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Model for the evolution of the telencephalon a, Proposed model of the evolution of the vertebrate telencephalon. The telencephalon arose from the incorporation of anterior placodal gene-regulatory module into forebrain regions of the neural tube. Key regulatory components in Ciona palp sensory cells (including Dlx, FoxG and Sp8) and in the pro-anterior sensory vesicle (including Six3/6, FoxJ1, Lhx2/9 and Otp) are conserved in the vertebrate telencephalon. b, The FoxG reporter gene (Kaede, green) exhibits restricted expression in palp sensory cells but not anterior regions of the sensory vesicle of Ciona larvae (n = 2 electroporation experiments). c, In killifish, GFP driven by the Ciona FoxG regulatory sequences and a zebrafish minimal promoter is expressed in a subset of cells in the olfactory bulb of the telencephalon (arrowheads, left). n = 3 independent transgenic lines (Methods). T, telencephalon. dph, days post-hatching. Scale bars, 20 μm (b), 250 μm (c).
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Ascidian embryos highlight the importance of cell lineages in animal development. As simple proto-vertebrates, they also provide insights into the evolutionary origins of cell types such as cranial placodes and neural crest cells. Here we have determined single-cell transcriptomes for more than 90,000 cells that span the entirety of development—fro...
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... To better understand these processes, we require differential gene expression data at cellular resolution during these stages of embryogenesis. Single-cell RNA sequencing (scRNA-seq) transcriptomics can provide this to allow cell state characterisation and differentiation, and unbiased identification of key marker genes [24,[27][28][29][30]. Indeed, scRNA-seq has successfully been applied to embryos of a rapidly growing number of animals [31][32][33][34][35][36][37][38], including stage 5 and 6 P. tepidariorum embryos, which identified cells corresponding to the three germ layers and reconstructed A-P polarity and initial patterning based on known and new markers genes [39], as well as recent single cell analysis of embryonic stages 10 to 12 for this spider [40]. ...
Spiders are a diverse order of chelicerates that diverged from other arthropods over 500 million years ago. Research on spider embryogenesis, particularly studies using the common house spider Parasteatoda tepidariorum, has made important contributions to understanding the evolution of animal development, including axis formation, segmentation, and patterning. However, we lack knowledge about the cells that build spider embryos, their gene expression profiles and fate. Single-cell transcriptomic analyses have been revolutionary in describing these complex landscapes of cellular genetics in a range of animals. Therefore, we carried out single-cell RNA sequencing of P. tepidariorum embryos at stages 7, 8 and 9, which encompass the establishment and patterning of the body plan, and initial differentiation of many tissues and organs. We identified 20 cell clusters, from 18.5 k cells, which were marked by many developmental toolkit genes, as well as a plethora of genes not previously investigated. We found differences in the cell cycle transcriptional signatures, suggestive of different proliferation dynamics, which related to distinctions between endodermal and some mesodermal clusters, compared with ectodermal clusters. We identified many Hox genes as markers of cell clusters, and Hox gene ohnologs were often present in different clusters. This provided additional evidence of sub- and/or neo-functionalisation of these important developmental genes after the whole genome duplication in an arachnopulmonate ancestor (spiders, scorpions, and related orders). We also examined the spatial expression of marker genes for each cluster to generate a comprehensive cell atlas of these embryonic stages. This revealed new insights into the cellular basis and genetic regulation of head patterning, hematopoiesis, limb development, gut development, and posterior segmentation. This atlas will serve as a platform for future analysis of spider cell specification and fate, and studying the evolution of these processes among animals at cellular resolution.
Supplementary Information
The online version contains supplementary material available at 10.1186/s13227-024-00224-4.
... s. ZnMc-astacin-like domain (green) and Zinc-binding metalloprotease motif (orange) are presented in Nas15 in diverse species. (C) Protein sequence alignment of Nas15 ZnMc-astacin-like domain from diverse species. In the Zinc-binding metalloprotease motif region of Nas15 from diverse species, the amino acid sequence HEXXHXXGXXH is highly conserved.(Cao et al., 2019;Tarhan et al., 2023) ( ...
