Blastoderm formation represents a key transition from a syncytial to a cellular embryo and provides the basis for subsequent embryonic patterning in insects. In most insect models, this transition occurs through synchronous cellularization, producing a uniform blastoderm that is patterned only afterward. Whether this sequence represents a common developmental principle across insects remains unclear. Here, we show that in the carpenter ant Camponotus floridanus, cellularization proceeds progressively rather than synchronously and establishes spatially differentiated blastoderm domains. Cellularization initiates at the anterior and progresses posteriorly, while a second front from the posterior advances in the opposite direction. These opposing fronts converge at the site where the germline capsule subsequently forms. At the same time, a regionalized blastoderm rather than a uniform blastoderm is established. The extraembryonic tissues amnion, serosa, and trophocytes emerge along progressive cellularization through coordinated morphogenetic dynamics and exhibit a distinct mode of organization. Our data present a revised understanding of cellularization of blastoderm in insects and broaden the comparative framework of embryogenesis described in model organisms.
Rupert Riedl showed in his "Order in Living Organisms" that morphology can produce law statements and is, therefore, a proper, that is, nomothetic, science. Furthermore, he coined useful terms (interphene and metaphene) and concepts (burden, cadre and minimal homology). Notwithstanding certain flaws-he used "laws in evolution" and "verification of a theory" although claiming to be in accordance with Popper's hypothetico-deductivism-and an almost complete absence of synecological relationships in his evolutionary explanations, his scientific legacy still forms a basis of a "logic of morphology" and an "evolutionary morphology," especially through the works of his students.
Asymmetry in a bilateral organism refers to the difference in the expression of a trait between right and left sides, which may result from genetic or environmental disturbances. Using geometric morphometrics and a phylogenetic approach, we studied asymmetry in dorsal and ventral views of the skulls of anuran species, representing 22 families, 16 of which belong to the Hyloidea clade. The cranial regions with the most pronounced shape asymmetry were identified. To discern species with elevated levels of asymmetry and to hypothesize its evolutionary trends across phylogeny we implemented the asymmetry index. Significant asymmetric skull shape variation was found between right and left sides, associated with the upper area of the mandibular joint and the anterior area of the nasals in dorsal view. In ventral view, the greatest variation was in the vomers and the ala of the parasphenoid. The degree of size-related asymmetry varied among species. Character mapping results indicate that cranial asymmetry is a conserved and widespread trait in the Hyloidea clade, representing an ancestral condition across both dorsal and ventral regions of the skull. The observed asymmetries were found in traits that develop later in their ontogeny, which would imply that as they establish fewer developmental dependencies with other traits and, consequently, are less phylogenetically constrained. The asymmetries we found belong to this category and would be more prone to change during evolution.
The ParaHox homeobox genes Gsx, Xlox, and Cdx are evolutionarily related to Hox genes and form part of the ANTP-class homeobox gene repertoire. Comparative genomic data indicate that ParaHox genes were already present before the cnidarian-bilaterian split. Across metazoans, ParaHox genes often show conserved associations with the anteroposterior body axis and have been implicated in both gut patterning and neural development, although gene complements and genomic organization vary substantially among lineages. To investigate ParaHox gene deployment in Gnathifera, we identified orthologs of Gsx and Cdx across gnathiferan lineages, including Chaetognatha, Monogononta, Bdelloidea, Seisonidea, and Acanthocephala, but found no evidence of Xlox, indicating a reduced ParaHox gene complement. We analyzed the genomic organization and embryonic expression of Gsx and Cdx in the monogonont rotifer Brachionus manjavacas (Bm). Genomic mapping revealed a dispersed ParaHox configuration, with Bm-Gsx and Bm-Cdx separated by 4.4 Mb. Using whole-mount in situ hybridization, we detected Bm-Gsx expression in neurons of the foot region, as well as in a small number of cells with neuronal characteristics and probable involvement in stomatogastric system development. Bm-Cdx was expressed in FMRFamide-positive cells associated with the bladder, consistent with a neuroepithelial identity. Together, these data indicate that in rotifers, ParaHox gene expression is predominantly associated with neural structures. We propose that this pattern represents a derived condition reflecting the compact body plan and reduced gut organization characteristic of rotifers, highlighting the evolutionary flexibility of ParaHox gene deployment under lineage-specific developmental constraints.
