RESUMO
Functional melanocortin receptor (MCR) genes have been identified in the genomes of early chordates, e.g., the cyclostomata. Whether they appear in the most ancient chordates such as cephalochordate and urochordata, however, remains unclear due to missing genetic data. Herein, we studied five putative (from NCBI database), sequence-based predicted MCR-like receptors from urochordata and cephalochordate, including Styela clava, Ciona intestinalis, Branchiostoma floridae, and Branchiostoma belcheri. The BLAST and phylogenetic analyses suggested a relationship between these specific receptors and vertebrate MCRs. However, several essential residues for MCR functions in vertebrates were missing in these putative chordata MCRs. To test receptor functionality, several experimental studies were conducted. Binding assays and functional analyses showed no specific binding and no ligand-induced cAMP or ERK1/2 signaling (with either endogenous α-MSH or synthetic ligands for MC4R), despite successfully expressing four receptors in HEK 293T cells. These four receptors showed high basal cAMP signaling, likely mediated by ligand-independent Gs coupling. In summary, our results suggest that the five predicted MCR-like receptors are, indeed, class A G protein-coupled receptors (GPCRs), which in four cases show high constitutive activity in the Gs-cAMP signaling pathway but are not MCR-like receptors in terms of ligand recognition of known MCR ligands. These receptors might be ancient G protein-coupled receptors with so far unidentified ligands.
Assuntos
Receptores de Melanocortina , Animais , Humanos , Sequência de Aminoácidos , Ciona intestinalis/genética , Ciona intestinalis/metabolismo , AMP Cíclico/metabolismo , Células HEK293 , Filogenia , Receptores de Melanocortina/metabolismo , Receptores de Melanocortina/genética , Urocordados/genética , Urocordados/metabolismoRESUMO
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
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 identified several homeobox transcription factor genes expressed during early OE patterning. Among these are two Pax3/7 copies 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 intestinalis, Pax3/7 plays a role in the development of cells deriving from the neural plate border, including trunk epidermal sensory neurons and tail nerve cord neurons, as well as in the neural tube closure. Here we have investigated the roles of Oikopleura dioica pax37A and pax37B in the development of the OE, by using CRISPR-Cas9 mutant lines and analyzing scRNA-seq data from wild-type animals. We found 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. Trajectory analysis supported a neuroepithelial-like or a preplacodal ectoderm transcriptional signature in these cells. We propose that the highly specialized secretory epithelial cells of the Fol region either maintained or evolved neuroepithelial features. This is supported by a fragmented gene regulatory network involved in their development that also operates in ascidian epidermal neurons.
Assuntos
Fator de Transcrição PAX3 , Fator de Transcrição PAX7 , Urocordados , Animais , Urocordados/embriologia , Urocordados/genética , Fator de Transcrição PAX7/genética , Fator de Transcrição PAX7/metabolismo , Fator de Transcrição PAX3/genética , Fator de Transcrição PAX3/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Epitélio/metabolismo , Ciona intestinalis/genética , Ciona intestinalis/embriologia , Diferenciação Celular/genética , Crista Neural/metabolismo , Crista Neural/embriologiaRESUMO
Keaton Schuster completed his PhD in the lab of Rachel Smith-Bolton at the University of Illinois, USA, investigating Drosophila wing disc regeneration before joining Lionel Christiaen's lab at New York University, USA, for his postdoc studying heart regeneration in the chordate tunicate Ciona robusta (formerly Ciona intestinalis type A). Keaton is part of the second cohort of Development's Pathway to Independence Programme fellows and we spoke to him over Teams to learn more about his career to date and his future plans for starting his own group continuing to use emerging model systems to study cardiac regeneration.