Lumen formation, as a key process of biological tube construction, is essential in various physiological processes such as nutrient and waste transporting, gas exchanging, and structural supporting. However, the mechanisms underlying tubular lumen development are still not fully understood. In the present study, we identified a matrix metalloproteinase, Nas15, which is enriched in the apical domain of the Ciona embryonic notochord. The expression level of the Nas15 gene significantly increased during notochord lumen formation and expansion. Nas15 loss-of-function resulted in abnormal notochord lumen expansion in Ciona embryos. Besides, yeast two-hybrid screening and CO-IP results indicated a Phosphatase 2 Catalytic Subunit Alpha (PPP2CA) physically interacted with Nas15. PPP2CA also involved in notochord lumen formation via localizing Nas15. Furthermore, we investigated the distribution of laminin in Nas15 disrupted embryos. In conclusion, our results revealed a mechanisms of how notochord cells regulating lumen expansion via metalloproteinase-mediated ECM localization. This findings provide insight into the mechanisms of tubular organ lumen formation and serve as a reference for research on human abnormal lumenogenesis diseases.
... Although homology of ascidian cells producing sensory bipolar tail neurons and vertebrate neural-crest cells is controversial 10 , it is likely that the last common ancestor of vertebrates and ascidians had cells resembling vertebrate neural-crest cells. Ascidian neural-crest-like cells identified so far do endodermal-strand cells in single-cell RNA sequencing (scRNA-seq) data published previously 34 (Supplementary Table 1). Among these genes, we confirmed that Hebp-r.a is indeed expressed in putative pLNPC-derived nerve-cord and endodermal-strand cells by in situ hybridization (Fig. 2b). ...
... Subsequently, some of them changed their location to the ventral side and these cells were finally located in the posterior part of the endodermal strand ( Fig. 2d and Extended Data Fig. 2). Using scRNA-seq data 34 , we compared expression profiles of pLNPC-derived nerve-cord cells and endodermal-strand cells with those of other cell types during the larval stage and found that the transcriptome of pLNPC-derived nerve-cord cells and endodermal-strand cells was quite different from those of the other endodermal-strand and nerve-cord cells and of other tissues (Fig. 2e). This was further supported by the observation that pLNPC-derived nerve-cord cells and endodermal-strand cells did not express a pan-neural marker, Celf3.a ...
... Next, we examined how Tbx6-r.b is activated in b9. 34. Previous studies have shown that orthologues for genes specifying neural plate border cells and neural-crest cells in vertebrate embryos are expressed in cells including LNPCs of ascidian embryos 30,36,37,40,41 . ...
Neural-crest cells and neuromesodermal progenitors (NMPs) are multipotent cells that are important for development of vertebrate embryos. In embryos of ascidians, which are the closest invertebrate relatives of vertebrates, several cells located at the border between the neural plate and the epidermal region have neural-crest-like properties; hence, the last common ancestor of ascidians and vertebrates may have had ancestral cells similar to neural-crest cells. However, these ascidian neural-crest-like cells do not produce cells that are commonly of mesodermal origin. Here we showed that a cell population located in the lateral region of the neural plate has properties resembling those of vertebrate neural-crest cells and NMPs. Among them, cells with Tbx6-related expression contribute to muscle near the tip of the tail region and cells with Sox1/2/3 expression give rise to the nerve cord. These observations and cross-species transcriptome comparisons indicate that these cells have properties similar to those of NMPs. Meanwhile, transcription factor genes Dlx.b, Zic-r.b and Snai, which are reminiscent of a gene circuit in vertebrate neural-crest cells, are involved in activation of Tbx6-related.b. Thus, the last common ancestor of ascidians and vertebrates may have had cells with properties of neural-crest cells and NMPs and such ancestral cells may have produced cells commonly of ectodermal and mesodermal origins.
... Cells from 64-cell, 112-cell, and early-neurula stage embryos of forward cross hybrids were clustered into 9, 12, and 19 clusters, respectively, using uniform manifold approximation and projection (UMAP) for dimension reduction method. Cell cluster identities were annotated as endoderm, notochord, mesenchyme, muscle, nerve cord, germ, neural, and epidermis ( 30,31 . ...
