RESUMO
Vertebrate calcitonin-producing cells (C-cells) are neuroendocrine cells that secrete the small peptide hormone calcitonin in response to elevated blood calcium levels. Whereas mouse C-cells reside within the thyroid gland and derive from pharyngeal endoderm, avian C-cells are located within ultimobranchial glands and have been reported to derive from the neural crest. We use a comparative cell lineage tracing approach in a range of vertebrate model systems to resolve the ancestral embryonic origin of vertebrate C-cells. We find, contrary to previous studies, that chick C-cells derive from pharyngeal endoderm, with neural crest-derived cells instead contributing to connective tissue intimately associated with C-cells in the ultimobranchial gland. This endodermal origin of C-cells is conserved in a ray-finned bony fish (zebrafish) and a cartilaginous fish (the little skate, Leucoraja erinacea). Furthermore, we discover putative C-cell homologs within the endodermally-derived pharyngeal epithelium of the ascidian Ciona intestinalis and the amphioxus Branchiostoma lanceolatum, two invertebrate chordates that lack neural crest cells. Our findings point to a conserved endodermal origin of C-cells across vertebrates and to a pre-vertebrate origin of this cell type along the chordate stem.
Assuntos
Calcitonina , Linhagem da Célula , Ciona intestinalis , Endoderma , Crista Neural , Células Neuroendócrinas , Animais , Endoderma/metabolismo , Endoderma/citologia , Calcitonina/metabolismo , Células Neuroendócrinas/metabolismo , Células Neuroendócrinas/citologia , Ciona intestinalis/metabolismo , Ciona intestinalis/embriologia , Crista Neural/metabolismo , Crista Neural/citologia , Embrião de Galinha , Camundongos , Vertebrados/embriologia , Vertebrados/metabolismo , Peixe-Zebra/embriologia , Anfioxos/embriologia , Anfioxos/metabolismo , Anfioxos/genética , Corpo Ultimobranquial/metabolismoRESUMO
During embryonic development, tissues and organs are gradually shaped into their functional morphologies through a series of spatiotemporally tightly orchestrated cell behaviors. A highly conserved organ shape across metazoans is the epithelial tube. Tube morphogenesis is a complex multistep process of carefully choreographed cell behaviors such as convergent extension, cell elongation, and lumen formation. The identity of the signaling molecules that coordinate these intricate morphogenetic steps remains elusive. The notochord is an essential tubular organ present in the embryonic midline region of all members of the chordate phylum. Here, using genome editing, pharmacology and quantitative imaging in the early chordate Ciona intestinalis we show that Ano10/Tmem16k, a member of the evolutionarily ancient family of transmembrane proteins called Anoctamin/TMEM16 is essential for convergent extension, lumen expansion, and connection during notochord morphogenesis. We find that Ano10/Tmem16k works in concert with the plasma membrane (PM) localized Na+/Ca2+ exchanger (NCX) and the endoplasmic reticulum (ER) residing SERCA, RyR, and IP3R proteins to establish developmental stage specific Ca2+ signaling molecular modules that regulate notochord morphogenesis and Ca2+ dynamics. In addition, we find that the highly conserved Ca2+ sensors calmodulin (CaM) and Ca2+/calmodulin-dependent protein kinase (CaMK) show an Ano10/Tmem16k-dependent subcellular localization. Their pharmacological inhibition leads to convergent extension, tubulogenesis defects, and deranged Ca2+ dynamics, suggesting that Ano10/Tmem16k is involved in both the "encoding" and "decoding" of developmental Ca2+ signals. Furthermore, Ano10/Tmem16k mediates cytoskeletal reorganization during notochord morphogenesis, likely by altering the localization of 2 important cytoskeletal regulators, the small GTPase Ras homolog family member A (RhoA) and the actin binding protein Cofilin. Finally, we use electrophysiological recordings and a scramblase assay in tissue culture to demonstrate that Ano10/Tmem16k likely acts as an ion channel but not as a phospholipid scramblase. Our results establish Ano10/Tmem16k as a novel player in the prevertebrate molecular toolkit that controls organ morphogenesis across scales.
