The vertebrate ahsg (alpha 2-HS glycoprotein, also coined fetuin-a) homologs are highly expressed in the liver, and their secreted protein products exert complex systemic effects, including the regulation of biomineralization of soft and skeletal tissues. Here, we report a previously uncharacterized ahsg retrocopy in the allotetraploid frog species Xenopus laevis. We show that this young retrocopy was born from the ahsg.L homeologue less than 10 Mya, and landed in the S subgenome in a locus located between asic2.S and smarcd2.S. The ahsg.L-retrocopy ends with a poly(A) tail, is intronless, and is flanked by target site duplications. While the ahsg.L-retrocopy's ORF is devoid of frameshifts and nonsense mutations, it suffers from a short 5' deletion, eliminating the original start codon and the signal peptide. Remarkably, this truncated ORF lies in frame with an ATG codon contributed by the neighboring genomic sequence, suggesting that the ahsg.L-retrocopy might potentially be expressed and translated into a protein product. Nevertheless, examination of RNA-Seq and proteomic experiments respectively performed on liver and bone tissues did not provide expression evidence for the ahsg.L-retrocopy. We propose that, in spite of its rescued ORF, the ahsg.L-retrocopy is non-functional and can be considered a young pseudogene born from recent retrotransposon activity in the Xenopus laevis lineage.
Homeodomain transcription factors share an ancient helix-turn-helix DNA-binding core yet regulate diverse developmental and reproductive programs across eukaryotes. We curated 355 nonredundant homeodomain structures spanning fungi, animals, and plants, including 118 experimental PDB entries and 237 high-confidence AlphaFold2 models, and performed large-scale structural comparisons using DaliLite5. Structural distances were summarized as a clustering tree visualized in iTOL and interpreted through topology-guided PyMOL superpositions and sequence-logo mapping of conserved positions. Across kingdoms, the canonical three-helix HTH geometry and the recognition-helix framework were strongly conserved, whereas structural diversification was concentrated in interhelical loops, terminal regions, linker segments, and lineage-specific auxiliary modules. Fungal mating-type homeodomains and major animal classes retained conserved recognition-helix architecture despite sequence divergence, while plant WOX, ZF-HD, PHD-associated, and SAWADEE-associated architectures illustrated modular expansion for developmental and chromatin-context integration.
Gcm1 and Gcm2 are paralogous transcription factors in vertebrates that play key roles in the development of pharyngeal-derived epithelia, yet their deployment across vertebrate lineages remains incompletely understood. While Gcm2 shows deeply conserved pharyngeal expression across gnathostomes, Gcm1 has been mainly characterized in mammals, where it exhibits broader expression patterns. How Gcm1 is deployed in non-mammalian vertebrates has remained unexplored. Here, we performed a comparative analysis of Gcm1 expression in cartilaginous fishes, non-teleost actinopterygians, and amphibians. RNA in situ hybridization revealed conserved Gcm1 expression in gill epithelia across these taxa. Parallel analyses showed that Gcm2 is also expressed in gill epithelia, with overlapping but distinct spatial patterns. In addition, Gcm1 showed lineage-specific expression in bichir embryos, including strong expression in external gills and scattered epithelial cells in the yolk-sac membrane. In the external gills, Gcm1-positive cells possess vacuole-like cytoplasmic structures, suggesting a previously unrecognized epithelial cell population. Together, our findings indicate that Gcm1 and Gcm2 share ancestral expression in pharyngeal epithelia but have followed distinct evolutionary trajectories, with Gcm1 exhibiting greater lineage-specific diversification.
