae. We propose that this lncRNA is a miRNA sponge that modulates chitin synthesis.

Developmental systems may preferentially produce certain types of variation and, thereby, bias phenotypic evolution. This is a central issue in evolutionary developmental biology, albeit somewhat understudied. Here we focus on the shape of the first upper molar which shows a clear, repeated tendency for anterior elongation at different scales from within mouse populations to between species of the Mus genus. In contrast, the lower molar displays more evolutionary stability. We compared upper and lower molar development of mouse strains representative of this fine variation (DUHi: elongated molars and FVB: short molars). Using a novel quantitative approach to examine small-scale developmental variation, we identified temporal, spatial and functional differences in tooth signaling centers between the two strains, likely due to different tuning of the activation-inhibition mechanisms ruling signaling center patterning. Based on the spatio-temporal dynamics of signaling centers and their lineage tracing, we show an intrinsic difference in the fate of signaling centers between lower and upper jaw of both strains. This can explain why variations in activation-inhibition parameters between strains are turned into anterior elongation in the upper molar only. Finally, although the \"elongated\" DUHi strain was inbred, first molar elongation was variable in adults, and we found high levels of intra-strain developmental variation in upper molar development. This is consistent with the inherent developmental instability of the upper molar system enabling the morphological variability of the tooth phenotype.\n\nIn conclusion, we have uncovered developmental properties that underlie the molars capacity for repeated phenotypic change, or said differently, that underlie a \"line of least resistance\". By focusing on the developmental basis of fine phenotypic variation, our study also challenges some common assumptions and practices in developmental and evolutionary developmental biology.

The gastrointestinal tract has evolved in numerous ways to allow animals to optimally assimilate energy from different foods. The morphology and physiology of the gut is plastic and can be greatly altered by diet in some animals. In this study, we investigated the evolution and plasticity of gastrointestinal tract morphology by comparing laboratory-raised cave- and river-adapted forms of the Mexican tetra, Astyanax mexicanus, reared under different dietary conditions. In the wild, river-dwelling populations (surface fish) consume plants and insects throughout the year, while cave-dwelling populations (cavefish) live in a perpetually dark environment and depend on nutrient-poor food brought in by bats or seasonal floods. We found that multiple cave populations converged on a reduced number of digestive appendages called pyloric caeca and that some cave populations have a lengthened gut while others have a shortened gut. Moreover, we identified differences in how gut morphology and proliferation respond to diet between surface fish and cavefish. Using a combination of quantitative genetic mapping, population genetics, and RNA sequencing, we found that changes to the molecular and genetic pathways that influence cell proliferation, differentiation, and immune system function may underlie evolution of the cavefish gut.

Echinoderms possess a complex immune system, primarily relying on coelomocytes - immune cells circulating in coelomic fluids. Over the last few decades, various coelomocytes have been described based on morphological features, with holothuroids exhibiting the highest diversity of cell morphotypes among the different echinoderm classes. However, while the overall immune function of these cells is broadly accepted, their respective functions remain unclear, and molecular data specific to the different cell types are still limited in the literature. In this study, we address this gap in functional information and molecular data by using single-cell RNA sequencing (scRNA-seq) on coelomocytes from the perivisceral fluid of Holothuria forskali. We identified 10 distinct clusters, each assumed to correspond to a distinct transcriptional coelomocyte population. Among these, cluster 0 occupies a central position relative to the others, suggesting it may represent "progenitor cells", whereas cluster 6 is markedly divergent from all other clusters. Functional enrichment analyses revealed that some clusters ensure key immune functions, including pathogen recognition, phagocytosis, complement activation and redox balance regulation. In addition, examination of the processed samples under a microscope confirms the presence of a small proportion of recently discovered carotenocytes (7.0%) in the perivisceral fluid, a cell type rich in carotenoids. By using transcriptomics data previously obtained for this cell type by bulk RNA sequencing (bRNA-seq), it was possible to confidently identify cluster 6 as carotenocytes and provide further insights into their gene expression. While further analyses are needed to link other clusters to the different morphotypes previously described in the literature, this pioneer study presents preliminary data on the functional diversity of holothuroid coelomocytes, which could be of broad interest for a better understanding of holothuroid immunity as well as for the study of immune cell lineage evolution across deuterostomes.