The diverse and colorful patterns found in organisms serve various functions. In Pachyrhynchus weevils, these colors and patterns composed of 3D photonic architectures function as signals for sexual selection and aposematism. Previous study of photonic structure suggests the involvement of specific genes in color regulation in Pachyrhynchus weevils. However, the genes responsible for scale coloring in Pachyrhynchus weevils have remained unknown. In this study, RNA-seq was conducted in P. nobilis at 6 h and 24 h after emergence, comparing elytra (no-scale) and scale tissues during the period of coloring. qPCR validation was performed across three species ( P. nobilis , P. sarcitis , and P. orbifer ). RNA-seq analysis identified 3172 and 2890 DEGs at 6 h and 24 h after emergence, respectively, with up-regulated DEGs increasing over time. The candidate genes were selected based on functional annotations related to scale development (C-type lectins, cuticle proteins, and Yellow family proteins), and KEGG pathway analysis. qPCR validation revealed species-specific expression patterns: C-type lectin-encoding gene was expressed in P. nobilis and P. sarcitis but absent in P. orbifer , while Hedgehog-like protein-encoding gene was exclusively detected in P. orbifer . Temporal analysis showed significant differences in Wnt signaling pathway between developmental stages in P. orbifer , and marginal significance in calcium signaling pathway gene in P. sarcitis . This study suggests that these specific genes may play roles in color regulation during scale development in Pachyrhynchus weevils, with varying expression at different time points after emergence or among species.
Transcription factors are typically thought to play a limited role in developmental evolution due to their high pleiotropic nature. However, such constraints may be relaxed following gene duplication or when proteins are organized into structural and functional modules, opening avenues for evolutionary innovation. Here, we integrate expression and genomic data to investigate the evolutionary dynamics of Hox gene duplicates in the allotetraploid frog Xenopus laevis. Despite overall conservation across the Hox clusters, we find that HoxB4L has acquired expression during maternally regulated stages and is evolving under positive selection. Protein-level changes include the number, length, and sequence of functionally important protein regions. Our results indicate that HoxB4L has escaped ancestral constraints and is undergoing maternal neofunctionalization as a result of cis-regulatory divergence and structural protein modifications. These findings illustrate how transcription factors can overcome developmental constraints and contribute to novel functions during early development.
Bivalve planktonic development is a critical phase during which larvae must secrete the first calcium carbonate shell, the prodissoconch I (PD I). As PD I formation is in close contact with seawater, this process can be negatively affected by adverse seawater carbonate chemistry. It is hypothesized that bivalves can regulate shell formation under environmental stress through biologically controlled biomineralization involving a complex extracellular shell proteome. However, the plasticity of this regulatory mechanism during PD I development is unknown. We assessed the PD I shell proteome of the Hong Kong oyster (Magallana hongkongensis) in carbonate chemistry that was adverse or favorable for biomineralization to understand the regulatory capacity of larval shell formation. While survival rates were not affected in adverse carbonate chemistry, there were significant changes, including the upregulation of several calcium-binding proteins and downregulation of proton-generating processes and putative calcification inhibitors. With 198 sequences, the oyster larval shell proteome was twice to over six times larger than those reported for other bivalve species at the same developmental stage. However, in adverse carbonate chemistry, the oyster larval shells were thinner and smaller, and protein diversity decreased to 131 sequences, with overall lower functional redundancy and reduced expression of structural proteins, indicating potential trade-offs. The proteomic and shell structural data also suggest that direct cellular control and biologically induced mechanisms, which will require further investigation, may be involved in PD I formation.