Reproductive division of labour is a key feature underlying the remarkable success of ants, where a reproductive queen caste and non-reproductive worker caste develop from a totipotent egg or larva. The number of ovarioles that develop after the egg or larva is determined to be a queen or worker caste ultimately determines their reproductive potential. Yet since very early descriptions of ant larval ovaries made by Ezhikov (1923) and Brian (1950s), the developmental basis of ovariole number dimorphism in ants has received little attention. Here, we revisit their work in light of more recent knowledge from Drosophila melanogaster, where terminal filaments (TFs) in the larval ovary determine ovariole number on a one-to-one basis. Ezhikov and Brian observed that queen and worker larval ovaries develop the same number of TFs, but in the worker caste, they 'bundle' together to form a single ovariole. Using in situ hybridisation chain reaction (HCR) on multiple queen and worker larval stages, we confirm this bundling process is present in two derived ant species with ovariole dimorphism, but is absent in a species that lacks it. Our findings therefore suggest that this terminal filament bundling reflects a mechanism evolved within ants to regulate differences in worker ovariole number between castes independently of overall number of terminal filaments. This work sheds fresh light on historical ant research and sets up future work investigating the origins and regulation of this putative mechanism.
Early amniote development includes a striking but underappreciated event: rightward torsion ("twisting") of the head and trunk followed by axial rotation that positions the embryo's left side against the yolk. Although well documented in the domestic chicken, the evolutionary distribution and biological significance of this rotational pattern have never been evaluated systematically. Reports of supposed "leftward" rotation in some reptiles and birds, as well as a reported absence of rotation in some reptilian species, have further suggested interspecific variability. Here we present a comprehensive comparative synthesis of embryological evidence across Amniota, supplemented by original observations. Our analysis suggests that dextral (rightward) torsion and axial rotation occur in birds and all major extant reptile lineages-lizards, snakes, tuatara, turtles, and crocodilians-consistently yielding embryos that rest on their left sides. Despite extremely rare anomalies and inconsistent terminology, no well-documented reptilian or avian species examined to date is known to depart from this pattern. Elements of the same developmental configuration also occur in mammals. The phylogenetic distribution of this conserved architecture appears to be consistent with the hypothesis that a pattern involving dextral torsion and axial rotation represents a developmental synapomorphy that predates the divergence of crown amniote clades. Axial rotation has been postulated to have facilitated embryonic organization within the spatial constraints of the ancestral terrestrial egg. The persistence of this pattern, including aspects found in viviparous lineages with reduced egg sizes, suggests an intriguing possibility: canalization of a developmental program established more than 310 million years ago and conserved across more than 32,000 living amniote species.
Evolution of central nervous systems is a long-debated topic. Similar mechanisms of conditional neural specification linked to dorsal-ventral (D-V) axis formation have been used to support homology across several taxa. We tested for autonomous versus conditional neural specification in two distantly related annelids, Capitella teleta and Platynereis dumerilii, using blastomere isolations and ablations. Our results demonstrate autonomous specification of anterior neural tissue in isolated first-quartet (1q) micromeres for both annelids. In both annelids, partial larvae derived from isolation of the somatoblast, micromere 2d, also resulted in the formation of trunk neural tissue (ventral nerve cord or VNC), suggesting that this cell has intrinsic neural fate. However, at least in C. teleta, we found evidence that additional external proneural and anti-neural signals affect specification of trunk neural fate in 2d. Ablation of the vegetal macromeres at the 16-cell stage (leaving 1q + 2q) resulted in partial larvae that failed to form trunk neural tissue although they did have anterior neural tissue and a D-V axis, suggesting that micromeres other than 2d may repress the intrinsic neural fate of 2d. Conversely, ablation of only three of four vegetal macromeres at the 16-cell stage (e.g., leaving 1 macromere with 1q + 2q) "rescues" trunk neural fate, suggesting the presence of a vegetal proneural signal in all four quadrants. Taken together, these results imply that VNC fate is specified by a complex interplay of signaling that is decoupled from D-V axis formation in C. teleta. Our results suggest that the distinct neural specification mechanisms between the head and trunk may be conserved in Annelida and that annelid CNS specification may operate independently of D-V axis formation. These findings will broaden our understanding of how neural specification mechanisms evolved and diversified in Spiralia in comparison to other bilaterians.