Assuntos
Ciona intestinalis , Animais , História do Século XXI , Ciona intestinalis/fisiologia , Regeneração/fisiologia , História do Século XX , Biologia do Desenvolvimento/história , Drosophila , Coração/fisiologiaRESUMO
The widely reported increase of terrestrial dissolved organic matter (terrDOM) in northern latitude coastal areas ("coastal darkening") can impact contaminant dynamics in affected systems. One potential impact is based on differences in chemical adsorption processes of the molecularly larger terrDOM compared to marine DOM (marDOM) that leads to increased emulsification of lipophilic contaminants with terrDOM. Filter feeders filter large amounts of water and DOM daily and thus are directly exposed to associated contaminants through both respiration and feeding activity. Thus, increased exposure to terrDOM could potentially lead to an increase in bioaccumulation of lipid soluble contaminants in filter feeders. To assess the effect of DOM on bioaccumulation in filter feeders, we exposed the mucous based filter feeding ascidian Ciona intestinalis (formerly known as Ciona intestinalis Type B), to the lipophilic veterinary drug teflubenzuron (log KOW: 5.39) in combination with four DOM treatments: TerrDOM, marDOM, a mix of the two called mixDOM, and seawater without DOM addition. The exposure lasted for 15 days, after which the individuals in all DOM treatments showed a trend towards higher bioaccumulation of Teflubenzuron than those in the seawater control. However, there was considerable overlap in posterior distributions. Against our expectations, marDOM resulted in the highest bioaccumulation factor (BAF), followed by mixDOM, with terrDOM resulting in the lowest BAF except for seawater (kinetic BAF L/kg median, 2.5 %-97.5 % percentile marDOM 94, 74-118; mixDOM 82, 63-104; terrDOM 79; 61-99; seawater 61, 44-79). All BAFs were below the level of concern according to the EU REACH regulation (BAF < 2000 L / kg) and, therefore, likely not environmentally problematic in the examined context. However, the results show that DOM can act as a dietary vector; thus, different combinations of contaminants, DOM, and filter feeding organisms should be tested further.
Assuntos
Ciona intestinalis , Poluentes Químicos da Água , Animais , Poluentes Químicos da Água/metabolismo , Ciona intestinalis/metabolismo , Tamanho da Partícula , Água do Mar/química , Bioacumulação , Lipídeos/química , Benzamidas/químicaRESUMO
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
The urochordate Ciona robusta exhibits numerous functional and morphogenetic traits that are shared with vertebrate models. While prior investigations have identified several analogies between the gastrointestinal tract (i.e., gut) of Ciona and mice, the molecular mechanisms responsible for these similarities remain poorly understood. This study seeks to address this knowledge gap by investigating the transcriptional landscape of the adult stage gut. Through comparative genomics analyses, we identified several evolutionarily conserved components of signaling pathways of pivotal importance for gut development (such as WNT, Notch, and TGFß-BMP) and further evaluated their expression in three distinct sections of the gastrointestinal tract by RNA-seq. Despite the presence of lineage-specific gene gains, losses, and often unclear orthology relationships, the investigated pathways were characterized by well-conserved molecular machinery, with most components being expressed at significant levels throughout the entire intestinal tract of C. robusta. We also showed significant differences in the transcriptional landscape of the stomach and intestinal tract, which were much less pronounced between the proximal and distal portions of the intestine. This study confirms that C. robusta is a reliable model system for comparative studies, supporting the use of ascidians as a model to study gut physiology.
Assuntos
Transdução de Sinais , Animais , Trato Gastrointestinal/metabolismo , Ciona/genética , Ciona/metabolismo , Ciona intestinalis/genética , Ciona intestinalis/metabolismo , Receptores Notch/metabolismo , Receptores Notch/genética , Perfilação da Expressão GênicaRESUMO
Septins are filamentous nucleotide-binding proteins which can associate with membranes in a curvature-dependent manner leading to structural remodelling and barrier formation. Ciona intestinalis, a model for exploring the development and evolution of the chordate lineage, has only four septin-coding genes within its genome. These represent orthologues of the four classical mammalian subgroups, making it a minimalist non-redundant model for studying the modular assembly of septins into linear oligomers and thereby filamentous polymers. Here, we show that C. intestinalis septins present a similar biochemistry to their human orthologues and also provide the cryo-EM structures of an octamer, a hexamer and a tetrameric sub-complex. The octamer, which has the canonical arrangement (2-6-7-9-9-7-6-2) clearly shows an exposed NC-interface at its termini enabling copolymerization with hexamers into mixed filaments. Indeed, only combinations of septins which had CiSEPT2 occupying the terminal position were able to assemble into filaments via NC-interface association. The CiSEPT7-CiSEPT9 tetramer is the smallest septin particle to be solved by Cryo-EM to date and its good resolution (2.7 Å) provides a well-defined view of the central NC-interface. On the other hand, the CiSEPT7-CiSEPT9 G-interface shows signs of fragility permitting toggling between hexamers and octamers, similar to that seen in human septins but not in yeast. The new structures provide insights concerning the molecular mechanism for cross-talk between adjacent interfaces. This indicates that C. intestinalis may represent a valuable tool for future studies, fulfilling the requirements of a complete but simpler system to understand the mechanisms behind the assembly and dynamics of septin filaments.