Cell proliferation is a key driver of morphogenesis and body plan transformation in multicellular animals, yet its spatial organization remains poorly understood in many non-segmented spiralians. In this study, we examine the dynamics of cell division during larval growth and metamorphosis in the larvae and early juveniles of the phoronid Phoronopsis harmeri, using EdU incorporation, anti-phospho-histone H3 immunostaining, confocal laser scanning microscopy, and electron microscopy. Early larval proliferation is partly regionalized from the outset and becomes progressively more localized toward metamorphosis. We identify a tripartite organization of proliferative activity: (1) posterior ring-shaped domains in the telotroch that persist through metamorphosis and support elongation and anal chamber formation; (2) regional proliferative zones at tentacle bases, preoral and postoral regions; and (3) scattered proliferation driving the expansion of the trunk epidermis. This coexistence of posterior, regional, and scattered patterns underscores the developmental plasticity of phoronids and the diversity of growth strategies within Spiralia. Posterior proliferative domains in phoronids contribute important context to homology-convergence debates on posterior growth across spiralians, but are not decisive by themselves; viewed with the distributed epithelial proliferation, they underscore the coexistence of multiple proliferative programs within a single life cycle. In addition, we identify atypical mitotic characteristics in this species, including unconventional metaphase organization and signs of interkinetic nuclear migration in larval epithelia. Our results suggest that phoronids provide a valuable model for exploring how diverse architectures of cell proliferation contribute to larval growth, body elongation, and morphogenetic compartmentalization in Lophotrochozoa.
Experimental studies have demonstrated that nutritional changes during development can result in phenotypic changes to mammalian cheek teeth. This developmental plasticity of tooth morphology is an example of phenotypic plasticity. Because tooth development occurs through complex interactions between manifold processes, there are many potential mechanisms which can contribute to a tooth's norm of reaction. Determining the identity of those mechanisms and the relative importance of each of them is one of the main challenges to understanding phenotypic plasticity. Quantitative proteomics combined with experimental studies allow for the identification of potential molecular contributors to a plastic response through quantification of expressed gene products. Here, we present the results of a quantitative proteomics analysis of mature upper first molars in Mus musculus from a controlled feeding experiment. Pregnant and nursing mothers were fed either a low-dietary protein (10%) treatment diet or control (20%) diet. Low-dietary protein was not associated with reduced molar size or skull length. However, expression of tooth-related proteins, immune system proteins, and actin-based myosin proteins were significantly altered in our low-dietary protein proteomics sample. The differential expression of immune proteins along with systematic reduction in actin-based myosin protein expression are novel discoveries for tooth proteomics studies. We propose that studies that aim to elucidate specific mechanisms of molar phenotypic plasticity should prioritize investigations into the relationships between IGF regulation and tooth development and actin-based myosin expression and tooth development.
Taxonomically restricted genes are increasingly understood to play major roles in evolution. However, a significant body of work has taken issue with the notion of widespread "novel" genes and argued that such genes have homologs in distant clades that can be found with either sufficiently powerful alignment techniques or by using synteny to find their ancestral sequences. Here, we argue that such work is misguided. Moreover, we argue that the whole notion of genetic assignment of function (and annotation) based on historical origin violates the levels of analysis distinction between origin and current utility. The evolutionary history of a gene is so often not reflective of its current utility that naming genes based on the function of their homologs is bad practice. This is nowhere more apparent than in the case of genes that have changed so radically from their ancestors that they bear no similarity to them at the sequence or protein folding levels. We coin the term, overwriting, for this process in which selection creates novel genes by completely changing the coding sequence of a gene in a manner that does not conserve function , and argue for the general importance of this mechanism.
Fibroblast growth factor signaling plays a crucial role in various developmental processes and is a key driver of regeneration. In annelids, this pathway is active from the earliest stages of reparative morphogenesis, yet its specific function remains unclear. Here we have functionally examined FGF signaling following the amputation of posterior segments in the marine annelid Alitta virens. We utilized the pharmacological agent SU5402 to inhibit the FGF receptor kinase at different time points. With whole-mount in situ hybridization, we analyzed the expression of regulatory genes that pattern posterior territories (cdx, evx, post2), multipotent/germ cells (vasa, piwi), mesodermal tissues (twist), and segmental boundaries (engrailed). Our findings reveal that FGF signaling is essential for blastema induction by promoting dedifferentiation and proliferation of cells at the wound site, but is not involved in posteriorization. On the contrary, this pathway is crucial for differentiation in the proximal (anterior) part of the regenerative bud, impacting its mesodermal derivatives and segment boundary formation. Comparative analysis suggests that while certain functions of FGF signaling, particularly in mesodermal patterning, are conserved across taxa, its role in posterior axis elongation appears to have evolved specifically within the vertebrate lineage. This study enhances our understanding of the evolutionary origins and functional diversification of FGF signaling in regeneration, positioning A. virens as a valuable model for exploring the complexities of regenerative biology.