Enamel and enameloid are hypermineralized tissues that can be found on the surface of vertebrates' dental structures. Shared developmental features suggest that minor changes could have driven multiple transitions between dentine-enamel and dentine-enameloid structures. It has been hypothesized that delayed (H1) or prolonged epithelial cell activity (H2), as well as modifications in epithelial cell production rates (H3), could explain such transitions. To test these evo-devo hypotheses, we built a cell-based histogenetic model using simplified properties common to current vertebrates. By varying secreting cell parameters, the simulations can reproduce a wide range of dentine, enameloid, and enamel proportions, allowing exploration of their role in the enameloid-enamel transition. Our exploration of 12 cellular parameters showed that H1, and H3 to a lesser extent, could account for such a transition, whereas H2 likely needs to be associated with one other modification. Our results also suggest that changes in the position and timing of the mineralization front formation are key to constraining enamel-enameloid-dentine development. Thus, beyond the gain or loss of gene function, minor developmental modifications may also have played a key role in the emergence and evolution of dental tissues.
Developmental modularity allows specific tissues to adjust their developmental timing to meet stage-specific functional demands. During insect eclosion, the biomechanical requirements for emergence often conflict with adult defensive adaptations. This study investigates the evolutionary and molecular mechanisms of the enlarged pronotum in the ladybird beetle, Harmonia axyridis. We found that pronotal melanization and sclerotization are completed approximately 3 h before eclosion, asynchronous with the elytra, which mature post-emergence. RT-qPCR confirmed that the key tanning gene HaLac2 peaks early in the pronotum but is delayed in the elytra, allowing the latter to remain flexible for expanding. RNAi knockdown of HaLac2 resulted in a 72.5% eclosion failure rate due to a soft pronotum being unable to rupture the pupal cuticle, demonstrating its critical role in this process. Furthermore, larval knockdown of the Hox gene HaScr induced a homeotic transformation of the prothorax into a mesothorax-like identity, causing a complete loss of this accelerated developmental modularity program. Together, our findings provide functional and molecular evidence demonstrating how upstream Hox genes couple segmental identity with modified developmental schedules to optimize distinct, stage-specific survival strategies.
The evolution of species is tightly linked to the generation of new molecular and structural features through variation and selection. Here we have performed a detailed analysis of the jaw apparatus of the spiny-tailed lizard Uromastyx maliensis. Our comparative anatomy study highlights a closed tooth row and a monophyodont set of teeth, indicative of an evolved dentition beyond lepidosaur standards. On a microscopic level, Uromastyx enamel was thicker than the enamel of other lepidosaurs, featuring bundles of elongated crystallites ordered into prisms. The Uromastyx amelogenin protein revealed a lengthy polyproline tripeptide repeat insert consisting of 10 tripeptide repeats and similar to the bovine amelogenin ruminant insert. On a protein level, Uromastyx amelogenin self-assembled into 20 nm subunits, half the size of the 40 nm subunits in Iguana and similar to the diameter of mammalian amelogenin protein subunit compartments. Formation of 20 nm subunit compartments has been previously shown to be essential for enamel prism growth. These data demonstrate that Uromastyx lizards feature an evolved dentition indicative of convergent evolution adapted to extreme arid conditions, as demonstrated by enamel crystallites and prisms, an elongated "herbivore" motif in the enamel amelogenin protein sequence and compacted enamel protein subunits facilitating elongated apatite crystal growth. In a broader evolutionary biology context, our results suggest that jaws and teeth may evolutionarily converge to surpass traditional Lepidosaur molecular and enamel microanatomy features to facilitate feeding specialization and survival.