Zygotic genome activation (ZGA) is a universal process in early embryogenesis of metazoan, when the quiescent zygotic nucleus initiates global transcription. However, the mechanisms related to massive genome activation and allele-specific expression (ASE) remain not well understood. Here, we develop hybrids from two deeply diverged (120 Mya) ascidian species to symmetrically document the dynamics of ZGA. We identify two coordinated ZGA waves represent early developmental and housekeeping gene reactivation, respectively. Single-cell RNA sequencing reveals that the major expression wave exhibits spatial heterogeneity and significantly correlates with cell fate. Moreover, allele-specific expression occurs in a species- rather than parent-related manner, demonstrating the divergence of cis-regulatory elements between the two species. These findings provide insights into ZGA in chordates.
... We searched Ciona robusta (i.e., intestinalis Type A) whole-larva scRNAseq data [39] for evidence of the cell types described by transmission electron microscopy (TEM) of the papillae [9]. While a cell cluster annotated as "Palp Sensory Cells" (PSCs) appeared enriched for known markers of ACCs like CryBG (KH.S605.3) ...
... " Fig 2A). Co-electroporation with the papilla-specific Foxg>mCherry reporter [39] showed clear, mutually exclusive expression between the 2 reporters. We propose that L96.43 marks a population of "peri-papillary" and/or "inter-papillary" cells previously identified as "basal cells" that are part of the larger papilla region but excluded from the 3 protruding, Foxg+ papillae sensu stricto [9]. ...
The papillae of tunicate larvae contribute sensory, adhesive, and metamorphosis-regulating functions that are crucial for the biphasic lifestyle of these marine, non-vertebrate chordates. We have identified additional molecular markers for at least 5 distinct cell types in the papillae of the model tunicate Ciona , allowing us to further study the development of these organs. Using tissue-specific CRISPR/Cas9-mediated mutagenesis and other molecular perturbations, we reveal the roles of key transcription factors and signaling pathways that are important for patterning the papilla territory into a highly organized array of different cell types and shapes. We further test the contributions of different transcription factors and cell types to the production of the adhesive glue that allows for larval attachment during settlement, and to the processes of tail retraction and body rotation during metamorphosis. With this study, we continue working towards connecting gene regulation to cellular functions that control the developmental transition between the motile larva and sessile adult of Ciona .
... Labeled ANP cells are well organized in regular rows with a sharp border and no visible protrusions. A pair of large cells that resemble migratory Eminens-like neurons (recently described in [70]; arrowheads, Fig 5Ei) begin to emerge from the posterior portion of the developing ASV (Fig 5Eii, arrowhead). The bilateral posterior columns have merged to form a single, well-organized narrow band. ...
... As mentioned above, it does not appear that Lhx3/4 misexpression was sufficient to drive full execution of the CPP specification program and downstream expression of Foxf target genes involved in CPP migration, such as RhoD/F. To further explore ectopic CPP gene expression, we extracted the gene expression profile of mid-tailbud stage CPPs from a singlecell embryonic database of C. robusta gene expression [70]. We chose this stage because it represents CPP gene expression 4 hours after the initial FGF-dependent induction, mirroring the 4-hour interval between Lhx3/4 misexpression/FGF induction in the ANP and the RNA sequencing time point in our comparative analysis. ...
... Genes with potential roles in cellular adhesion displayed in blue, actin dynamics in red, cell polarity in yellow, and extracellular matrix in black. (H) Plot of genes expressed at above average levels in the CPP lineage at stage 21 (mid-tailbud), as characterized through extensive, stage-specific single-cell RNA-sequencing [70]. The top 10 downregulated genes and the top 30 up-regulated genes are highlighted. ...