The impact of land-to-water transition on chemosensory genes has been explored in marine tetrapod vertebrates, with scarce data on aquatic insect lineages. Diving beetles (Dytiscidae) are predaceous freshwater insects with strictly aquatic larvae and amphibious adults. Using RNA-seq, we compared the expression of odorant receptors (ORs), gustatory receptors (GRs), ionotropic receptors (IRs), and odorant-binding proteins (OBPs) in the cephalic appendages of larval and adult Cybister lateralimarginalis . Overall, larvae expressed fewer chemosensory genes than adults but larva-specific genes displayed a unique expression pattern, not previously observed in any other holometabolous insect, with five larva-specific ORs all having a close paralogue which is adult-specific, 14 larva-specific IRs all belonging to a single gene expansion in the IR tree, and no larva-specific GR. Expression profiles across appendage types mirrored those in aerial insects, with ORs mainly in antennae, GRs in labial palps, “Antennal class” IRs in antennae, and “Divergent class” IRs in palps. This suggests that the land-to-freshwater transition in this lineage did not involve major changes in deployment of the major families of chemosensory genes among cephalic appendages. Notably, the expression of a substantial repertoire of ORs specifically in the antennae of the larva suggests that hydrophobic chemical cues are important for long-range chemodetection in freshwater, contrary to prevailing views about constraints for chemosensation within a water medium.
The present study reveals the immunolocalization of the MARCKS-like protein in two urodeles and an anuran during the initial stages of appendage regeneration. This acidic protein of 22-32 kDa interacts with the dynamic cytoskeleton of activated keratinocytes and blastema cells and is believed to be among the initial signaling factors stimulating limb regeneration in the axolotl. Bioinformatics controls indicate presence of homologous MARCKS-like proteins also in other amphibian species. The present study aims to generalize the presence of this protein during the first 2-8 days of appendage regeneration in amphibians. In the wound epidermis of the axolotl, the protein is prevalently localized in pale Leydig cells, a mucous cell type described in amphibian epidermis, many of which are present in the regenerating epidermis. A lower immunolabeling is found in the newt wound epidermis but is high in regenerating nerves. In the regenerating tail of frog tadpoles MARCKS-like immunolabeling is present in the wound epidermis, regenerating spinal cord, ganglia and nerves but also with lower intensity in myotubes and in the external layer of notochord. Low to absent MARCKS-like immunoreactivity is instead observed in the normal epidermis and in the wound epidermis of the non-regenerating tadpole limb. Although mainly cytoplasmic, also some nuclear labeling is detected in immunoreactive cells of different tissues, especially in the spinal cord, suggesting the activation of nuclear transcriptional process. The protein is present in tissues with high proliferative activity, but is low to absent in most blastema cells and connective tissues during regeneration. The study indicates that the presence of MARCKS-like protein is a general reaction that favors regeneration in amphibians and possibly also in other vertebrates.
Cetaceans exhibit remarkable wound-healing abilities, an adaptation critical for survival in their aquatic environment. This study provides the first identification of leucine-rich alpha-2-glycoprotein 1 (LRG1) in the bottlenose dolphin (Tursiops truncatus), focusing on its potential role in wound healing. LRG1, a member of the leucine-rich repeat family, is involved in immune regulation, angiogenesis, and tissue remodeling in terrestrial mammals. We extracted and analyzed LRG1 from T. truncatus to detect basic evolutionary insights on cetacean wound healing. Comparative analyses indicated that T. truncatus LRG1 exhibits similarities with its terrestrial counterparts but may offer distinct adaptational characters. These findings represent a pivotal first step toward elucidating the molecular evolutionary mechanisms of cetacean wound healing and highlight LRG1 as a promising target for future veterinary clinical applications.