Assuntos
Ciona intestinalis , Microscopia Crioeletrônica , Modelos Moleculares , Multimerização Proteica , Septinas , Ciona intestinalis/metabolismo , Ciona intestinalis/química , Ciona intestinalis/genética , Septinas/metabolismo , Septinas/química , Septinas/genética , Animais , Humanos , Nucleotídeos/metabolismo , Nucleotídeos/química , Conformação Proteica , Ligação ProteicaRESUMO
Loss of mitochondrial electron transport complex (ETC) function in the retinal pigment epithelium (RPE) in vivo results in RPE dedifferentiation and progressive photoreceptor degeneration, and has been implicated in the pathogenesis of age-related macular degeneration. Xenogenic expression of alternative oxidases in mammalian cells and tissues mitigates phenotypes arising from some mitochondrial electron transport defects, but can exacerbate others. We expressed an alternative oxidase from Ciona intestinalis (AOX) in ETC-deficient murine RPE in vivo to assess the retinal consequences of stimulating coenzyme Q oxidation and respiration without ATP generation. RPE-restricted expression of AOX in this context is surprisingly beneficial. This focused intervention mitigates RPE mTORC1 activation, dedifferentiation, hypertrophy, stress marker expression, pseudohypoxia, and aerobic glycolysis. These RPE cell autonomous changes are accompanied by increased glucose delivery to photoreceptors with attendant improvements in photoreceptor structure and function. RPE-restricted AOX expression normalizes accumulated levels of succinate and 2-hydroxyglutarate in ETC-deficient RPE, and counteracts deficiencies in numerous neural retinal metabolites. These features can be attributed to the activation of mitochondrial inner membrane flavoproteins such as succinate dehydrogenase and proline dehydrogenase, and alleviation of inhibition of 2-oxyglutarate-dependent dioxygenases such as prolyl hydroxylases and epigenetic modifiers. Our work underscores the importance to outer retinal health of coenzyme Q oxidation in the RPE and identifies a metabolic network critical for photoreceptor survival in the context of RPE mitochondrial dysfunction.
Assuntos
Mitocôndrias , Oxirredutases , Proteínas de Plantas , Epitélio Pigmentado da Retina , Animais , Mitocôndrias/metabolismo , Camundongos , Oxirredutases/metabolismo , Oxirredutases/genética , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Ciona intestinalis/metabolismo , Ubiquinona/metabolismo , Ubiquinona/análogos & derivados , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Degeneração Retiniana/genética , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologiaRESUMO
Tunicates are the sister group to the vertebrates, yet most species have a life cycle split between swimming larva and sedentary adult phases. During metamorphosis, larval neurons are replaced by adult-specific ones. The regulatory mechanisms underlying this replacement remain largely unknown. Using tissue-specific CRISPR/Cas9-mediated mutagenesis in the tunicate Ciona, we show that orthologs of conserved hindbrain and branchiomeric neuron regulatory factors Pax2/5/8 and Phox2 are required to specify the 'neck', a cellular compartment set aside in the larva to give rise to cranial motor neuron-like neurons post-metamorphosis. Using bulk and single-cell RNA-sequencing analyses, we characterize the transcriptome of the neck downstream of Pax2/5/8. We present evidence that neck-derived adult ciliomotor neurons begin to differentiate in the larva and persist through metamorphosis, contrary to the assumption that the adult nervous system is formed after settlement and the death of larval neurons during metamorphosis. Finally, we show that FGF signaling during the larval phase alters the patterning of the neck and its derivatives. Suppression of FGF converts neck cells into larval neurons that fail to survive metamorphosis, whereas prolonged FGF signaling promotes an adult neural stem cell-like fate.
