EvoDevo

Despite an abundance of gene expression surveys, comparatively little is known about Hox gene function in Chelicerata, with emphasis on the Hox logic of the anterior prosomal segments, which bear the mouthparts. Previous investigations of individual paralogs of labial (lab) and Deformed (Dfd) in the spider Parasteatoda tepidariorum have shown that these play a role in tissue maintenance of the pedipalpal segment (labial-1) and in patterning the first walking leg identity (Deformed-1), respectively. However, broader extrapolations of these data points across chelicerates are hindered by the existence of duplicated copies of Hox genes in arachnopulmonates (e.g., spiders and scorpions), which have resulted from an ancient whole genome duplication event. Here, we investigated the function of single-copy orthologs of lab in the harvestman Phalangium opilio, an exemplar of a lineage that was not subject of this whole genome duplication. Embryonic RNAi against lab resulted in homeotic transformations of pedipalps to chelicerae, as well as reduction and fusion of the pedipalpal segment with adjacent segments. To test for combinatorial function, we performed double knockdown of lab and Dfd, which results in homeotic transformation of both pedipalps and first walking legs into cheliceral identity, whereas the second walking leg is transformed into a pedipalpal identity. Taken together, these results elucidate a model for the Hox logic of head segments in Chelicerata. To substantiate the validity of this model, we additionally performed expression surveys for duplicated copies of lab and Dfd in scorpions and horseshoe crabs, toward understanding the genetic basis of a heteronomous prosoma. We show that repetition of morphologically similar appendages is correlated with uniform expression levels of the Hox genes lab and Dfd, irrespective of the number of gene copies.

Botrylloides diegensis is a colonial ascidian that has been the focus of developmental, evolutionary, and regeneration research. In this study, we performed single-cell RNA sequencing (scRNA-seq) of an entire B. diegensis colony, including zooids, buds, and vascular tunics, to resolve cellular heterogeneity and identify cell and tissue markers. We identified 29 major cell clusters within the colony and used in situ hybridization to examine the spatial expression of cluster marker genes. Numerous tissue types were identified at the molecular level, including blood cells and zooid tissues such as the branchial epithelium, stomach, and endostyle. Distinct cluster markers were identified for specific regions of the stomach epithelium, highlighting the specialization of these regions and the strength of using scRNA-seq to explore their functionality. Cell trajectory projections highlighted the early appearance of progenitor clusters, whereas more differentiated zooid-related tissues appeared later in the developmental path. This study provides a valuable resource for understanding the development, tissue function, and regeneration of B. diegensis. This demonstrates the power of scRNA-seq to define cell types and tissues in complex colonial organisms. Summary statementSingle-cell RNA sequencing of Botrylloides diegensis revealed cellular heterogeneity, identified 29 major cell clusters, and provided insights into tissue specialization and blastogenesis.

Odontodes, i.e., teeth and tooth-like structures, consist of a pulp cavity and dentine covered by a mineralized cap. These structures first appeared on the outer surface of vertebrate ancestors and were repeatedly lost and gained across vertebrate clades; yet, the underlying genetic mechanisms and trajectories of this recurrent evolution remain long-standing mysteries. Here, we established suckermouth armored catfish (Ancistrus sp.; Loricariidae), which have uniquely evolved dermal odontodes (dermal denticles) all over most of their body surface, as an experimental model animal amenable to genetic manipulation for studying odontode development. Our histological analysis showed that suckermouth armored catfish develop dermal denticles through the previously defined odontode developmental stages. De novo transcriptomic profiling identified the conserved odontode genetic regulatory network (oGRN) as well as unique expression of paired like homeodomain 2 (pitx2), previously characterized as an early regulator of oGRN in teeth, in developing dermal denticles. Knockdown of pitx2 perturbed formation of the epithelial placode of dermal denticles and altered expression oGRN genes. By comprehensively identifying the genetic program for dermal odontode development in suckermouth armored catfish, this work illuminates how dermal odontodes independently evolved and diverged in distinct teleost lineages. Summary statementCranial dermal denticles in suckermouth armored catfish develop via an evolutionarily conserved and unique odontode genetic regulatory network.