Assuntos
Anoctaminas , Ciona intestinalis , Morfogênese , Notocorda , Animais , Notocorda/metabolismo , Notocorda/embriologia , Anoctaminas/metabolismo , Anoctaminas/genética , Ciona intestinalis/metabolismo , Ciona intestinalis/embriologia , Ciona intestinalis/genética , Morfogênese/genética , Sinalização do Cálcio , Regulação da Expressão Gênica no Desenvolvimento , Retículo Endoplasmático/metabolismo , Cálcio/metabolismoRESUMO
Throughout embryonic development, the shaping of the functional and morphological characteristics of embryos is orchestrated by an intricate interaction between transcription factors and cis-regulatory elements. In this study, we conducted a comprehensive analysis of deuterostome cis-regulatory landscapes during gastrulation, focusing on four paradigmatic species: the echinoderm Strongylocentrotus purpuratus, the cephalochordate Branchiostoma lanceolatum, the urochordate Ciona intestinalis, and the vertebrate Danio rerio. Our approach involved comparative computational analysis of ATAC-seq datasets to explore the genome-wide blueprint of conserved transcription factor binding motifs underlying gastrulation. We identified a core set of conserved DNA binding motifs associated with 62 known transcription factors, indicating the remarkable conservation of the gastrulation regulatory landscape across deuterostomes. Our findings offer valuable insights into the evolutionary molecular dynamics of embryonic development, shedding light on conserved regulatory subprograms and providing a comprehensive perspective on the conservation and divergence of gene regulation underlying the gastrulation process.
Assuntos
Ciona intestinalis , Gastrulação , Regulação da Expressão Gênica no Desenvolvimento , Animais , Gastrulação/genética , Ciona intestinalis/genética , Ciona intestinalis/embriologia , Peixe-Zebra/genética , Peixe-Zebra/embriologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Strongylocentrotus purpuratus/genética , Strongylocentrotus purpuratus/embriologia , Sequência Conservada/genética , Sequências Reguladoras de Ácido Nucleico/genética , Anfioxos/genética , Anfioxos/embriologia , Evolução MolecularRESUMO
In chordates, the central nervous system arises from precursors that have distinct developmental and transcriptional trajectories. Anterior nervous systems are ontogenically associated with ectodermal lineages while posterior nervous systems are associated with mesoderm. Taking advantage of the well-documented cell lineage of ascidian embryos, we asked to what extent the transcriptional states of the different neural lineages become similar during the course of progressive lineage restriction. We performed single-cell RNA sequencing (scRNA-seq) analyses on hand-dissected neural precursor cells of the two distinct lineages, together with those of their sister cell lineages, with a high temporal resolution covering five successive cell cycles from the 16-cell to neural plate stages. A transcription factor binding site enrichment analysis of neural specific genes at the neural plate stage revealed limited evidence for shared transcriptional control between the two neural lineages, consistent with their different ontogenies. Nevertheless, PCA analysis and hierarchical clustering showed that, by neural plate stages, the two neural lineages cluster together. Consistent with this, we identified a set of genes enriched in both neural lineages at the neural plate stage, including miR-124, Celf3.a, Zic.r-b, and Ets1/2. Altogether, the current study has revealed genome-wide transcriptional dynamics of neural progenitor cells of two distinct developmental origins. Our scRNA-seq dataset is unique and provides a valuable resource for future analyses, enabling a precise temporal resolution of cell types not previously described from dissociated embryos.