Ecomorphology examines how species' morphology adapt to their environments, providing insights into biodiversity and evolution. This field relies on three main components: a morphological matrix, an ecological matrix, and phylogeny. A major challenge in contemporary anuran ecomorphology is constructing the ecological matrix, as categorizing species' ecological roles lacks a standardized methodology, leading to inconsistencies across studies and complicating comparisons. In this study, we discuss the challenges of systematizing criteria for constructing the ecological matrix in anurans. To this end, we conducted a literature search, focusing on studies that consider microhabitats as ecological categories and locomotor abilities, using relevant keywords to the topic. A total of 31 studies from the last 46 years were selected for analysis, and information was extracted on the following aspects: analyzed species; microhabitat and locomotor mode categories; and whether or not own criteria for assigning ecological categories (i.e., microhabitat and locomotor modes) were specified. The analyzed studies reveal a high degree of consistency in the assignment of ecological categories for microhabitat classification but not for locomotor modes designation. The main discrepancies occur in the burrowing and/or fossorial categories, as well as climbing. Interestingly, these categories appear both as microhabitats and as locomotor modes. Key criteria include direct field observations and assignments based on primary literature sources. The variability in category assignments and data collection criteria underscores the need to develop more standardized protocols for ecological categorization to improve the accuracy and reproducibility of ecomorphological studies.
The mechanical loads from muscle contraction and gravity affect the biomechanical properties of long-bone limbs, varying according to the functional demands of each limb. In anurans, both limbs are used for locomotion, but the hindlimbs generate higher energy for jumping or swimming, and the forelimbs serve additional purposes (e.g., landing, amplexus, feeding, etc). This study examines the bone architecture of the forelimb bones (humerus and radioulna) and the hindlimb bones (femur, tibiafibula, tibiale, and fibulare) of 24 anuran species with different habitat uses within a phylogenetic context. Also, because of functional divergence among limbs, we investigate possible divergence in morphological integration among long bones depending on habitat use. Across all species, forelimb bones show significantly higher bone biomechanical properties values than hindlimbs, with aquatic and semiaquatic species exhibiting the most resistant bones to bending and fracture. The femur and tibiafibula of aquatic, semiaquatic, and terrestrial species showed similar and higher values, while arboreal species had the lowest values. The tibiale and fibulare bones show a unique stratified pattern across habitats, and in most species, these bones have higher values than the femur and tibiafibula. Although morphological integration varies across habitats-with terrestrial species showing the highest and aquatic and arboreal species the lowest, reflecting differences in limb specialization-the tibiale and fibulare uniquely exhibit significant covariation across all species. While phylogenetic factors may contribute to the observed variability, ecological factors play a crucial role in shaping bone geometry, highlighting the evolutionary adaptations of long bone resistance across ecological niches.
During the origin of new niches, animals face novel situations and must adapt to access new resources. Innovative individuals may develop strategies and behaviors to take advantage of these resources, although these individuals often lack striking adaptations for the new niche. In these individuals, adequate performance must be achieved, even in cases where behaviors are not typical or usual, which does not necessarily imply optimal performance in terms of energy or speed, but rather the flexibility to choose a different scenario to pursue a biological purpose. Through experience, animals can improve their ability to perform complex movements and adapt to new conditions. We evaluated the existence of additional locomotor skills in a widespread anuran amphibian, Rhinella arenarum. This toad has a terrestrial niche, probably the ancestral condition within the genus. Therefore, it allows us to know the limits of these capacities to execute novel behaviors. Specifically, we analyzed whether the climbing abilities demonstrated by this terrestrial toad can be improved through learning. Adult male and female toads were tested in a climbing device during eight daily sessions. After training, animals improved climbing performance, measured by climbing latency, climbing speed, and stride frequency. The improvement by learning the ability to climb could thus represent an adaptation that allows the exploitation of arboreal niches. Our results indicate that it is possible that innovative individuals who manage to acquire and perfect the ability to climb could expand their range of available niches and, potentially, give rise to new evolutionary lines.