This study presents the first detailed histological description of wing primordium development in Mecoptera and demonstrates a late-forming, non-invaginated developmental mode lacking a morphologically distinct peripodial epithelium. Wing primordium development in holometabolous insects follows two major modes, early-forming and late-forming. Late-forming primordia are epidermal cell populations that remain integrated within the larval epithelium, contribute to cuticle production during each larval molt, and begin morphogenesis only in the final instar. The order Mecoptera remains among the least studied groups of Holometabola with respect to wing primordium development. Using a newly established rearing method and histological analysis, we investigated wing development in the scorpionfly Mavropanorpa japonica and found the following: (1) wing primordia are absent in actively feeding larvae; (2) they originate as localized epidermal thickenings beneath the lateral pinacula approximately 20 days after cocoon formation; (3) thickening of wing primordia begins concurrently with apolysis, and the epithelium extends outward without pouch-like invagination; and (4) no morphologically distinct peripodial epithelium was observed at all stages. Our findings show that wing primordia in Mecoptera develop externally, without invagination and without formation of a morphologically distinct peripodial epithelium. This study provides new comparative insight into heterochronic shifts and evolutionary diversity in holometabolous wing development.
Vertebrate limb proportions have diversified in association with species-specific locomotion and life-history strategies. Understanding the ontogenetic origins of this diversity is key to explaining the processes of limb evolution, yet these origins remain poorly investigated. This study explored the developmental timing of limb proportion diversification in birds using published data on limb element lengths (humerus, radius/ulna, femur, and tibiotarsus) at two crucial stages, hatching and adulthood, across 84 species (Wetherbee 1961). We found that while interspecific variation in limb proportions emerges prenatally, these initial patterns further develop into their adult proportions during postnatal growth. Specifically, intralimb stylopod-to-zeugopod ratios slightly decreased or remained constant after hatching, whereas interlimb segmental ratios (i.e., comparisons of homologous elements between the forelimb and hindlimb) changed substantially with the increase in forelimb element lengths. The contribution of prenatal development to limb proportion diversity, as evaluated from the strength of correlation between hatchling and adult proportions, appeared to be comparable among the limb proportion types. However, only for the intra-forelimb proportion, the balance between prenatal and postnatal modification was significantly affected by the developmental mode (precocial, altricial, or intermediate): the correlation was strongest in precocial species and weakest in altricial species, indicating a major contribution from prenatal development and postnatal growth, respectively. These patterns are consistent with the pre- and postnatal ontogenetic trajectory data available for several species, likely reflecting species-specific developmental constraints and postnatal ecological demands. Furthermore, the observed developmental flexibility appeared to be facilitated by a decoupling of postnatal growth between the forelimbs and hindlimbs. Overall, our findings indicate that avian limb proportion variation arises from a variable combination of prenatal patterning and postnatal growth, the balance of which is partly associated with developmental mode. By analyzing an extensive dataset, this study highlights the importance of an ontogenetic perspective for understanding the evolutionary factors driving avian limb diversity.
How a single signaling pathway patterns the morphologically distinct body forms within one organism remains an open question in developmental biology. Colonial hydrozoans, which transition through metamorphosis from a free-living planula to an architecturally complex branching colony, provide a compelling system to address this problem. Wnt signaling is well established as a key regulator of cnidarian axis patterning, yet the expression patterns and roles of Wnt components across the successive morphological transitions of the life cycle, particularly during colony growth, remain largely unknown. Here we provide a comprehensive expression map of Wnt ligands, Frizzled receptors, downstream components, and endogenous inhibitors throughout the entire life cycle of the thecate hydrozoan Dynamena pumila, from larval axis patterning through metamorphosis to colony growth. We show that in the larva, multiple Wnt genes are expressed in spatially distinct, partially overlapping domains spanning the entire oral-aboral axis, suggesting that combinatorial Wnt signaling underlies primary axis patterning in D. pumila. Rather than being directly inherited by the colony, this larval patterning system is extensively reorganized during metamorphosis, with individual ligands disappearing, newly activated, or redeployed in new spatial contexts. In the growing colony, combinatorial expression of Wnt genes marks distinct parts of the colony, while an activator-inhibitor logic involving canonical Wnt components and likely the antagonist sFRP3/4 drives the cyclic subdivision of the shoot growth tip into hydranth primordia and a self-renewing central primordium. Together, our results reveal a dynamic, flexible Wnt patterning system that is redeployed in new developmental contexts to generate the architectural complexity of the hydrozoan colony.
