RESUMO
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
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
Discovering macromolecules and understanding the associated mechanisms involved in underwater adhesion are essential for both studying the fundamental ecology of benthos in aquatic ecosystems and developing biomimetic adhesive materials in industries. Here, we employed quantitative proteomics to assess protein expression variations during the development of the distinct adhesive structure - stolon in the model fouling ascidian, Ciona robusta. We found 16 adhesive protein candidates with increased expression in the stolon, with ascidian adhesive protein 1 (AAP1) being particularly rich in adhesion-related signal peptides, amino acids, and functional domains. Western blot and immunolocalization analyses confirmed the prominent AAP1 signals in the mantle, tunic, stolon, and adhesive footprints, indicating the interfacial role of this protein. Surface coating and atomic force microscopy experiments verified AAP1's adhesion to diverse materials, likely through the specific electrostatic and hydrophobic amino acid interactions with various substrates. In addition, molecular docking calculations indicated the AAP1's potential for cross-linking via hydrogen bonds and salt bridges among Von Willebrand factor type A domains, enhancing its adhesion capability. Altogether, the newly discovered interfacial protein responsible for permanent underwater adhesion, along with the elucidated adhesion mechanisms, are expected to contribute to the development of biomimetic adhesive materials and anti-fouling strategies. STATEMENT OF SIGNIFICANCE: Discovering macromolecules and studying their associated mechanisms involved in underwater adhesion are essential for understanding the fundamental ecology of benthos in aquatic ecosystems and developing innovative bionic adhesive materials in various industries. Using multidisciplinary analytical methods, we identified an interfacial protein - Ascidian Adhesive Protein 1 (AAP1) from the model marine fouling ascidian, Ciona robusta. The interfacial functions of AAP1 are achieved by electrostatic and hydrophobic interactions, and the Von Willebrand factor type A domain-based cross-linking likely enhances AAP1's interfacial adhesion. The identification and validation of the interfacial functions of AAP1, combined with the elucidation of adhesion mechanisms, present a promising target for the development of biomimetic adhesive materials and the formulation of effective anti-fouling strategies.
Assuntos
Incrustação Biológica , Animais , Adesividade , Urocordados/metabolismo , Simulação de Acoplamento Molecular , Adesivos/química , Sequência de Aminoácidos , Ciona/metabolismoRESUMO
Studying the generation of biomechanical force and how this force drives cell and tissue morphogenesis is challenging for understanding the mechanical mechanisms underlying embryogenesis. Actomyosin has been demonstrated to be the main source of intracellular force generation that drives membrane and cell contractility, thus playing a vital role in multi-organ formation in ascidian Ciona embryogenesis. However, manipulation of actomyosin at the subcellular level is impossible in Ciona because of the lack of technical tools and approaches. In this study, we designed and developed a myosin light chain phosphatase fused with a light-oxygen-voltage flavoprotein from Botrytis cinerea (MLCP-BcLOV4) as an optogenetics tool to control actomyosin contractility activity in the Ciona larva epidermis. We first validated the light-dependent membrane localization and regulatory efficiency on mechanical forces of the MLCP-BcLOV4 system as well as the optimum light intensity that activated the system in HeLa cells. Then, we applied the optimized MLCP-BcLOV4 system in Ciona larval epidermal cells to realize the regulation of membrane elongation at the subcellular level. Moreover, we successfully applied this system on the process of apical contraction during atrial siphon invagination in Ciona larvae. Our results showed that the activity of phosphorylated myosin on the apical surface of atrial siphon primordium cells was suppressed and apical contractility was disrupted, resulting in the failure of the invagination process. Thus, we established an effective technique and system that provide a powerful approach in the study of the biomechanical mechanisms driving morphogenesis in marine organisms.
Assuntos
Fibrilação Atrial , Ciona intestinalis , Ciona , Animais , Humanos , Actomiosina/metabolismo , Ciona/metabolismo , Optogenética , Células HeLa , Morfogênese/fisiologia , Células Epidérmicas/metabolismo , Epiderme/metabolismoRESUMO
The ascidian Ciona intestinalis type A (or Ciona robusta) is an important organism for elucidating the mechanisms that make the chordate body plan. CRISPR/Cas9 and TAL effector nuclease (TALEN) are widely used to quickly address genetic functions in Ciona. Our previously reported method of CRISPR/Cas9-mediated mutagenesis in this animal has inferior mutation rates compared to those of TALENs. We here describe an updated way to effectively mutate genes with CRISPR/Cas9 in Ciona. Although the construction of TALENs is much more laborious than that of CRISPR/Cas9, this technique is useful for tissue-specific knockouts that are not easy even by the optimized CRISPR/Cas9 method.