To cope with extreme environmental conditions diverse marine species have developed mechanisms that allow them to permanently or temporarily attach to substrates. In the intertidal zone of marine habitats, where tidal ranges and currents may drift organisms away from their habitat, temporary adhesive systems such as the one inherent the arrow worm Spadella cephaloptera (Chaetognatha) constitute an essential trait for the survival of this taxon. The underlying molecular mechanism of this system has not been described yet, and the existing morphological information is limited to adults. Furthermore, a relationship between the nervous system and the attachment in S. cephaloptera remains to be demonstrated. In this study, single-nuclei sequencing of S. cephaloptera hatchlings was performed, using as a reference a newly sequenced and assembled genome to identify the transcriptomic profiles of the cells mediating attachment, neuronal populations, and the main cell types of chaetognath hatchlings. Our findings, supported by previous studies, suggest that the chaetognath adhesive system evolved convergently to those of other other metazoans. Moreover, diverse cell types were identified in the ventral nerve center and multiple ciliated cell types previously described from anatomical observations were validated. Ongoing in-depth investigation of these data, together with datasets from other developmental stages, will provide further insights into the evolutionary origins of the unique chaetognath body plan.

Minicollagens are major structural components in the biogenesis of nematocysts in Cnidaria. Sequence mining and recent proteomic analysis of polar capsules, homologues of cnidarian nematocysts, have confirmed the presence of minicollagens in this evolutionarily ancient cnidarian endoparasitic group. Nonetheless, the role of nematocyst-associated proteins in polar capsule morphogenesis has never been studied in myxozoans. Here, we report the gene expression of three myxozoan minicollagens, ncol-1, ncol-3, and the recently identified ncol-5, during the intrapiscine development of Myxidium lieberkuehni, the myxozoan parasite of Northern pike Esox lucius. Moreover, we determined the abundance and localisation of Ncol-1 and Ncol-5 proteins in the developing myxozoan stages by western blotting and by immunofluorescence and immunogold electron microscopy. We found that expression of minicollagens was spatiotemporally restricted to developing polar capsules in sporogonic stages. Intriguingly, Ncol-1 and Ncol-5 were localised as major components of the polar capsule wall and polar tubule. These results support the common origin of nematocysts and myxozoan polar capsules. Furthermore, our findings have practical implications for a more accurate identification of developmental stages of myxozoan parasites.

As a highly diverse phyla, Mollusca is both intriguing to study and difficult to define. No single common feature distinguishes the phyla, but the most common and recognizable is the radula. Throughout Molluscan history, there have been many developments to the radular structure. Evolutionary development of unique radular structures may have been made possible by developmental modularity. This histological study examines the development of polyplacophoran internal gut development. Here, I describe the developmental sequence of chiton feeding structures for the first time. Feeding structures are present by 10 days post hatch. Future research should examine larval stages prior to this in order to determine whether developmental modularity exists in polyplacophorans.

Evolution of nervous systems is a long debated topic, and similar mechanisms of conditional neural specification linked to dorsal-ventral (D-V) axis formation across some taxa have been used to support homology. We tested for autonomous versus conditional neural specification in two distantly related annelids, Capitella teleta and Platynereis dumerilii, using blastomere isolations. Our results support previous work in C. teleta and further demonstrate that the autonomous specification of anterior neural tissue and for the first time in trunk neural tissue for both annelids. In C. teleta, we found evidence for conditional pro-neural and anti-neural signals for the VNC. Animal caps lacking vegetal macromeres at the 16-cell stage form a brain and a D-V axis but not a VNC while the addition of any single macromere rescues VNC fate. This suggests that animal micromeres other than 2d produce an anti-neural signal while a pro-neural signal is produced vegetally and that VNC specification is decoupled from D-V axis formation. Taken together, our study suggests possible conservation of autonomous specification of the brain and VNC within Annelida, raising interesting questions of how mechanisms controlling neural specification evolved in Spiralia.