Assuntos
Linhagem da Célula , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Animais , Linhagem da Célula/genética , Desenvolvimento Embrionário/genética , Placa Neural/embriologia , Placa Neural/metabolismo , Placa Neural/citologia , Ciona intestinalis/embriologia , Ciona intestinalis/genética , Urocordados/embriologia , Urocordados/genética , Análise de Célula Única , Embrião não Mamífero/metabolismo , Embrião não Mamífero/citologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/citologiaRESUMO
Transcription control is a major determinant of cell fate decisions in somatic tissues. By contrast, early germline fate specification in numerous vertebrate and invertebrate species relies extensively on RNA-level regulation, exerted on asymmetrically inherited maternal supplies, with little-to-no zygotic transcription. However delayed, a maternal-to-zygotic transition is nevertheless poised to complete the deployment of pre-gametic programs in the germline. Here, we focus on early germline specification in the tunicate Ciona to study zygotic genome activation. We first demonstrate that a peculiar cellular remodeling event excludes localized postplasmic Pem-1 mRNA, which encodes the general inhibitor of transcription. Subsequently, zygotic transcription begins in Pem-1-negative primordial germ cells (PGCs), as revealed by histochemical detection of elongating RNA Polymerase II, and nascent Mef2 transcripts. In addition, we uncover a provisional antagonism between JAK and MEK/BMPRI/GSK3 signaling, which controls the onset of zygotic gene expression, following cellular remodeling of PGCs. We propose a 2-step model for the onset of zygotic transcription in the Ciona germline and discuss the significance of germ plasm dislocation and remodeling in the context of developmental fate specification.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas , Janus Quinases , Zigoto , Animais , Ciona/genética , Ciona/metabolismo , Ciona intestinalis/genética , Ciona intestinalis/embriologia , Células Germinativas/metabolismo , Janus Quinases/antagonistas & inibidores , Janus Quinases/metabolismo , Transdução de Sinais , Transcrição Gênica , Zigoto/metabolismoRESUMO
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.
Assuntos
Crista Neural , Vertebrados , Animais , Vertebrados/embriologia , Crista Neural/citologia , Crista Neural/embriologia , Urocordados/embriologia , Urocordados/citologia , Embrião não Mamífero/citologia , Ciona intestinalis/embriologia , Ciona intestinalis/genética , Ciona intestinalis/citologiaRESUMO
Physiological and pathological morphogenetic events involve a wide array of collective movements, suggesting that multicellular arrangements confer biochemical and biomechanical properties contributing to tissue-scale organization. The Ciona cardiopharyngeal progenitors provide the simplest model of collective cell migration, with cohesive bilateral cell pairs polarized along the leader-trailer migration path while moving between the ventral epidermis and trunk endoderm. We use the Cellular Potts Model to computationally probe the distributions of forces consistent with shapes and collective polarity of migrating cell pairs. Combining computational modeling, confocal microscopy, and molecular perturbations, we identify cardiopharyngeal progenitors as the simplest cell collective maintaining supracellular polarity with differential distributions of protrusive forces, cell-matrix adhesion, and myosin-based retraction forces along the leader-trailer axis. 4D simulations and experimental observations suggest that cell-cell communication helps establish a hierarchy to align collective polarity with the direction of migration, as observed with three or more cells in silico and in vivo. Our approach reveals emerging properties of the migrating collective: cell pairs are more persistent, migrating longer distances, and presumably with higher accuracy. Simulations suggest that cell pairs can overcome mechanical resistance of the trunk endoderm more effectively when they are polarized collectively. We propose that polarized supracellular organization of cardiopharyngeal progenitors confers emergent physical properties that determine mechanical interactions with their environment during morphogenesis.
Assuntos
Comunicação Celular , Movimento Celular , Polaridade Celular , Ciona intestinalis/embriologia , Células-Tronco/fisiologia , Animais , Embrião não Mamífero/embriologiaRESUMO
Precise control of lineage segregation is critical for the development of multicellular organisms, but our quantitative understanding of how variable signaling inputs are integrated to activate lineage-specific gene programs remains limited. Here, we show how precisely two out of eight ectoderm cells adopt neural fates in response to ephrin and FGF signals during ascidian neural induction. In each ectoderm cell, FGF signals activate ERK to a level that mirrors its cell contact surface with FGF-expressing mesendoderm cells. This gradual interpretation of FGF inputs is followed by a bimodal transcriptional response of the immediate early gene, Otx, resulting in its activation specifically in the neural precursors. At low levels of ERK, Otx is repressed by an ETS family transcriptional repressor, ERF2. Ephrin signals are critical for dampening ERK activation levels across ectoderm cells so that only neural precursors exhibit above-threshold levels, evade ERF repression, and "switch on" Otx transcription.