Individual signaling pathways, such as fibroblast growth factors (FGFs), can regulate a plethora of inductive events. According to current paradigms, signal-dependent transcription factors (TFs), such as FGF/MapK-activated Ets family factors, partner with lineage-determining factors to achieve regulatory specificity. However, many aspects of this model have not been rigorously investigated. One key question relates to whether lineage-determining factors dictate lineage-specific responses to inductive signals or facilitate these responses in collaboration with other inputs. We utilize the chordate model Ciona robusta to investigate mechanisms generating lineage-specific induction. Previous studies in C . robusta have shown that cardiopharyngeal progenitor cells are specified through the combined activity of FGF-activated Ets1/2 . b and an inferred ATTA-binding transcriptional cofactor. Here, we show that the homeobox TF Lhx3/4 serves as the lineage-determining TF that dictates cardiopharyngeal-specific transcription in response to pleiotropic FGF signaling. Targeted knockdown of Lhx3/4 leads to loss of cardiopharyngeal gene expression. Strikingly, ectopic expression of Lhx3/4 in a neuroectodermal lineage subject to FGF-dependent specification leads to ectopic cardiopharyngeal gene expression in this lineage. Furthermore, ectopic Lhx3/4 expression disrupts neural plate morphogenesis, generating aberrant cell behaviors associated with execution of incompatible morphogenetic programs. Based on these findings, we propose that combinatorial regulation by signal-dependent and lineage-determinant factors represents a generalizable, previously uncategorized regulatory subcircuit we term “cofactor-dependent induction.” Integration of this subcircuit into theoretical models will facilitate accurate predictions regarding the impact of gene regulatory network rewiring on evolutionary diversification and disease ontogeny.
... Ascidians are marine invertebrates that belong to the phylum Urochordata and superphylum Chordata, and are recognized as one of the closest relatives of vertebrates (Delsuc et al., 2006;Denoeud et al., 2010;Satoh et al., 2014). The cosmopolitan species Ciona intestinalis Type A (synonym for Ciona robusta) has been employed as a model organism in various fields, including developmental biology, which have been markedly enhanced by genome sequencing (Dehal et al., 2002;Satou et al., 2005), analysis of gene expression profiles (Imai et al., 2004(Imai et al., , 2006(Imai et al., , 2012Azumi et al., 2007;Kawada et al., 2017;Matsubara et al., 2021;Kawada et al., 2022), characterization of the developmental and/or functional transcriptional network in embryos and larvae (Satoh, 2003;Lemaire, 2011;, Horie et al., 2018Cao et al., 2019;Satou et al., 2019;Liu and Satou, 2020), and the development of transgenic and gene-edited Ciona (Sasakura et al., 2003Sasakura and Horie, 2023). These studies have contributed a great deal to the investigation of embryonic development and metamorphosis of Ciona and the evolutionary history of chordates. ...
Bullfrog (Rana catesbeiana) larvae inhabiting the main island of Japan overwinter as preclimax animals, whereas the larvae that reached climax in summer complete metamorphosis. We analyzed the mRNA expression levels of the adenohypophyseal hormones, hypothalamic hormones, and their receptors that are involved in controlling metamorphosis in tadpoles at various developmental stages available in summer and winter in order to understand the hormonal mechanism regulating metamorphosis progression. Corticotropin-releasing factor (CRF) and thyrotropin β-subunit (TSHβ) mRNA expression was enhanced as they reached the climax stage in metamorphosing summer tadpoles, although type 2 CRF receptor (CRFR2) mRNA levels demonstrated a tendency of elevation, indicating the activation of the hypothalamo–hypophyseal axis for stimulating the release of thyroid hormone in summer. Arginine vasotocin (AVT) mRNA levels were elevated as metamorphosis progressed, but mRNA expression levels were not synchronized with those of proopiomelanocortin (POMC) and V1b-type AVT receptor (V1bR). The elevation of mRNA levels of prolactin (PRL) 1A and type 3 thyrotropin-releasing hormone receptor (TRHR3), but not of thyrotropin-releasing hormone (TRH) precursor mRNA levels, was noted in climactic tadpoles, indicating that PRL mRNA levels are not simply dependent on the expression levels of TRH precursor mRNA. In the preclimactic larvae captured in winter, which are in metamorphic stasis, mRNA levels of pituitary hormones, hypothalamic factors, and their receptors remained low or at levels similar to those of the larvae captured in summer. These results indicate the relationship between the mRNA expression of metamorphosis-related factors and the seasonal progression/stasis of metamorphosis.
