The weapon trait is typically characterized by an exaggerated body part employed in intrasexual competition, such as male-male combat over mating partners. In the last two decades, EvoDevo studies have engaged in understanding the genetic basis and the evolutionary origin of this sexually selected weapon. However, it remains unclear to what extent factors in the molecular mechanism controlling organ growth regulate the sex- and organ-specific exaggeration of the weapon traits. To this end, we investigate the functions of two well-known organ-growth controlling pathways, Fat-signaling and Crumbs-Expanded modules, for weaponized mandible growth in the Japanese common stag beetle, Dorcus rectus. Here, we find that nine genes of these pathways are involved in mandible extension and morphogenesis along the proximodistal (PD) axis. Some of these genes control the morphogenesis of the distal portion of the leg and antenna and leg size as well. These results suggest that the common role of these genes in appendage growth may be recruited to the exaggerated growth of the Dorcus mandible as a functional module. In addition, our morphometrics shows that dachsous and expanded contribute to sex-linked growth of male mandibles, suggesting that these genes adjust mandible size and shape according to sex through integrating sexual information with organ growth mechanisms. Overall, the weaponized nature of the Dorcus mandible might have originated via the co-option of the Fat-signaling and Crumbs-Expanded module-related genes into mandible exaggeration along the PD axis.
Skull simplification describes the convergent loss of bones in the tetrapod skull over evolutionary time. Much of this trend remains elusive, in particular, the apparent difference in evolutionary conservation across the skull. While some bones (e.g. nasal) have been retained for over 400 million years, others (e.g. tabular) were repeatedly lost. The drivers of this preferential loss remain unknown. We explore whether the discrepancy in evolutionary conservation in the lissamphibian lineage was related to differences in evolvability of skull bones in their stem group, the temnospondyls. Linear morphometrics were applied to temnospondyl skull roof bones to quantify and compare the morphological disparity and evolutionary rates of bones that are lost and retained by lissamphibians. Analyses were conducted at inter- and intraspecific levels to capture the effects of development and natural selection on bone disparity. Bones lost in lissamphibians exhibited greater morphological disparity and evolutionary rates in temnospondyls than retained bones. This indicates that developmental bias may have promoted the retention of conserved bones. The higher disparity and evolutionary rates in bones absent in lissamphibians suggest that they were less constrained by development, potentially facilitating their loss in response to functional demands associated with buccal-pump breathing and eye retraction during feeding.
The transformation of paired appendage structure from aquatic fins to terrestrial limbs represents a pivotal event in vertebrate evolution, underpinning the colonization of land by early tetrapods. This transition involved profound morphological and genetic modifications, particularly in the distal limb region known as the autopod and in the dorsoventral plane of paired appendages. Recent advances in paleontology, comparative and functional genomics, as well as evo-devo studies have shed light on several key events and evolutionary pathways and have improved our understanding of the direction of changes in regulatory mechanisms underlying the fin-to-limb transition. In this review, we aim to summarize current knowledge on limb evolution, with particular emphasis on studies of phylogenetically pivotal vertebrate groups - cartilaginous fishes and chondrosteans, which represent basally diverging evolutionary lineages of extant vertebrates, as well as sarcopterygians, the group of lobe-finned fishes most closely related to tetrapods. We consider the principal hypotheses concerning the prerequisites for vertebrate terrestrialization, key aspects in the search for structural homology between the morphological elements of fins and limbs, as well as the genetic mechanisms of spatial limb bud development described to date and the possible modifications of these mechanisms associated with the transformation of ancestral fins into pentadactyl terrestrial limbs.
RNA interference (RNAi) is a natural antiviral defense mechanism in plants and animals. As a counter defense strategy, most viruses have evolved viral suppressors of RNAi (VSRs) to antagonize the RNAi pathway. Here, we utilized transgenic misexpression of a VSR from Cricket Paralysis virus (CrPV1A) to dampen RNAi in a temporal and life-stage specific way in order to overcome limitations of knocking down pleiotropic genes by the strong systemic RNAi response in the red flour beetle Tribolium castaneum. We found that ubiquitously driven VSR rescued the sterility of the females injected with Tc-axin or Tc-decapentaplegic double-stranded RNA, where sterility had previously hampered analysis. By overcoming sterility using this tool and by rescuing zygotic function with a heat-shock driven VSR, we were able to separate maternal from zygotic function for the Wnt pathway inhibitor Tc-Axin. Thereby, we could provide evidence that maternal Wnt signalling alone is responsible for axis formation in Tribolium. Our tool opens new experimental possibilities such as studying genes by parental RNAi, which would normally lead to sterility and separating maternal from zygotic gene functions. Further improvements are required to allow for studying zygotic gene function while rescuing maternal functions and for spatially restricting the RNAi effect.