Assuntos
Padronização Corporal/genética , Desenvolvimento Embrionário/fisiologia , Indução Embrionária/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Ciona intestinalis/citologia , Ciona intestinalis/embriologia , Ectoderma/citologia , Embrião não Mamífero/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismoRESUMO
Recent innovations in genetics and imaging are providing the means to reconstruct cell lineages, either by tracking cell divisions using live microscopy, or by deducing the history of cells using molecular recorders. A cell lineage on its own, however, is simply a description of cell divisions as branching events. A major goal of current research is to integrate this description of cell relationships with information about the spatial distribution and identities of the cells those divisions produce. Visualizing, interpreting and exploring these complex data in an intuitive manner requires the development of new tools. Here we present CeLaVi, a web-based visualization tool that allows users to navigate and interact with a representation of cell lineages, whilst simultaneously visualizing the spatial distribution, identities and properties of cells. CeLaVi's principal functions include the ability to explore and manipulate the cell lineage tree; to visualise the spatial distribution of cell clones at different depths of the tree; to colour cells in the 3D viewer based on lineage relationships; to visualise various cell qualities on the 3D viewer (e.g. gene expression, cell type) and to annotate selected cells/clones. All these capabilities are demonstrated with four different example data sets. CeLaVi is available at http://www.celavi.pro.
Assuntos
Linhagem da Célula , Software , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/crescimento & desenvolvimento , Ciona intestinalis/citologia , Ciona intestinalis/embriologia , Crustáceos/citologia , Crustáceos/embriologia , Gástrula/citologia , Expressão Gênica , Larva/citologiaRESUMO
During the developmental processes of embryos, cells undergo massive deformation and division that are regulated by mechanical cues. However, little is known about how embryonic cells change their mechanical properties during different cleavage stages. Here, using atomic force microscopy, we investigated the stiffness of cells in ascidian embryos from the fertilised egg to the stage before gastrulation. In both animal and vegetal hemispheres, we observed a Rho kinase (ROCK)-independent cell stiffening that the cell stiffness exhibited a remarkable increase at the timing of cell division where cortical actin filaments were organized. Furthermore, in the vegetal hemisphere, we observed another mechanical behaviour, i.e., a ROCK-associated cell stiffening, which was retained even after cell division or occurred without division and propagated sequentially toward adjacent cells, displaying a characteristic cell-to-cell mechanical variation. The results indicate that the mechanical properties of embryonic cells are regulated at the single cell level in different germ layers.
Assuntos
Citoesqueleto de Actina/metabolismo , Ciona intestinalis/embriologia , Embrião não Mamífero/enzimologia , Mecanotransdução Celular , Quinases Associadas a rho/metabolismo , Animais , Pontos de Checagem do Ciclo Celular , Módulo de Elasticidade , Embrião não Mamífero/citologia , Desenvolvimento Embrionário , Microscopia de Força Atômica , Mitose , Miosinas/metabolismo , Análise de Célula Única , Fatores de TempoRESUMO
In early embryos of Ciona, an invertebrate chordate, the animal-vegetal axis is established by the combinatorial actions of maternal factors. One target of these maternal factors, Foxd, is specifically expressed in the vegetal hemisphere and stabilizes the animal-vegetal axis by activating vegetal hemisphere-specific genes and repressing animal hemisphere-specific genes. This dual functionality is essential for the embryogenesis of early ascidian embryos; however, the mechanism by which Foxd can act as both a repressor and an activator is unknown. Here, we identify a Foxd binding site upstream of Lhx3/4, which is activated by Foxd, and compare it with a repressive Foxd binding site upstream of Dmrt.a. We found that activating sites bind Foxd with low affinity while repressive sites bind Foxd with high affinity. Reporter assays confirm that this qualitative difference between activating and repressive Foxd binding sites is sufficient to change Foxd functionality. We therefore conclude that the outcome of Foxd transcriptional regulation is encoded in cis-regulatory elements.