Hydra has the unique ability to regenerate from aggregates of dissociated single cells that lack positional information. We compared two strains of hydra, a strain of hydra that was capable of regenerating from aggregates and a strain of hydra that was deficient in this type of regeneration. We observed unique actin cytoskeletal arrangements that were present in the regenerates of regeneration-competent strain but not in the regeneration-deficient strain. Concomitantly, the regeneration-deficient strain failed to organise the extracellular cytoskeleton of laminin and collagen between ectodermal and endodermal epithelial cells. These interesting preliminary observations highlight the importance of the cytoskeletal organisation in regeneration of hydra and suggest that regeneration from the aggregates of dissociated cells through de novo patterning requires correct structural organisation of cytoskeletal elements.

Specific character traits of mineralized endoskeletal tissues need to be clearly defined and comprehensively examined among extant chondrichthyans (elasmobranchs, such as sharks and skates, and holocephalans, such as chimaeras) to understand their evolution. For example, tiles of mineralized polygonal structures called tesserae occur at cartilage surfaces in chondrichthyans, but recent studies showing trabecular mineralization at elasmobranch cartilage surfaces suggest that tesserae are not as common as previously thought. Also, while areolar mineralized tissue in elasmobranchs is generally considered a unique, shared chondrichthyan feature, some chondrichthyan species demonstrate bone-like tissues in both a specific region of tesserae termed the cap zone and continuous (not tiled) mineralized neural arches. To clarify the distribution of specific endoskeletal features among extant chondrichthyans, adult skeletal tissues in a holocephalan chimaera (spotted ratfish) and two elasmobranchs (small-spotted catshark and little skate) were characterized using synchrotron radiation and desktop micro-CT imaging, and histological and immunofluorescent assays. Endoskeletal mineralization in the ratfish, catshark, and little skate varied both quantitively in tissue mineral density (TMD), and qualitatively in the morphology and localization of mineralized structures and tissues. For example, TMD of several skeletal elements was significantly lower in ratfish, compared to catshark and little skate. Trabecular and areolar mineralization were shared among these extant chondrichthyan species, but tesserae and bone-like tissues were not. Interestingly, three separate analyses argued that the adult chimaera endoskeleton has features of the embryonic little skate endoskeleton. Generally, this study proposes specific terminology for character states of the extant chondrichthyan endoskeleton and infers those states in ancestral chondrichthyans with reference to fossil data.

Dietary restriction is a putative key to a healthier and longer life, but these benefits may come at a trade-off with reproductive fitness and may affect the following generation(s). The potential inter- and transgenerational effects of starvation are particularly poorly understood in vertebrates when they originate from the paternal line. We utilised the externally fertilising zebrafish amenable to a split-egg clutch design to explore the male-specific effects of starvation on fertility and fitness of offspring independently of maternal contribution. Eighteen days of fasting resulted in reduced fertility in exposed males. While average offspring survival was not affected, we detected higher larval growth in offspring from starved males and increased malformation rates at 24 hours post fertilisation in the F2 embryos produced by the offspring of the starved males. The transcriptome analysis of embryos from starved and fed fathers revealed robust and reproducible induction of muscle composition genes and a contrasting repressive effect on lipid metabolism and lysosome genes. A large proportion of these genes showed enrichment in the yolk syncytial layer suggesting gene regulatory responses associated with metabolism of nutrients through paternal impact on extra embryonic tissues which are loaded with maternally deposited factors. We compared the embryo transcriptome to adult transcriptome datasets and demonstrated comparable repressive effects on metabolism-associated genes. These similarities suggest a physiologically relevant, directed and potentially adaptive response transmitted by the father, independently from the offsprings nutritional state, which was defined by the mother.