Assuntos
Larva , Metamorfose Biológica , Animais , Larva/crescimento & desenvolvimento , Neurônios/metabolismo , Neurônios/citologia , Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica no Desenvolvimento , Neurônios Motores/metabolismo , Neurônios Motores/citologia , Transdução de Sinais/genética , Ciona intestinalis/genética , Sobrevivência Celular , Transcriptoma/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Sistemas CRISPR-Cas/genéticaRESUMO
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
The respiratory chain alternative enzymes (AEs) NDX and AOX from the tunicate Ciona intestinalis (Ascidiacea) have been xenotopically expressed and characterized in human cells in culture and in the model organisms Drosophila melanogaster and mouse, with the purpose of developing bypass therapies to combat mitochondrial diseases in human patients with defective complexes I and III/IV, respectively. The fact that the genes coding for NDX and AOX have been lost from genomes of evolutionarily successful animal groups, such as vertebrates and insects, led us to investigate if the composition of the respiratory chain of Ciona and other tunicates differs significantly from that of humans and Drosophila, to accommodate the natural presence of AEs. We have failed to identify in tunicate genomes fifteen orthologous genes that code for subunits of the respiratory chain complexes; all of these putatively missing subunits are peripheral to complexes I, III and IV in mammals, and many are important for complex-complex interaction in supercomplexes (SCs), such as NDUFA11, UQCR11 and COX7A. Modeling of all respiratory chain subunit polypeptides of Ciona indicates significant structural divergence that is consistent with the lack of these fifteen clear orthologous subunits. We also provide evidence using Ciona AOX expressed in Drosophila that this AE cannot access the coenzyme Q pool reduced by complex I, but it is readily available to oxidize coenzyme Q molecules reduced by glycerophosphate oxidase, a mitochondrial inner membrane-bound dehydrogenase that is not involved in SCs. Altogether, our results suggest that Ciona AEs might have evolved in a mitochondrial inner membrane environment much different from that of mammals and insects, possibly without SCs; this correlates with the preferential functional interaction between these AEs and non-SC dehydrogenases in heterologous mammalian and insect systems. We discuss the implications of these findings for the applicability of Ciona AEs in human bypass therapies and for our understanding of the evolution of animal respiratory chain.
Assuntos
Ciona intestinalis , Proteínas Mitocondriais , Fosforilação Oxidativa , Animais , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Ciona intestinalis/genética , Ciona intestinalis/enzimologia , Humanos , Oxirredutases/genética , Oxirredutases/metabolismo , Subunidades Proteicas/metabolismo , Subunidades Proteicas/genética , Drosophila melanogaster/genética , Drosophila melanogaster/enzimologia , Urocordados/genética , Urocordados/enzimologia , Transporte de Elétrons , Complexo I de Transporte de Elétrons/metabolismo , Complexo I de Transporte de Elétrons/genética , Filogenia , Proteínas de PlantasRESUMO
The heart of ascidians (marine invertebrate chordates) has a tubular structure, and heartbeats propagate from one end to the other. The direction of pulsation waves intermittently reverses in the heart of ascidians and their relatives; however, the underlying mechanisms remain unclear. We herein performed a series of experiments to characterize the pacemaker systems in isolated hearts and their fragments, and applied a mathematical model to examine the conditions leading to heart reversals. The isolated heart of Ciona robusta autonomously generated pulsation waves at â¼20 to 25 beats min-1 with reversals at â¼1 to 10â min intervals. Experimental bisections of isolated hearts revealed that independent pacemakers resided on each side and also that their beating frequencies periodically changed as they expressed bimodal rhythms, which comprised an â¼1.25 to 5.5 min acceleration/deceleration cycle of a beating rate of between 0 and 25 beats min-1. Only fragments including 5% or shorter terminal regions of the heart tube maintained autonomous pulsation rhythms, whereas other regions did not. Our mathematical model, based on FitzHugh-Nagumo equations applied to a one-dimensional alignment of cells, demonstrated that the difference between frequencies expressed by the two independent terminal pacemakers determined the direction of propagated waves. Changes in the statuses of terminal pacemakers between the excitatory and oscillatory modes as well as in their endogenous oscillation frequencies were sufficient to lead to heart reversals. These results suggest that the directions of pulsation waves in the Ciona heart reverse according to the changing rhythms independently expressed by remotely coupled terminal pacemakers.