Assuntos
Ciona intestinalis/embriologia , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Animais , Sítios de Ligação/genética , Padronização Corporal/genética , Ciona intestinalis/genética , Ciona intestinalis/metabolismo , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/genética , Fatores de Transcrição Forkhead/metabolismo , Expressão Gênica/genética , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/metabolismo , Urocordados/genética , Urocordados/metabolismoRESUMO
Tris(1-chloro-2-propyl)phosphate (TCPP) is the most common chlorinated organophosphorus flame retardant in seawater. Due to its chemical features and abundance, TCPP has been classified as a high hazard, and restrictions of use have been set in multiple countries. Despite TCPP being highly present in the marine environment, only a few studies have explored the TCPP impact on the development of marine invertebrates. Ascidians are important invertebrate members of benthic marine communities and reliable model systems for ecotoxicological research. The aim of this study was to assess the adverse effects of TCPP exposure on the embryogenesis of the ascidian Ciona intestinalis. Our results showed that this pollutant affected both muscles and nervous system development. Malformations appeared similar to those reported in other animal models for other flame retardants, suggesting that these molecules could share a common mechanism of action and induce a mixture effect when simultaneously present in the aquatic environment even at sub-teratogenic concentrations.
Assuntos
Ciona intestinalis/efeitos dos fármacos , Ciona intestinalis/embriologia , Embrião não Mamífero/efeitos dos fármacos , Retardadores de Chama/toxicidade , Compostos Organofosforados/toxicidade , AnimaisRESUMO
Endosomal trafficking of receptors and associated proteins plays a critical role in signal processing. Until recently, it was thought that trafficking was shut down during cell division. Thus, remarkably, the regulation of trafficking during division remains poorly characterized. Here we delineate the role of mitotic kinases in receptor trafficking during asymmetric division. Targeted perturbations reveal that Cyclin-dependent Kinase 1 (CDK1) and Aurora Kinase promote storage of Fibroblast Growth Factor Receptors (FGFRs) by suppressing endosomal degradation and recycling pathways. As cells progress through metaphase, loss of CDK1 activity permits differential degradation and targeted recycling of stored receptors, leading to asymmetric induction. Mitotic receptor storage, as delineated in this study, may facilitate rapid reestablishment of signaling competence in nascent daughter cells. However, mutations that limit or enhance the release of stored signaling components could alter daughter cell fate or behavior thereby promoting oncogenesis.
Assuntos
Aurora Quinases/fisiologia , Proteína Quinase CDC2/fisiologia , Mitose/fisiologia , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Animais , Animais Geneticamente Modificados , Aurora Quinases/genética , Proteína Quinase CDC2/genética , Proteínas de Ciclo Celular/metabolismo , Ciona intestinalis/embriologia , Ciona intestinalis/genética , Embrião não Mamífero , Mitose/genética , Transporte Proteico/genética , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento/genética , Receptores de Fatores de Crescimento/metabolismo , Transdução de Sinais/genética , Distribuição Tecidual/genéticaRESUMO
Establishment of presynaptic mechanisms by proteins that regulate neurotransmitter release in the presynaptic active zone is considered a fundamental step in animal evolution. Rab3 interacting molecule-binding proteins (Rimbps) are crucial components of the presynaptic active zone and key players in calcium homeostasis. Although Rimbp involvement in these dynamics has been described in distantly related models such as fly and human, the role of this family in most invertebrates remains obscure. To fill this gap, we defined the evolutionary history of Rimbp family in animals, from sponges to mammals. We report, for the first time, the expression of the two isoforms of the unique Rimbp family member in Ciona robusta in distinct domains of the larval nervous system. We identify intronic enhancers that are able to drive expression in different nervous system territories partially corresponding to Rimbp endogenous expression. The analysis of gene expression patterns and the identification of regulatory elements of Rimbp will positively impact our understanding of this family of genes in the context of Ciona embryogenesis.