1.The evolutionary origins and body plan diversification within bilaterians hinge on our understanding of conserved developmental gene networks across diverse taxa. While significant advances have been made in elucidating the anterior-posterior (AP) axis patterning across well-studied bilaterian lineages, our understanding of the conservation of AP-patterning gene expression in underexplored protostomes with a phylogenetically informative position remains limited. Chaetognaths, a group of marine invertebrates, form a clade with Gnathifera as a sister to the remaining Lophotrochozoa, occupying an early-diverging branch of the spiralian lineage. Their phylogenetic position provides potentially valuable evolutionary insights into whether the AP patterning reflects conserved bilaterian mechanisms or reveals distinct lineage-specific adaptations. Here, we investigate the expression patterns of anterior nervous system markers (otx, nk2.1, six3/6) and Hox genes in post-embryonic stages of the chaetognath Spadella cephaloptera using fluorescence whole-mount in situ hybridization. We identify expression domains of anterior-patterning genes in the cerebral ganglion and head structures, consistent with their conserved role in anterior central nervous system (CNS) specification in bilaterians. Additionally, we describe a staggered expression pattern of Hox genes, including previously undescribed central (Sce-med6) and posterior class (Sce-postC and Sce-postD), along the ventral nerve cord (VNC) and post-anal tail. Our results demonstrate that chaetognaths exhibit the most extensive repertoire of Hox genes among protostomes, within metazoans only surpassed by chordates. All AP patterning genes are expressed in a staggered manner, with Hox gene expression absent in the head region. This pattern resembles the conserved expression profile inferred for the last common bilaterian ancestor and is only rudimentarily visible in other spiralians, including annelids and mollusks. Posterior Hox genes including the newly discovered postC and postD genes are absent in the hitherto investigated gnathiferan sister groups such as rotifers. The absence of a postanal tail in rotifers and other gnathiferans, combined with the expression of posterior Hox genes in the elongated postanal tail region, suggests their involvement in the formation of this unique chaetognath structure. Posterior flexibility of Hox genes, as previously hypothesized for chordates, likely contributed to the formation of the chaetognath tail during the early Cambrian period.

Gametogenesis is the process by which germ cells differentiate into mature sperm and oocytes, cells essential for sexual reproduction. The sex-specific molecular programs that drive spermatogenesis and oogenesis can also serve as sex identification markers. Platynereis dumerilii is a research organism that has been studied in many areas of developmental biology. However investigations often disregard sex, as P. dumerilii juveniles lack sexual dimorphism. The molecular mechanisms of gametogenesis in the segmented worm P. dumerilii are also largely unknown. In this study, we used RNA sequencing to investigate the transcriptomic profiles of gametogenesis in P. dumerilii juveniles. Our analysis revealed that sex-biased gene expression becomes increasingly pronounced during the advanced developmental stages, particularly during the meiotic phases of gametogenesis. We identified conserved genes associated with spermatogenesis, such as dmrt1, and a novel gene psmt, that is associated with oogenesis. Additionally, putative long non-coding RNAs were upregulated in both male and female gametogenic programs. This study provides a foundational resource for germ cell research in P. dumerilii, markers for sex identification, and offers comparative data to enhance our understanding of the evolution of gametogenesis mechanisms across species. Summary statementThis study provides insights into the mechanisms of gametogenesis in Platynereis dumerilii through comparative transcriptomics, unveiling sex-biased genes, including conserved and novel genes, governing this largely unexplored process.

Larvacean tunicates feature a spectacular innovation not seen in other animals - the trunk oikoplastic epithelium (OE). This epithelium produces a house, a large and complex extracellular structure used for filtering and concentrating food particles. Previously we have shown that several homeobox transcription factors may play a role in patterning the OE. Among these are two Pax3/7 duplicates that we named Pax37A and Pax37B. The vertebrate homologs, PAX3 and PAX7, are involved in developmental processes related to neural crest and muscles. In the ascidian tunicate Ciona robusta, Pax3/7 has been given a role in development of cells deriving from the neural plate border including trunk epidermal sensory neurons and tail nerve cord neurons as well as in neural tube closure. Here we have investigated the roles of Pax37A and Pax37B in the development of the OE using CRISPR-Cas9, analyzing scRNA-seq data from wild-type animals that were compared with scRNA-seq data from C. robusta. We revealed that Pax37B but not Pax37A is essential for the differentiation of cell fields that produce the food concentrating filter of the house: the anterior Fol, giant Fol and Nasse cells. Lineage analysis supports that expression of Pax37 is under influence of Wnt signaling and that Fol cells have a neuroepithelial-like transcriptional signature. We propose that the highly specialized secretory epithelial cells of the Fol region either maintained or evolved neuroepithelial features as do "glue" secreting collocytes of ascidians. Their development seems to be controlled by a GRN that also operates in some ascidian neurons.