How animals thrive in extreme deep-sea environments remains a key question in marine evolutionary biology. While adaptation is often studied through the lens of genetic sequence, the epigenetic mechanisms that allow for phenotypic plasticity and rapid environmental response in these organisms remain a major "black box". DNA N6-methyladenine (6mA) is an emerging epigenetic mark in eukaryotes, but its presence and role in deep-sea animals remain unknown. Here, we present the first genome-wide map of DNA 6mA in a deep-sea animal, the cold seep limpet Bathyacmaea lactea, generated using PacBio single-molecule real-time (SMRT) sequencing. We identified 281,772 high-confidence 6mA sites, comprising ~0.13% of all adenines in the genome. Over 60% of genes harbor 6mA, with sites significantly enriched in exons. Integration with RNA-seq data revealed that gene body 6mA correlates with active transcription, whereas promoter 6mA is associated with gene repression. Genes with abundant 6mA are enriched in pathways related to energy metabolism, protein homeostasis, and osmoregulation, suggesting that 6mA methylation may facilitate adaptation to high pressure, low temperature, and dark deep-sea conditions. Our findings uncover 6mA as a component of the B. lactea epigenome and provide novel insights into epigenetic regulation under extreme deep-sea environments.
Phenotypic variation is the raw material for evolutionary diversification and adaptation. However, a critical gap remains in evolutionary theory between developmental and statistical representations of phenotypic variation, limiting our ability to understand and predict evolutionary change. In this paper, we close this gap by establishing a formal bridge between developmental and statistical accounts of phenotypic variation. Representing development as a dynamical system, we derive explicit relationships between perturbations to developmental systems and quantitative-genetic parameters. Through this framework, we obtain two important results. First, we show that the full developmental trajectory contains information that can improve the estimation of statistical parameters relevant to evolution. Second, we explain how different sources of variation-genetic, environmental, and stochastic-shape the distribution of phenotypic variation. This reveals conditions under which covariance matrices are expected to align, offering a developmental explanation for statistical patterns of phenotypic variation at both micro- and macroevolutionary scales. These findings advance our understanding of how developmental processes structure phenotypic variation, shape evolutionary dynamics, and influence evolvability.
Trichoplax adhaerens, one of the simplest multicellular organisms belonging to the phylum Placozoa, occupies a basal position within Metazoa and serves as a critical model for investigating early multicellular evolution. Despite its significance, the absence of functional genetic tools has limited research to comparative genomics, ultrastructural characterization, and behavioral observation. Developing methodologies such as transgenesis and gene editing would substantially expand experimental capabilities. However, the absence of sexual reproduction precludes the use of conventional approaches like single-cell microinjection. To overcome these limitations, the present study established a transgenic method for expressing exogenous genes in T. adhaerens. Systematic evaluation of multiple transfection techniques identified electroporation as the most effective strategy for inducing ectopic gene expression in this species. Electroporation parameters were optimized to maximize efficiency while minimizing cytotoxicity. Using this approach, robust expression of green fluorescent protein (GFP) was achieved using various promoters. Ectopic expression of the puromycin acetyltransferase ( PAC) gene conferred increased resistance to puromycin exposure, suggesting a potential strategy for generating stable transgenic lines in the future. This work introduces a reliable framework for ectopic gene expression in T. adhaerens, enabling molecular and functional analyses in one of the earliest-diverging metazoan lineages. 丝盘虫（ Trichoplax adhaerens）属于扁盘动物门（Placozoa）,是最简单的多细胞生物之一。由于其位于系统发育树的基部，扁盘动物门已成为研究多细胞生命起源的重要模型。然而，遗传操作技术的缺乏使得大多数研究仅限于基因组学、形态学和行为学研究。发展转基因和基因编辑等遗传工具将极大地推动对该生物的研究。鉴于其缺乏有性生殖，传统的单细胞显微注射等方法并不可行。该研究建立了一种在丝盘虫中表达外源基因的转基因方法。通过评估不同的转染策略，我们证明电穿孔（electroporation）是在该物种中进行异位基因表达的有效方法。我们进一步优化了电穿孔条件，并利用不同启动子成功表达了GFP（绿色荧光蛋白）。此外，我们发现异位表达嘌呤霉素乙酰转移酶（puromycin acetyltransferase, PAC）基因能提高动物对嘌呤霉（puromycin）的抗性，这为未来构建稳定的转基因品系提供了一种潜在策略。我们的研究结果建立了一种在丝盘虫中进行基因表达的方法，为研究这一基础后生动物提供了宝贵的分子生物学工具。.