Assuntos
Biomarcadores/análise , Ciona intestinalis/metabolismo , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sistema Nervoso/metabolismo , Sequências Reguladoras de Ácido Nucleico , Animais , Ciona intestinalis/embriologia , Ciona intestinalis/genética , Evolução Molecular , Peptídeos e Proteínas de Sinalização Intracelular/genética , Sistema Nervoso/embriologia , FilogeniaRESUMO
Nitric oxide synthase is ubiquitously present in metazoans and is involved in a wide range of biological processes. Three distinct Nos genes have been so far identified in vertebrates exhibiting a complex expression pattern and transcriptional regulation. Nevertheless, although independent events of Nos duplication have been observed in several taxa, only few studies described the regulatory mechanisms responsible for their activation in non-vertebrate animals. To shed light on the mechanisms underlying neuronal-type Nos expression, we focused on two non-vertebrate chordates: the cephalochordate Branchiostoma lanceolatum and the tunicate Ciona robusta. Here, throughout transphyletic and transgenic approaches, we identified genomic regions in both species acting as Nos functional enhancers during development. In vivo analyses of Nos genomic fragments revealed their ability to recapitulate the endogenous expression territories. Therefore, our results suggest the existence of evolutionary conserved mechanisms responsible for neuronal-type Nos regulation in non-vertebrate chordates. In conclusion, this study paves the way for future characterization of conserved transcriptional logic underlying the expression of neuronal-type Nos genes in chordates.
Assuntos
Ciona intestinalis/genética , Sequência Conservada , Regulação da Expressão Gênica no Desenvolvimento , Anfioxos/genética , Neurônios/metabolismo , Óxido Nítrico Sintase/genética , Animais , Animais Geneticamente Modificados , Evolução Biológica , Ciona intestinalis/embriologia , Ciona intestinalis/crescimento & desenvolvimento , Elementos Facilitadores Genéticos , Genoma , Anfioxos/embriologia , Anfioxos/crescimento & desenvolvimento , Larva/genética , Óxido Nítrico Sintase/metabolismo , Filogenia , Sequências Reguladoras de Ácido NucleicoRESUMO
Metamorphosis is the dramatic conversion of an animal body from larva to adult. In ascidians, tadpole-shaped, swimming larvae become sessile juveniles by losing their tail during metamorphosis. This study investigated the cellular and molecular mechanisms underlying this metamorphic event called tail regression, in the model ascidian Ciona. The ascidian tail consists of internal organs such as muscle, notochord, nerve cord, and the outer epidermal layer surrounding them. We found that the epidermis and internal organs show different regression strategies. Epidermal cells are shortened along the anterior-posterior axis and gather at the posterior region. The epidermal mass is then invaginated into the trunk by apical constriction. The internal tissues, by contrast, enter into the trunk by forming coils. During coiling, notches are introduced into the muscle cells, which likely reduces their rigidness to promote coiling. Actin filament is the major component necessary for the regression events in both the epidermis and internal tissues. The shortening and invagination of the epidermis depend on the phosphorylation of the myosin regulatory light chain (mrlc) regulated by rho-kinase (ROCK). The coiling of internal tissues does not require ROCK-dependent phosphorylation of mrlc, and they can complete coiling without epidermis, although epidermis can facilitate the coiling of internal tissues. We conclude that tail regression in ascidians consists of active morphogenetic movements in which each tissue's independent mechanism is orchestrated with the others to complete this event within the available time window.