BackgroundThe evolutionary origins of animal nervous systems remain contentious because we still have a limited understanding of neural development in most major animal clades. Annelids -- a species-rich group with centralised nervous systems -- have played central roles in hypotheses about the origins of animal nervous systems. However, most studies have focused on adults of deeply nested species in the annelid tree. Recently, Owenia fusiformis has emerged as an informative species to reconstruct ancestral traits in Annelida, given its phylogenetic position within the sister clade to all remaining annelids. MethodsCombining immunohistochemistry of the conserved neuropeptides FVamide-lir, RYamide-lir, RGWamide-lir and MIP-lir with gene expression, we comprehensively characterise neural development from larva to adulthood in Owenia fusiformis. ResultsThe early larval nervous system comprises a neuropeptide-rich apical organ connected through peripheral nerves to a prototroch ring and the chaetal sac. There are seven sensory neurons in the prototroch. A bilobed brain forms below the apical organ and connects to the ventral nerve cord of the developing juvenile. During metamorphosis, the brain compresses, becoming ring-shaped, and the trunk nervous system develops several longitudinal cords and segmented lateral nerves. ConclusionsOur findings reveal the formation and reorganisation of the nervous system during the life cycle of O. fusiformis, an early-branching annelid. Despite its apparent neuroanatomical simplicity, this species has a diverse peptidergic nervous system, exhibiting morphological similarities with other annelids, particularly at the larval stages. Our work supports the importance of neuropeptides in animal nervous systems and the evolution of biphasic life cycles.

Comparing head development among arthropods has helped identify ancestral aspects of brain patterning and structure in these animals and more broadly. Most understanding of arthropod head patterning has been learned from insects and the myriapod Strigamia maritima. Chelicerates represent an outgroup to mandibulate arthropods and can provide a valuable perspective to arthropod evolution and development. We assayed the expression of key markers of head patterning and neurosecretory centres in mandibulates in the pre-cheliceral region of embryos of the spider Parasteatoda tepidariorum. We found that, like mandibulates, this spider likely has a pars intercerebralis, marked by six3.2 and visual system homeobox/chx. We also found some evidence for another neurosecretory centre, the pars lateralis, marked by six3.2 and fasciclin 2. Furthermore, we identified anterior-medial cells in the spider pre-cheliceral region that express six3.2, foxQ2, and collier1, suggesting they may be pioneer neurons. However, these spider cells do not appear to be equivalent to the central pioneer neuronal cells identified in S. maritima because they lack expression of other key markers. Taken together, our study of spider pre-cheliceral region patterning adds a new chelicerate perspective to understanding the development and evolution of the arthropod head.

Planarian flatworms undergo continuous internal turnover, wherein old cells are replaced by the division progeny of adult pluripotent stem cells known as neoblasts. How dynamic cell turnover is executed at the organismal scale remains an intriguing question in planarians and biological systems in general. While previous studies have predominantly focused on neoblast proliferation, little is known about the processes that mediate cell loss during tissue homeostasis. Here, we use the planarian epidermis as a model to study the mechanisms of cell removal in Schmidtea mediterranea. We established a covalent dye-labeling assay and image analysis pipeline to quantify the cell turnover rate in the planarian epidermis. Our findings indicate that the ventral epidermis is highly dynamic, with a half-life of the constituent cells of approximately 4.5 days. Using live-imaging and pulse-chase assays, we find that epidermal cells undergo internalization via basal extrusion, followed by a migration towards the intestine and ultimately digestion by intestinal phagocytes. Overall, our studies reveal an intricate homeostatic cell clearance process that may reduce the metabolic costs of high turnover tissues in planarians.