Multicellular morphogenesis must balance organismal cohesion with local tissue differentiation. In avian beaks, conserved epithelial-mesenchymal crosstalk underlies the formation of condensations of migratory neural crest mesenchymal (NCM) cells, yet how these cells acquire precise positional information without compromising stemness is unclear. Using high-throughput quantification of protein expression and morphology of 2.1 million cells and 16 stereotypical condensations across upper and lower beaks, we resolve the temporal sequence of condensation anchoring. We find that a subset of mesenchymal cells at each condensation site transiently matches protein expression in the overlying epithelium, which diverges as development proceeds. Propagation of these location-specific expression profiles into mesenchyme establishes signaling boundaries that anchor forming condensations. As NCM cells accumulate within these boundaries, they progressively erase location-specific protein profiles and restore their region- and tissue-specific protein expression. These transient location-matching and cell-homogenization phases show how migrating NCM cells achieve precise positional anchoring while retaining stemness needed for regional specifications. Ultimately, spatiotemporal modulations of a conserved regulatory network by predictable patterns of cell proliferation and migration can underpin the remarkable evolutionary diversification of avian beaks.
Terrestrialization in anurans is associated with the evolution of endotrophy. It is hypothesized that heterochrony, or changes in the time or rate of developmental events, is associated with the evolution of development in endotrophic species. To analyse heterochrony, we investigated and revised the description of the development in Arthroleptella villiersi, a small frog species of the family Pyxicephalidae, found in the Cape fold mountain region of the Western Cape, South Africa. We also compared the development of A. villiersi with that of species with exotrophic aquatic larvae, endotrophic terrestrial indirect, and direct development, using heterochrony plots to identify heterochronic shifts during development. As a result, we found that the terrestrial endotrophic larva of A. villiersi shares external similarities with exotrophic, aquatic larvae in having a long, muscularized tail with a fin, a lateral line system, and an opercular fold that completely covers the forelimbs. However, other developmental events like the reduction of larval mouthparts and the pre-displaced fore- and hindlimb development are comparatively similar to direct-developing taxa. The results of our study show that the timing of early developmental events can be shifted profoundly, while the timing of later events seems to be more conserved in anuran development. We interpret that some of these heterochronic shifts might be consequences of functional and developmental constraints underlying the establishment of the adult body plan.
Summary Funding for basic research in the United States and elsewhere is under threat. In light of this, compelling arguments have been made in support of the long‐term economic value of basic research. Here, I stress a more basic psychological value, and argue that evo‐devo is uniquely suited to address a fundamental human need to better understand the world through the telling of origin stories.
Despite their diversity in habitats, nematodes are often considered to have a highly conserved neuroanatomy. This premise is based on only a subset of the nematode phylogenetic tree within the subclass Chromadoria, which includes the model organism Caenorhabditis elegans, thereby limiting our understanding of macroevolutionary trends in nervous system structure. To approach this problem, we used nuclear morphology to quantify the number of neurons in the nematode ventral nerve cord (VNC) across the phylum and identified evolutionary patterns in neuroanatomical organization. Nuclear staining revealed that Dorylaimia has significantly more VNC neuronal nuclei than other taxa in Enoplia and Chromadoria, with some species having four times the number of neurons as C. elegans. These results suggest at least two independent transitions in VNC neuron number across subclasses. To further examine developmental patterns and potential variation in nervous system architecture of species with substantially more neurons than C. elegans, we established an isogenic culture of Mononchus aquaticus (Dorylaimia). We found that while M. aquaticus contained four times as many VNC neuronal nuclei as C. elegans, the VNC had a similar developmental timeline during post-embryonic stages. However, dye-filling assays also revealed an extensive distribution of neurons along the lateral body wall of M. aquaticus, which have no obvious homologs in C. elegans. We further found that M. aquaticus is capable of sustained movement following bisection and speculate that this ability results from a more decentralized neuronal network. Our results provide a roadmap for understanding phylum-wide nervous system evolution and demonstrate large-scale differences in neuroanatomy across the phylum.