... Ascidians are marine invertebrates that belong to the phylum Urochordata and superphylum Chordata, and are recognized as one of the closest relatives of vertebrates (Delsuc et al., 2006;Denoeud et al., 2010;Satoh et al., 2014). The cosmopolitan species Ciona intestinalis Type A (synonym for Ciona robusta) has been employed as a model organism in various fields, including developmental biology, which have been markedly enhanced by genome sequencing (Dehal et al., 2002;Satou et al., 2005), analysis of gene expression profiles (Imai et al., 2004(Imai et al., , 2006(Imai et al., , 2012Azumi et al., 2007;Kawada et al., 2017;Matsubara et al., 2021;Kawada et al., 2022), characterization of the developmental and/or functional transcriptional network in embryos and larvae (Satoh, 2003;Lemaire, 2011;, Horie et al., 2018Cao et al., 2019;Satou et al., 2019;Liu and Satou, 2020), and the development of transgenic and gene-edited Ciona (Sasakura et al., 2003Sasakura and Horie, 2023). These studies have contributed a great deal to the investigation of embryonic development and metamorphosis of Ciona and the evolutionary history of chordates. ...
Grass puffer is a semilunar-synchronized spawner: spawning occurs on beaches only for several days of spring tide around new moon (lunar age 0) and full moon (lunar age 15) every 2 weeks from spring to early summer. To investigate the role of kisspeptin and gonadotropin-inhibitory hormone (GnIH) in the semilunar-synchronized spawning, lunar age-dependent expression of the genes encoding kisspeptin (kiss2), kisspeptin receptor (kissr2), GnIH (gnih), GnIH receptor (gnihr), gonadotropin-releasing hormone 1 (GnRH1) (gnrh1), and three gonadotropin (GTH) subunits (gpa, fshb, lhb) was examined in the male grass puffer, which was kept in an aquarium under natural light condition in a lunar month during the spawning period. In the brain, both kiss2 and kissr2 showed lunar variations with a peak at lunar age 10, while both gnih and gnihr showed semilunar variations with two peaks at lunar age 0 and 20. On the other hand, gnrh1 showed semilunar variation with two peaks at lunar age 0 and 15. In the pituitary, kiss2, kissr2, gnih, and gnihr showed similar variations to those shown in the brain. The fshb and lhb mRNA levels showed semilunar variations with two peaks at lunar age 0 and 15. The present study shows lunar and semilunar oscillations of kiss2/kissr2 and gnih/gnihr expressions, respectively, with their peaks around spring tide in the brain and pituitary along with the semilunar expressions of gnrh1 and the pituitary GTH subunit genes. These results suggest that the lunar age-dependent expressions of the kisspeptin, GnIH, and their receptor genes may be primarily important in the control of the precisely timed semilunar spawning of the grass puffer.
... This process is coordinated with tail morphogenesis, which involves intercalation of the notochord cells and elongation of muscle cells (30,31). To obtain better insights into the developmental role of Crbn, we used a single-cell RNA-seq atlas (scRNA-seq atlas) (32) to identify the sites and timing of Crbn expression during development. We present evidence that Crbn plays a role in controlling the temporal precision of muscle differentiation, possibly by targeting Mrf proteins for proteasomal degradation. ...
... As in humans, the Ciona thalidomide binding domain contains a conserved glutamate 377 (E377) residue, which is important for interactions of CRBN-thalidomide complexes with specific zinc finger transcription factors (SI Appendix, Fig. S1A) (5)(6)(7). In contrast to the ubiquitous profiles of crbn expression reported in zebrafish (19,33), the Ciona scRNA-seq atlas suggests restricted expression in developing muscles after clearance of maternal mRNAs (32). Zygotic expression is first detected in the muscle cells during neurula stages, reaches a peak of expression in early to mid tailbud stages, and then diminishes in late tailbud embryos prior to the hatching of swimming tadpoles ( Fig. 1 A and B and SI Appendix, To understand the regulation of Crbn expression in developing tail muscles, we sought to identify regulatory sequences in the vicinity of the Crbn locus that can recapitulate its endogenous expression profile (SI Appendix, Fig. S2A). ...