The colonial tunicate Botryllus schlosseri regenerates weekly through a cyclical process in which adult zooids are replaced by a new generation of buds. While this dynamic asexual development is a hallmark of the species, its molecular regulation remains poorly understood. This study presents the first comprehensive proteomic analysis of B. schlosseri blastogenesis at the individual zooid level, using data-independent acquisition mass spectrometry to quantify protein abundance across developmental stages. The results reveal extensive proteome remodeling between proliferating buds and degenerating zooids. Co-expression analysis identified stage-specific protein modules enriched for biosynthesis and cell cycle pathways in buds, and for apoptosis, catabolism, and metabolic remodeling in zooids. A focused comparison between takeover buds and takeover zooids uncovered distinct regulatory programs controlling proliferation and senescence. Key proteins, including CDK1, CDK2, HDAC2, and PCNA, were identified as candidate regulators of cell cycle progression. These findings provide a molecular framework for understanding regeneration in a basal chordate and offer protein targets that may enable cell cycle re-entry and long-term culture of tunicate primary cells. Summary StatementThis study maps proteome dynamics during the blastogenic cycle in Botryllus schlosseri, identifying candidate proteins that regulate cell proliferation and offer targets for tunicate cell line development.

Tapeworms are parasitic flatworms that lack key features conventionally used to define the head-tail axis in free-living organisms, resulting in long standing questions regarding the true orientation of their main body axis. As adults, most tapeworms also exhibit a segmented body which has been considered an adaptation unique to the group. Anteroposterior (AP) patterning in free-living flatworms is controlled by {beta}-catenin-dependent Wnt signalling and positional control genes are expressed by their musculature in highly regionalised domains. Here we investigate the expression of Wnt and Hedgehog components during the strobilar phase of the tapeworm life cycle during which larval tissues are lost and replaced through the continuous production of new tissues. Results reveal previously unidentified centres of signalling associated with their neuromuscular system and show that segments are marked by secondary, AP axes in agreement with the polarity of the primary body axis. Neuromuscular expression and communication between Wnt and Hedgehog signalling is consistent with embryonic and regenerative growth in planarians and is a common mechanism for establishing AP-polarised boundaries in a diverse range of segmented animals. Taken together our results suggest that segmentation in tapeworms represents a modified form of posterior regeneration, a common feature among flatworms.

Hox genes are transcriptional regulators that elicit cell positional identity along the anterior-posterior region of the body plan across different lineages of Metazoan. Comparison of Hox gene expression across distinct species reveals their evolutionary conservation, however their gains and losses in different lineages can correlate with body plan modifications and morphological novelty. We compare the expression of eleven Hox genes found within Streblospio benedicti, a marine annelid that produces two types of offspring with distinct developmental and morphological features. For these two distinct larval types, we compare Hox gene expression through ontogeny using HCR (hybridization chain reaction) probes for in-situ hybridization and RNA-seq data. We find that Hox gene expression patterning for both types is typically similar at equivalent developmental stages. However, some Hox genes have spatial or temporal differences between the larval types that are associated with morphological and life-history differences. This is the first comparison of developmental divergence in Hox genes expression within a single species and these changes reveal how body plan differences may arise in larval evolution.

The evolution of visual systems has compelled numerous investigations of developmental processes underlying eye patterning across Bilateria. It is well-established that homologs of the transcription factor Pax6 play a highly conserved role in eye fate specification and are at the top of the retinal determination gene network (RDGN) hierarchy. In insects, the two Pax6 homologs eyeless (ey) and twin of eyeless (toy) are required for the development of the two visual systems broadly found within the phylum (i.e., median and lateral eyes). Curiously, Pax6 homologs do not appear to maintain this function in well-studied chelicerate models, with emphasis on spiders, a lineage of arachnids with great diversity of eye form and acuity. It was recently proposed that the gene Pax2 (shaven; sv) may have subsumed the role of eye fate specification in chelicerates, a hypothesis predicated upon the observation that one of two spider Pax2 copies is strongly expressed in the developing lateral eyes during embryogenesis. However, no functional data are available for any Pax homologs across Chelicerata. We examined the incidence of Pax family genes across Chelicerata, as well as interrogated the expression and function of Pax2 and Pax6 homologs in the daddy-longlegs Phalangium opilio, an arachnid recently discovered to bear a highly plesiomorphic arrangement of visual systems. Here, we show that ey and toy are expressed early in the developing head lobes of P. opilio, whereas sv is not expressed until well after stages when downstream RDGN members (eyes absent and sine oculis) are already activated. Gene silencing of ey, toy, and sv individually had no discernible effect on eye development. By contrast, double knockdown of ey and toy resulted in an array of median eye defects, spanning loss of some cells of the eye to total loss of the median eyes. Gene expression assays also showed that depletion of the two Pax6 copies resulted in failure of the vestigial median and vestigial lateral eyes. These data are consistent with a conserved role for Pax6 homologs in patterning both visual systems and all three eye pairs in the daddy-longlegs. Our results comprise the first functional data for Pax6 genes in any chelicerate and suggest that heterochronic shifts in expression, rather than changes in function, underlie the atypical dynamics of Pax genes in derived arachnid groups such as spiders.