Even though the canonical role of the transcription factor LEAFY (LFY) is in establishing the identity of angiosperm floral meristems, homologs of this gene are found across all land plants, predating the evolution of flowers. It has been hypothesized that the ancestral role of LFY was more broadly meristematic, regulating cell division, and that it acquired its reproductive function more recently. Here, we review mounting evidence from LFY orthologs in non-flowering plants that support the hypothesis that the reproductive role of LFY arose earlier in the land plant lineage than previously thought, in the gametophyte of haploid-dominant plants, and that it was co-opted to the sporophyte as land plants evolved towards diploid-dominant life cycles. Additionally, we examine how recent insights into LFY's mechanism of action inform the reconstruction of its functional evolution, including its recognition as a member of the elite class of plant pioneer transcription factors.
Coral reefs are biodiversity hotspots increasingly threatened by climate-induced bleaching, yet profiling the coral holobiont-the host and its associated microbiota-remains technically challenging due to high host-DNA contamination (often >95%) and the lack of comprehensive reference databases. Here, we present holo-2bRAD, a type IIB restriction site-associated DNA sequencing approach. This method, strategically integrated with a meticulously curated hologenome database (comprising 404,946 microbial genomes and 56 coral-derived metagenome-assembled genomes), effectively overcomes overwhelming host contamination (~99%). We demonstrate its exceptional species specificity (99.92%) in profiling Galaxea fascicularis (Linnaeus, 1767; Order Scleractinia, Family Euphylliidae) holobionts across bleaching severities, thereby validating its technical feasibility. Leveraging this high-resolution tool, our hologenome analysis revealed significant restructuring of coral-associated microbiota during bleaching, where microbial shifts (e.g., depletion of beneficial Thermoanaerobacterium thermosaccharolyticum and enrichment of stress-responsive bacteria) correlated more strongly with bleaching phenotypes than host genetic variation. By providing cost-effective, multi-domain hologenome profiling at unprecedented resolution, holo-2bRAD offers a practical tool for investigating holobiont dynamics and developing microbiome-informed coral conservation strategies.
The transcription factor Sox30 (SoxH), a conserved regulator of vertebrate spermatogenesis, remains poorly characterized in mollusks, despite emerging evidence of its potential role in sex regulation. In this study, we demonstrate that AiSoxH is a functional ortholog of vertebrate Sox30 in the hermaphroditic scallop Argopecten irradians. Developmental profiling reveals that AiSoxH expression is negligible during embryogenesis and larval stages. In adults, its expression is strictly testis-specific, confined to later developmental phases (growth and maturation stages) and localized to spermatids and spermatozoa under the control of a testis-enriched promoter. Knockdown of AiSoxH significantly disrupts spermiogenesis, leading to a reduction in mature germ cells and the downregulation of key spermiogenesis-related genes, including those encoding flagellar components, microtubule-associated proteins, and protein kinases. DNA affinity purification sequencing (DAP-seq) identifies 220 putative AiSoxH targets involved in microtubule binding, protein phosphorylation, and membrane trafficking. Dual-luciferase reporter assays confirm AiSoxH's direct activation of Limk1 and Eip74EF, two essential regulators of spermiogenesis. Collectively, our findings establish SoxH as a specific regulator of spermiogenesis, with limited evidence supporting a role in sex determination, positioning it as a potential target for fertility control in hermaphroditic scallops and possibly other mollusks.