Concepts of developmental timing have traditionally been framed under heterochrony as evolved (genetically based) differences in timing, while environmentally induced shifts in timing within genotypes have been treated more loosely. In this article, heterokairy is presented as plasticity in the timing of developmental events, and the term "heterokairic genes" is proposed for environmentally modulated heterochronic genes that underlie this plasticity. Evidence from nematodes, insects, plants, and vertebrates is assembled, with emphasis placed on systems where environmental cues are relayed through endocrine or metabolic pathways to known timing modules/genes. On this basis, a distinction is drawn between validated heterokairic genes, supported by direct mechanistic data, and a broader set of candidates inferred from gene-environment interactions in developmental timing. The eco-evolutionary consequences of such genes are considered, and experimental and genomic strategies for their identification are outlined. It is argued that heterokairic genes provide a useful bridge between environmental variation, developmental mechanisms, and evolutionary change in timing.
Cryptic genetic variation-heritable genetic variation that is only expressed under stressful or novel environments-can potentially fuel the evolution of novel traits. While previous work has demonstrated that novel environments can expose cryptic genetic variation, whether and how multiple environments interact to shape such variation in natural populations is poorly understood. To determine how multiple environments may modulate cryptic genetic variation, we used tadpoles of the Eastern spadefoot, Scaphiopus holbrookii (Sc. holbrookii). Species of Scaphiopus have previously been used as outgroups to the genus Spea, which has evolved a novel carnivorous tadpole morph specialized for a shrimp diet. Here, we assess whether shrimp-induced cryptic genetic variation in Sc. holbrookii tadpole traits varies as a function of conspecific competition. Across all traits measured, we found that shrimp-induced cryptic genetic variation only occurred under specific competitive conditions. Specifically, the shrimp diet revealed cryptic genetic variation in body size, gut length, and jaw area when tadpoles experienced high intraspecific competition. Surprisingly, across these same traits, the shrimp diet suppressed the expression of heritable variation under low competition, suggesting that moderately stressful conditions can limit the expression of heritable variation. In contrast to the other traits, the expression of heritable variation in tadpole tail depth was largely unaffected by diet or competition. Together, our results indicate that interacting environmental factors jointly modulate how and in what traits cryptic genetic variation may be expressed, thereby affecting its potential to drive novel trait evolution.
Brain evolution in vertebrates has been conceptualized through two major hypotheses: the mosaic and concerted evolution models. The mosaic evolution model suggests that brain structures are primarily shaped by functional constraints, whereas the concerted evolution model emphasizes the role of developmental constraints. Our objectives in this study were (1) to describe brain shape and volume changes during Mexican axolotl (Ambystoma mexicanum) larvae development, and (2) to interpret possible functional and developmental constraints during post-hatching brain maturation. A total of 77 larvae, spanning four developmental stages, were examined using 3D geometric morphometrics and volumetric measurements derived from iodine micro-CT imaging. To understand the relationships among brain regions, we employed morphological integration and modularity analyses, providing a comprehensive assessment of changes in shape covariation patterns during post-hatching development. Our results reveal that the telencephalon-diencephalon boundary and the hypothalamus region exhibit a low level of morphological variation throughout larval development. This stability may influence the positioning of the coronal suture, a key feature in tetrapod skull morphogenesis. In contrast, sensory structures undergo significant changes. The olfactory bulbs and optic tectum display positive allometric growth during early post-hatching development, transitioning to isometric growth at later stages. These shifts suggest an early developmental emphasis on sensory-related brain areas, potentially driven by functional constraints. Results also revealed a general correspondence between brain region volume and total brain volume, which aligns with the concerted model. Modular, morphological integration, and volumetric analyses suggest that an interplay of functional and developmental constraints might be involved in axolotl brain development.