Assuntos
Relógios Biológicos , Ciona intestinalis , Coração , Animais , Coração/fisiologia , Relógios Biológicos/fisiologia , Ciona intestinalis/fisiologia , Frequência CardíacaRESUMO
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
Tissue-specific gene expression is fundamental in development and evolution, and is mediated by transcription factors and by the cis-regulatory regions (enhancers) that they control. Transcription factors and their respective tissue-specific enhancers are essential components of gene regulatory networks responsible for the development of tissues and organs. Although numerous transcription factors have been characterized from different organisms, the knowledge of the enhancers responsible for their tissue-specific expression remains fragmentary. Here we use Ciona to study the enhancers associated with ten transcription factors expressed in the notochord, an evolutionary hallmark of the chordate phylum. Our results illustrate how two evolutionarily conserved transcription factors, Brachyury and Foxa2, coordinate the deployment of other notochord transcription factors. The results of these detailed cis-regulatory analyses delineate a high-resolution view of the essential notochord gene regulatory network of Ciona, and provide a reference for studies of transcription factors, enhancers, and their roles in development, disease, and evolution.
Assuntos
Ciona intestinalis , Ciona , Animais , Ciona/genética , Redes Reguladoras de Genes , Ciona intestinalis/genética , Ciona intestinalis/metabolismo , Notocorda/metabolismo , Proteínas Fetais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
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.
Assuntos
Ciona intestinalis , Neuropeptídeos , Animais , Feminino , Filogenia , Ovulação , Folículo Ovariano , MamíferosRESUMO
Tunicates are evolutionary model organisms bridging the gap between vertebrates and invertebrates. A genomic sequence in Ciona intestinalis (CiOX) shows high similarity to vertebrate orexin receptors and protostome allatotropin receptors (ATR). Here, molecular phylogeny suggested that CiOX is divergent from ATRs and human orexin receptors (hOX1/2). However, CiOX appears closer to hOX1/2 than to ATR both in terms of sequence percent identity and in its modelled binding cavity, as suggested by molecular modelling. CiOX was heterologously expressed in a recombinant HEK293 cell system. Human orexins weakly but concentration-dependently activated its Gq signalling (Ca2+ elevation), and the responses were inhibited by the non-selective orexin receptor antagonists TCS 1102 and almorexant, but only weakly by the OX1-selective antagonist SB-334867. Furthermore, the 5-/6-carboxytetramethylrhodamine (TAMRA)-labelled human orexin-A was able to bind to CiOX. Database mining was used to predict a potential endogenous C. intestinalis orexin peptide (Ci-orexin-A). Ci-orexin-A was able to displace TAMRA-orexin-A, but not to induce any calcium response at the CiOX. Consequently, we suggested that the orexin signalling system is conserved in Ciona intestinalis, although the relevant peptide-receptor interaction was not fully elucidated.
Assuntos
Ciona intestinalis , Animais , Humanos , Receptores de Orexina/genética , Receptores de Orexina/metabolismo , Orexinas/genética , Orexinas/metabolismo , Ciona intestinalis/genética , Ciona intestinalis/metabolismo , Células HEK293 , Transdução de Sinais , Vertebrados/metabolismo , Proteínas de Transporte/metabolismoRESUMO
Voltage-clamp fluorometry (VCF) enables the study of voltage-sensitive proteins through fluorescent labeling accompanied by ionic current measurements for voltage-gated ion channels. The heterogeneity of the fluorescent signal represents a significant challenge in VCF. The VCF signal depends on where the cysteine mutation is incorporated, making it difficult to compare data among different mutations and different studies and standardize their interpretation. We have recently shown that the VCF signal originates from quenching amino acids in the vicinity of the attached fluorophores, together with the effect of the lipid microenvironment. Based on these, we performed experiments to test the hypothesis that the VCF signal could be altered by amphiphilic quenching molecules in the cell membrane. Here we show that a phenylalanine-conjugated flavonoid (4-oxo-2-phenyl-4H-chromene-7-yl)-phenylalanine, (later Oxophench) has potent effects on the VCF signals of the Ciona intestinalis HV1 (CiHv1) proton channel. Using spectrofluorimetry, we showed that Oxophench quenches TAMRA (5(6)-carboxytetramethylrhodamine-(methane thiosulfonate)) fluorescence. Moreover, Oxophench reduces the baseline fluorescence in oocytes and incorporates into the cell membrane while reducing the membrane fluidity of HEK293 cells. Our model calculations confirmed that Oxophench, a potent membrane-bound quencher, modifies the VCF signal during conformational changes. These results support our previously published model of VCF signal generation and point out that a change in the VCF signal may not necessarily indicate an altered conformational transition of the investigated protein.