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Abnormal morphogenesis causes dysfunction of biological tubes and thus is associated with various diseases. However, the underlying regulatory mechanisms for biological tubulogenesis are not fully understood, especially on the cooperation among organs during embryogenesis. In this study, we unexpectedly found that mutation of dac (the ortholog of dach1 in Drosophila), which is expressed in nervous system cells but not in the trachea, causes anterior dorsal trunk (DT) breakages in Drosophila tracheal tubulogenesis. Further investigations showed that Dac interacts with the transcription factor Lola, which was also required for tracheal DT integrity. More intriguingly, we demonstrated that the expression of the secreted glycoprotein Slit was genetically regulated by Dac-Lola axis. Furthermore, we observed that loss of Slit causes DT branch breakages, copying the phenotypes of dac and lola mutations. Finally, our data proved that dac, lola, and slit orchestrate within the same genetic pathway to maintain tracheal DT integrity. In summary, our study reveals a novel Dac-Lola-Slit axis that is required for fly tracheal tubulogenesis through inter-organ communications, providing a molecular theoretical basis for the therapy of DACH1-related tubular diseases.
Developmental biology and evolutionary theory have traditionally emphasized gene mutations as the primary drivers of new traits, with natural selection shaping the resulting variation. However, recent insights highlight the role of environmental factors during development in shaping trait evolution. In this Commentary, I introduce the 'environmentally dependent developmental induction' (EDDI) model, which proposes that phenotypic evolution is driven not only by genetic changes but also by environmentally induced modifications to the core developmental program. Using cardiogenesis as an example, I argue that environmental triggers such as oxygen levels and mechanical forces expand the genotypic toolkit available to heart development, activating new pathways that lead to the emergence of novel cardiac structures. These lineage-specific environmental changes might thus influence the differentiation of cardiac progenitor cells, resulting in modifications to the cardiac building plan. The EDDI model provides a novel explanation for how the basic cardiac plan was expanded during evolution while simultaneously explaining why cardiogenesis is vulnerable to malformations, even in the absence of genetic defects.
The evolutionary success of animals has been facilitated by the expansion and diversification of serially homologous elements. The vertebrate pharyngeal skeleton exemplifies this process. Evolved from a uniform series of cartilaginous rods, different elements have evolved distinct shapes and functions over time. This process implies that the evolution of the pharyngeal skeleton involved the decoupling of its constituent elements; however, to ensure functionality, a level of connectivity must be maintained. Thus, the evolution of the pharyngeal skeleton is likely influenced by a balance of diversifying and unifying forces. We probe these ideas using East African cichlid fishes. Shape analyses in a sample of 75 ecologically diverse species and a large F5 hybrid population showed a conspicuous lack of independence. Nearly all elements were correlated, a pattern reflected in the genotype-phenotype map and associated with co-expression of the candidate gene, smad7. Results are discussed in the context of the palimpsest model, whereby trait covariation is the result of numerous, hierarchical forces of covariation. Given its broad regulatory roles, we suggest that smad7 may be involved in several layers of the palimpsest, providing molecular insights into how serially homologous elements may diversify while also maintaining functionality.
Neurodegenerative diseases are traditionally viewed as age-associated conditions, characterized by distinct biochemical, cellular, and clinical features. However, emerging evidence suggests that their origins may trace back to much earlier stages of life. In this review, we synthesize insights from molecular genetics, developmental neurobiology, and systems neuroscience to examine the hypothesis that selective neuronal vulnerability can arise from developmental misprogramming. We explore how early-life processes-ranging from neurogenesis to synaptic maturation and circuit formation-can imprint long-lasting susceptibilities that manifest as degeneration decades later. Crucially, we highlight that many neurological disorders share early developmental commonalities that may predispose individuals to neurodegenerative vulnerability later in life. This is most apparent in familial forms of these diseases but may also emerge through embryonic or perinatal interactions with environmental or polygenic risk factors. Furthermore, we emphasize the importance of human-specific developmental features, which not only advance our understanding of brain formation but also reveal unique vulnerabilities to neurodegenerative diseases-insights that are increasingly accessible through advances in 3D organoid modeling. Together, these perspectives support a conceptual reframing of neurodegeneration as a late-onset neurodevelopmental disorder. This shift opens promising avenues for early diagnosis, prevention, and precision therapeutics, redirecting focus from late-stage intervention to fostering developmental resilience.