Assuntos
Ciona intestinalis/embriologia , Metamorfose Biológica/fisiologia , Cauda/embriologia , Animais , EpidermeRESUMO
Gastrulation is the first major morphogenetic event during animal embryogenesis. Ascidian gastrulation starts with the invagination of 10 endodermal precursor cells between the 64- and late 112-cell stages. This process occurs in the absence of endodermal cell division and in two steps, driven by myosin-dependent contractions of the acto-myosin network. First, endoderm precursors constrict their apex. Second, they shorten apico-basally, while retaining small apical surfaces, thereby causing invagination. The mechanisms that prevent endoderm cell division, trigger the transition between step 1 and step 2, and drive apico-basal shortening have remained elusive. Here, we demonstrate a conserved role for Nodal and Eph signalling during invagination in two distantly related ascidian species, Phallusia mammillata and Ciona intestinalis Specifically, we show that the transition to step 2 is triggered by Nodal relayed by Eph signalling. In addition, our results indicate that Eph signalling lengthens the endodermal cell cycle, independently of Nodal. Finally, we find that both Nodal and Eph signals are dispensable for endoderm fate specification. These results illustrate commonalities as well as differences in the action of Nodal during ascidian and vertebrate gastrulation.
Assuntos
Ciona intestinalis/embriologia , Endoderma/embriologia , Gastrulação/fisiologia , Proteína Nodal/metabolismo , Receptor EphA1/metabolismo , Animais , Endoderma/citologiaRESUMO
Ascidian embryos are used as a model system in developmental biology due to their unique properties, including their invariant cell division patterns, being comprised of a small number of cells and tissues, the feasibility of their experimental manipulation, and their simple and compact genome. These properties have provided an opportunity for examining the gene regulatory network at the single cell resolution and at a genome-wide scale. This article summarizes when and where each regulatory gene is expressed in early ascidian embryos, and the extent to which the gene regulatory network explains each gene expression.
Assuntos
Diferenciação Celular/genética , Ciona/genética , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Animais , Linhagem da Célula/genética , Ciona/classificação , Ciona/embriologia , Ciona intestinalis/embriologia , Ciona intestinalis/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Genes Reguladores/genética , Modelos GenéticosRESUMO
Single-cell RNA-seq has been established as a reliable and accessible technique enabling new types of analyses, such as identifying cell types and studying spatial and temporal gene expression variation and change at single-cell resolution. Recently, single-cell RNA-seq has been applied to developing embryos, which offers great potential for finding and characterising genes controlling the course of development along with their expression patterns. In this study, we applied single-cell RNA-seq to the 16-cell stage of the Ciona embryo, a marine chordate and performed a computational search for cell-specific gene expression patterns. We recovered many known expression patterns from our single-cell RNA-seq data and despite extensive previous screens, we succeeded in finding new cell-specific patterns, which we validated by in situ and single-cell qPCR.
Assuntos
Ciona intestinalis/embriologia , Ciona intestinalis/genética , Embrião não Mamífero/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , RNA-Seq/métodos , Análise de Célula Única/métodos , Animais , Linhagem da Célula , Embrião não Mamífero/citologia , Sequenciamento do ExomaRESUMO
The microRNAs are small RNAs that regulate gene expression at the post-transcriptional level and can be involved in the onset of neurodegenerative diseases and cancer. They are emerging as possible targets for antisense-based therapy, even though the in vivo stability of miRNA analogues is still questioned. We tested the ability of peptide nucleic acids, a novel class of nucleic acid mimics, to downregulate miR-9 in vivo in an invertebrate model organism, the ascidian Ciona intestinalis, by microinjection of antisense molecules in the eggs. It is known that miR-9 is a well-conserved microRNA in bilaterians and we found that it is expressed in epidermal sensory neurons of the tail in the larva of C. intestinalis. Larvae developed from injected eggs showed a reduced differentiation of tail neurons, confirming the possibility to use peptide nucleic acid PNA to downregulate miRNA in a whole organism. By identifying putative targets of miR-9, we discuss the role of this miRNA in the development of the peripheral nervous system of ascidians.