... Reporter genes containing 455 bp, 410 bp or 314 bp of 5′ flanking sequences upstream of its translation start site drove robust and restricted expression in the tail muscles ( Fig. 2 A-C and SI Appendix, Fig. S2 B-D and Table S1). The 455 bp regulatory sequence contains three putative binding motifs for the transcription factor Mrf but lack motifs for the other transcription factors present in developing muscles ( Fig. 2A and SI Appendix, Fig. S2A and Table S2) (27,32). To test their contributions to the regulation of Crbn, we mutagenized all three binding sites in otherwise normal Crbn reporter genes. ...
Thalidomide has a dark history as a teratogen, but in recent years, its derivates have been shown to function as potent chemotherapeutic agents. These drugs bind cereblon (CRBN), the substrate receptor of an E3 ubiquitin ligase complex, and modify its degradation targets. Despite these insights, remarkably little is known about the normal function of cereblon in development. Here, we employ Ciona , a simple invertebrate chordate, to identify endogenous Crbn targets. In Ciona, Crbn is specifically expressed in developing muscles during tail elongation before they acquire contractile activity. Crbn expression is activated by Mrf, the ortholog of MYOD1, a transcription factor important for muscle differentiation. CRISPR/Cas9-mediated mutations of Crbn lead to precocious onset of muscle contractions. By contrast, overexpression of Crbn delays contractions and is associated with decreased expression of contractile protein genes such as troponin. This reduction is possibly due to reduced Mrf protein levels without altering Mrf mRNA levels. Our findings suggest that Mrf and Crbn form a negative feedback loop to control the precision of muscle differentiation during tail elongation.
... Ascidians are marine invertebrates that belong to the phylum Urochordata and superphylum Chordata, and are recognized as one of the closest relatives of vertebrates (Delsuc et al., 2006;Denoeud et al., 2010;Satoh et al., 2014). The cosmopolitan species Ciona intestinalis Type A (synonym for Ciona robusta) has been employed as a model organism in various fields, including developmental biology, which have been markedly enhanced by genome sequencing (Dehal et al., 2002;Satou et al., 2005), analysis of gene expression profiles (Imai et al., 2004(Imai et al., , 2006(Imai et al., , 2012Azumi et al., 2007;Kawada et al., 2017;Matsubara et al., 2021;Kawada et al., 2022), characterization of the developmental and/or functional transcriptional network in embryos and larvae (Satoh, 2003;Lemaire, 2011;, Horie et al., 2018Cao et al., 2019;Satou et al., 2019;Liu and Satou, 2020), and the development of transgenic and gene-edited Ciona (Sasakura et al., 2003Sasakura and Horie, 2023). These studies have contributed a great deal to the investigation of embryonic development and metamorphosis of Ciona and the evolutionary history of chordates. ...
Ovarian follicle development is an essential process for continuation of sexually reproductive animals, and is controlled by a wide variety of regulatory factors such as neuropeptides and peptide hormones in the endocrine, neuroendocrine, and nervous systems. Moreover, while some molecular mechanisms underlying follicle development are conserved, others vary among species. Consequently, follicle development processes are closely related to the evolution and diversity of species. Ciona intestinalis type A (Ciona rubusta) is a cosmopolitan species of ascidians, which are the closest relative of vertebrates. However, unlike vertebrates, ascidians are not endowed with the hypothalamus-pituitary-gonadal axis involving pituitary gonadotropins and sexual steroids. Combined with the phylogenetic position of ascidians as the closest relative of vertebrates, such morphological and endocrine features suggest that ascidians possess both common and species-specific regulatory mechanisms in follicle development. To date, several neuropeptides have been shown to participate in the growth of vitellogenic follicles, oocyte maturation of postvitellogenic follicles, and ovulation of fully mature follicles in a developmental stage-specific fashion. Furthermore, recent studies have shed light on the evolutionary processes of follicle development throughout chordates. In this review, we provide an overview of the neuropeptidergic molecular mechanism in the premature follicle growth, oocyte maturation, and ovulation in Ciona, and comparative views of the follicle development processes of mammals and teleosts.