Biomineralization, the formation of mineralized tissues like skeletons and shells, is an essential developmental process in diverged phyla. Vertebrates' biomineralization involves the secretion of specialized extracellular matrix (ECM) proteins and the formation of Integrin-based focal adhesions, yet less is known about the role of such factors in invertebrates. A recent study has shown that focal adhesions form around the calcite spicule of the sea urchin larva, however, the skeletogenic expression and role of adhesion related proteins in this system are understudied. Here, we identified a set of ECM and adhesion genes that show enriched expression in the sea urchin skeletogenic cells and studied the role of the ECM protein, Npnt, in Paracentrotus lividus. The integrin alpha proteins, Pl-Ahi, Pl-Aji, Pl-Api, and the Pl-Talin protein are highly conserved between sea urchin and humans and the expression of these genes is enriched in the skeletogenic cells during early skeletogenesis. Pl-npnt is expressed specifically in skeletogenic cells throughout skeletogenesis and requires Vascular Endothelial Growth Factor (VEGF) signaling for its maintenance. Genetic perturbations of Pl-npnt result in skeletal defects, including reduced length of skeletal rods, ectopic spicule formation and branching, while skeletogenic cell migration remained unaffected. The activation of focal adhesion kinase (FAK) around the spicules is independent of Pl-Npnt activity in agreement with the loss of Integrin binding site in the sea urchin Npnt protein. Our findings set the stage for further analyses of ECM and adhesion-mediated mechanisms that drive sea urchin biomineralization, and most likely participate in skeletal development across metazoans.
Nemertea is a phylum of predominantly marine worms that exhibit various larval forms, including the iconic pilidium. Pelagic lecithotrophic pilidia are considered more derived than pelagic planktotrophic pilidia, but data on the structure of lecithotrophic larvae are limited to the light-optical level. Here, we study the lecithotrophic reversed Iwata's larvae of an undescribed heteronemertean, Nipponomicrura sp. Using transmission electron microscopy and confocal laser scanning microscopy with F-actin, acetylated α-tubulin, and serotonin (5-hydroxytryptamine) labeling, the provisional structures of the larva are described. The larval envelope of Nipponomicrura sp. consists of three layers: the epidermis, the circular musculature, and the epithelium of the amnion. The larval epidermis contains a considerable amount of yolk, only half of which is consumed by the end of metamorphosis. The apical plate consists of 5-hydroxytryptamine-negative cells, each bearing a cilium surrounded by a collar of eight to nine microvilli. Four monociliated 5-hydroxytryptamine-like-immunoreactivity sensory apical neurons are associated with the apical plate. For the first time, a pair of longitudinal muscles running along the body of the juvenile and joining the anterior and posterior parts of the provisional epithelium has been identified in nemertean larvae. These muscles serve as retractors of the apical plate and fix the position of the juvenile within the larva. The obtained data indicate a similar morphology of the apical organ in Pilidiophora larvae; however, in the Nipponomicrura sp. larva, there are more layers under the apical plate, and the muscle-retractor is derived from two longitudinal muscle cords that pass through the juvenile's body, and in posterior pole, attach at the base of the larval envelope.
Cilia are found on the epithelia of almost all metazoans, so their absence from the epithelia of all but one class of Porifera is puzzling. Homoscleromorph sponges possess ciliated epithelia, but their function and evolutionary history within Porifera are unclear. We compared the ciliary beat frequencies (CBFs) of cilia on outer epithelia of the homoscleromorph sponge Oscarella sp. with those of other animals to suggest possible functions for the cilia. Settled Stage 4 buds, or juveniles, were found to have a higher CBF than free-moving Stage 1 buds, and CBF was within the range of cilia that function in mucus transport in other aquatic invertebrates. Scanning Electron Microscopy (SEM) images of buds fixed with ruthenium red to detect the presence of mucus showed that mucus was associated with the cilia of the exopinacoderm and both SEM and immunofluorescence images revealed fields of homogeneously oriented cilia. Confocal imaging of fluorescent beads also showed that cilia beat in the same direction. Movement of beads was reduced by nocodazole treatment indicating that the movement of particles over the surface was caused by ciliary beat. These results suggest that cilia on the epithelia of Homoscleromorph sponges are involved in mucociliary-driven particle flux, and may be used to clean the surface using mucus.