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 development is characterized by widespread proliferative activity across ectodermal and mesodermal tissues, which becomes progressively compartmentalized as the larva matures. Two structured ring-shaped posterior proliferative zones, pre- and post-telotrochal, emerge within the telotroch and persist through metamorphosis, supporting both larval elongation and the juvenile development of ascending gut branch. In contrast, the metasomal sac and future adult trunk epidermis expand via broadly distributed epithelial proliferation, without forming a localized growth zone. This suggests that P. harmeri combines conserved features, such as a posterior growth zone, with lineage-specific innovations in regional growth. In addition, we identify atypical mitotic characteristics in this species, including unconventional metaphase organization and signs of interkinetic nuclear migration in larval epithelia. Our findings highlight the coexistence of ancestral and derived proliferative mechanisms in phoronids and provide new insights into the evolution of axial elongation and morphogenetic compartmentalization in Lophotrochozoa.

Arthropods (insects, "crustaceans", myriapods, and chelicerates) display a fascinating diversity of ectodermal structures like plates, horns, helmets, knobs, carapaces, mimicry outgrowths, and wings. The origins and relationships of these structures has tantalized researchers for over a century: did these structures arise de novo in each lineage, or do they emerge from shared, ancestral primordia present in some or even all arthropods? One way to begin answering this is to assess the position and context of these structures on arthropod bodies: do these structures emerge from proximal embryonic leg segments that were converted into the body wall, or do they represent dorsal, non-leg-derived structures? Here, the expression of pannier, araucan, and drumstick - genes previously shown to distinguish proximal leg segments in crustaceans and insects - are examined in a chelicerate representative, the tarantula Acanthoscurria. This gene expression comparison, together with over a century of gene functional, morphological, embryological, and paleontological data, suggests that all arthropod leg segments correspond to each other in a one-to-one fashion, but that in many arthropod lineages, the base of the ancestral leg in the embryo flattens and expands to form the body wall of the adult. This would mean that many arthropod outgrowths which appear to stand on the lateral body wall, such as wings, tergal plates, and gin traps, are derived from the ancestral/embryonic leg base, and likely arose from shared, ancestral primordia present on this leg base in all arthropods. The analysis detailed here means that a simple three- or four-gene in situ expression experiment with pannier, araucan, and drumstick can elucidate the homologies of any arthropod ectodermal structure of interest, including beetle horns, treehopper helmets, and more.

Venom is a remarkable innovation found across the animal kingdom, yet the evolutionary origins of venom systems in various groups, including spiders, remain enigmatic. Here, we investigated the organogenesis of the venom apparatus in the common house spider, Parasteatoda tepidariorum. The venom apparatus consists of a pair of secretory glands, each connected to an opening at the fang tip by a duct that runs through the chelicerae. We performed bulk RNA-seq to identify venom gland-specific markers and assayed their expression using RNA in situ hybridisation experiments on whole-mount time-series. These revealed that the gland primordium emerges during embryonic stage 13 at the chelicera tip, progresses proximally by the end of embryonic development and extends into the prosoma post-eclosion. The initiation of expression of an important toxin component in late postembryos marks the activation of venom-secreting cells. Our selected markers also exhibited distinct expression patterns in adult venom glands: sage and the toxin marker were expressed in the secretory epithelium, forkhead and sum-1 in the surrounding muscle layer, while Distal-less was predominantly expressed at the gland extremities. Our study provides the first comprehensive analysis of venom gland morphogenesis in spiders, offering key insights into their evolution and development.