Evolutionary changes in cortical development were instrumental in the emergence of the mammalian neocortex. Cajal-Retzius cells, a transient neuron type discovered over a century ago, are critical players in the development of mammalian-specific cortical features such as inside-out neurogenesis. However, it is unclear whether Cajal-Retzius cells exist only in mammals or whether they are ancestral in vertebrates but acquired new functions during mammalian brain development. To trace the evolution of this cell type, we probe the presence of Cajal-Retzius cells in chicken, salamander, zebrafish, and little skate. First, through comparative transcriptomics and spatial analysis, we show the presence of cells with a Cajal-Retzius molecular identity in non-mammalian vertebrates. However, only amniote Cajal-Retzius cells gained robust expression of Reelin, a secreted glycoprotein crucial for mammalian cortical development. Second, we find that Cajal-Retzius cells are part of a larger and diverse family of neuron types and are closely related to Tp73+ external tufted cells in the olfactory bulb, which express most of the "canonical" Cajal-Retzius transcription factors. Our results indicate that Cajal-Retzius cells emerged early in vertebrate history, using a regulatory program largely shared with neurons in the olfactory system, and that transcriptomic novelties underlie their specialized roles in mammalian cortical development. We uncover an extreme example of how cell types may diverge in function over the course of evolution, even when their core transcriptomic profile is broadly preserved.
Evolutionary morphology, including comparative embryology, flourished in the 19th century, epitomized in Haeckel's recapitulation theory. However, it subsequently declined, largely due to accumulating evidence of caenogenesis, while embryology's mainstream gradually shifted toward developmental mechanics-experimental embryology-which laid the foundation for modern developmental biology grounded in reductionist and mechanistic principles. A closer examination of Haeckel's scientific trajectory reveals that, even before formulating the Gastrea theory and the Biogenetisches Grundgesetz (recapitulation theory), he employed siphonophores in what may be regarded as one of the earliest examples of authentic experimental embryology, anticipating many conceptual foundations of contemporary evolutionary developmental biology (Evo-Devo). Despite its innovative contributions, Evo-Devo is not without methodological limitations. One such limitation is highlighted in Sewertzoff's theory of "secondary Archallaxis," logically coherent within the framework of heterochrony, which presupposes an evolutionary process characterized by a persistent search for stable creodes culminating in novel phenotypes-an idea resonant with Waddington's genetic assimilation and Schmalhausen's stabilizing selection. At the same time, the theory underscores the intrinsic difficulty, if not impossibility, of precisely identifying the timing of shifts in developmental programs, a challenge that may constitute a critical vulnerability of the Evo-Devo paradigm. The future advancement of Evo-Devo will depend on the development of methodologies capable of visualizing embryonic developmental pathways and their dynamic transformations.
Body axis formation was a pivotal innovation in animal evolution, providing the spatial framework necessary for organized multicellularity. While the genetic basis of axis specification is well established in bilaterians, how axial organization emerged in early-diverging metazoans remains unresolved. Here, we address this question in Trichoplax adhaerens, a placozoan representing one of the simplest extant animal body plans. We show that Wnt signaling, a conserved regulator of axial patterning, exhibits polarized expression enriched in the peripheral region of this morphologically simple organism. Functional perturbations demonstrate that Wnt activity promotes peripheral cell proliferation and maintains central-peripheral tissue balance. Transcriptomic profiling further reveals distinct molecular identities along this axis, resembling the oral-aboral polarity of cnidarians. Together, our findings uncover a Wnt-dependent axial system in placozoans and support the view that core components of metazoan body axis patterning were already established in early animal evolution.
Thyroid hormones (THs) are essential for development, growth, and metabolism in animals. Although TH synthesis is well described in vertebrates, it remains elusive in invertebrates due to the lack of identified thyroglobulin (TG) orthologs, the protein precursor for TH synthesis. Here, we identified a functional TG-like protein in ascidian Styela clava via immunoprecipitation-mass spectrometry combined with phylogenetic and expression analyses. In vitro iodination demonstrated that ScTG-like provides hormonogenic sites for TH synthesis. In vivo ScTg-like knockdown significantly reduced THs and inhibited larval metamorphosis. An invaginated follicle-like structure in the larval trunk, deposited with ScTG-like, was identified as the location for THs synthesis and storage. Furthermore, structural analysis of ScTG-like and predicted TG-like proteins across bilaterian phyla suggests that endogenous TH synthesis may be an ancestral and synapomorphic bilaterian trait. This study reports the identification of a TH precursor outside vertebrates, shedding lights on the evolution of the TH synthesis.