The blue mussel (Mytilus edulis) and the honeycomb tubeworm (Sabellaria alveolata) have evolved similar adhesive systems to cope with the hydrodynamic conditions of the intertidal environment where they live. Both organisms can establish a permanent adhesion through the secretion of adhesive proteins rich in DOPA (3,4-dihydroxyphenylalanine), a post-translationally modified amino acid playing essential roles in interfacial adhesion and bulk cohesion. DOPA is produced by the hydroxylation of tyrosine residues by tyrosinase enzymes, which can also in some cases oxidise it further into dopaquinone Compared to the detailed knowledge available on mussel and tubeworm adhesive proteins, little information exists about the tyrosinases involved in their adhesive systems. By combining different molecular analyses, a catalogue of tyrosinase candidates potentially involved in the adhesive systems of M. edulis and S. alveolata was identified. Some of these candidates were shown to be expressed in the adhesive glands by in situ hybridization, with a high gland-specificity in mussels but not in tubeworms. The diversity of tyrosinases highlighted in the two species suggests the coexistence of different functions (monophenol monooxygenase or catechol oxidase activity) or different substrate specificities. However, the exact role of the different enzymes needs to be further investigated. Phylogenetic analyses support the hypothesis of independent expansions and parallel evolution of tyrosinases involved in adhesive protein maturation in both lineages, supporting the convergent evolution of their DOPA-based adhesion.

Regeneration, regrowing lost and injured body parts, is an ability that generally declines with age or developmental transitions (i.e. metamorphosis, sexual maturation) in many organisms. Regeneration is also energetically a costly process, and trade-offs occur between regeneration and other costly processes such as somatic growth, or sexual reproduction. Here we investigate the interplay of regeneration, reproduction, and age in the segmented worm Platynereis dumerilii. P. dumerilii can regenerate its whole posterior body axis, along with its reproductive cells, thereby having to carry out the two costly processes (somatic and germ cell regeneration) after injury. We specifically examine how age affects the success of germ cell regeneration and sexual maturation in developmentally young versus old organisms. We hypothesized that developmentally younger individuals (i.e. lower investment state, with gametes in early mitotic stages) will have higher regeneration success and reach sexual maturation faster than the individuals at developmentally older stages (i.e. higher investment state, with gametes in the process of maturation). Surprisingly, older amputated worms grew faster and matured earlier than younger amputees, even though they had to regenerate more segments and recuperate the more costly germ cells which were already starting to undergo gametogenesis. To analyze germ cell regeneration across stages, we used Hybridization Chain Reaction for the germline marker vasa. We found that regenerated worms start repopulating new segments with germ cell clusters as early as 14 days post amputation. In addition, vasa expression is observed in a wide region of newly-regenerated segments, which appears different from expression patterns during normal growth or regeneration in worms before gonial cluster expansion. Future studies will focus on determining the exact sources of gonial clusters in regeneration. FundingNIGMS 1R35GM138008-01, Hibbitt Fellowship, WashU Startup funds

BackgroundThe evolvability of ontogenetic trajectories, driven by modular developmental processes and their evolutionary repatterning, plays a crucial role in the diversity of animal diversity. Anurans offer a model for exploring how developmental repatterning, including the loss of larval features and heterochronic shifts of metamorphic processes lead to the diversity of, often terrestrialized, life histories. Ancestrally, anurans exhibit an aquatic larva (tadpoles) that differs tremendously from their semi-terrestrial adult forms. The transition from this tadpole to the adult is a thyroid hormone-dependent metamorphosis that results in the rearrangement of almost all organ systems. One of the organ systems that experiences the most extensive rearrangements is the skin. Is the ancestral pattern of metamorphic skin rearrangement altered in anurans with a completely terrestrial tadpole phase? ResultsSkin development and metamorphic rearrangement in the terrestrial tadpole of Arthroleptella villiersi is investigated based on histological sections. It follows the major pattern observed in anurans with an aquatic tadpole phase and correlates with thyroid gland maturation. Neuromasts develop and are present during a short developmental time. Both, metamorphic skin rearrangement and neuromast degeneration correlate with histological thyroid gland maturation. A few minor heterochronic shifts are detectable compared to aquatic indirect and terrestrial direct developing anurans. ConclusionSkin development and metamorphic rearrangement in anurans with different reproductive modes is very similar and correlates with thyroid gland maturation indicating that skin development in anurans might be constrained by thyroid hormone activity. Neuromasts are the major exception being present in aquatic and terrestrial indirect developing species but not in terrestrial direct developing anurans. Heterochronic shifts detected during skin development indicate that at least the differentiation of multicellular gland complexes might represent a developmental module similar to neuromasts.