Assuntos
Ciona intestinalis , Ciona , Animais , Edição de Genes/métodos , Ciona/metabolismo , Ciona intestinalis/genética , Ciona intestinalis/metabolismo , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Efetores Semelhantes a Ativadores de Transcrição/genética , Sistemas CRISPR-Cas/genética , Técnicas de Inativação de GenesRESUMO
Ventral tail bending, which is transient but pronounced, is found in many chordate embryos and constitutes an interesting model of how tissue interactions control embryo shape. Here, we identify one key upstream regulator of ventral tail bending in embryos of the ascidian Ciona. We show that during the early tailbud stages, ventral epidermal cells exhibit a boat-shaped morphology (boat cell) with a narrow apical surface where phosphorylated myosin light chain (pMLC) accumulates. We further show that interfering with the function of the BMP ligand Admp led to pMLC localizing to the basal instead of the apical side of ventral epidermal cells and a reduced number of boat cells. Finally, we show that cutting ventral epidermal midline cells at their apex using an ultraviolet laser relaxed ventral tail bending. Based on these results, we propose a previously unreported function for Admp in localizing pMLC to the apical side of ventral epidermal cells, which causes the tail to bend ventrally by resisting antero-posterior notochord extension at the ventral side of the tail.
Assuntos
Ciona intestinalis , Ciona , Animais , Ciona intestinalis/metabolismo , Ciona/metabolismo , Cadeias Leves de Miosina/metabolismo , Ligantes , Células Epidérmicas/metabolismo , Cauda/metabolismoRESUMO
d-Serine, a free amino acid synthesized by serine racemase, is a coagonist of N-methyl-d-aspartate-type glutamate receptor (NMDAR). d-Serine in the mammalian central nervous system modulates glutamatergic transmission. Functions of d-serine in mammalian peripheral tissues such as skin have also been described. However, d-serine's functions in nonmammals are unclear. Here, we characterized d-serine-dependent vesicle release from the epidermis during metamorphosis of the tunicate Ciona. d-Serine leads to the formation of a pocket that facilitates the arrival of migrating tissue during tail regression. NMDAR is the receptor of d-serine in the formation of the epidermal pocket. The epidermal pocket is formed by the release of epidermal vesicles' content mediated by d-serine/NMDAR. This mechanism is similar to observations of keratinocyte vesicle exocytosis in mammalian skin. Our findings provide a better understanding of the maintenance of epidermal homeostasis in animals and contribute to further evolutionary perspectives of d-amino acid function among metazoans.
Assuntos
Ciona intestinalis , Ciona , Animais , Ciona/metabolismo , Ciona intestinalis/metabolismo , Epiderme/metabolismo , Mamíferos/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Serina/metabolismoRESUMO
Neural tube closure (NTC) is a complex multi-step morphogenetic process that transforms the flat neural plate found on the surface of the post-gastrulation embryo into the hollow and subsurface central nervous system (CNS). Errors in this process underlie some of the most prevalent human birth defects, and occur in about 1 out of every 1000 births. Previously, we discovered a mutant in the basal chordate Ciona savignyi (named bugeye) that revealed a novel role for a T-Type Calcium Channel (Cav3) in this process. Moreover, the requirement for CAV3s in Xenopus NTC suggests a conserved function among the chordates. Loss of CAV3 leads to defects restricted to anterior NTC, with the brain apparently fully developed, but protruding from the head. Here we report first on a new Cav3 mutant in the related species C. robusta. RNAseq analysis of both C. robusta and C. savignyi bugeye mutants reveals misregulation of a number of transcripts including ones that are involved in cell-cell recognition and adhesion. Two in particular, Selectin and Fibronectin leucine-rich repeat transmembrane, which are aberrantly upregulated in the mutant, are expressed in the closing neural tube, and when disrupted by CRISPR gene editing lead to the open brain phenotype displayed in bugeye mutants. We speculate that these molecules play a transient role in tissue separation and adhesion during NTC and failure to downregulate them leads to an open neural tube.
Assuntos
Caveolina 3/genética , Adesão Celular/fisiologia , Ciona/metabolismo , Animais , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/metabolismo , Caveolina 3/metabolismo , Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Morfogênese/genética , Placa Neural/metabolismo , Tubo Neural/metabolismo , Defeitos do Tubo Neural/genética , Neurulação/genéticaRESUMO
The transforming growth factor-ß (TGF-ß) family of cytokines performs a multifunctional signaling, which is integrated and coordinated in a signaling network that involves other pathways, such as Wintless, Forkhead box-O (FOXO) and Hedgehog and regulates pivotal functions related to cell fate in all tissues. In the hematopoietic system, TGF-ß signaling controls a wide spectrum of biological processes, from immune system homeostasis to the quiescence and self-renewal of hematopoietic stem cells (HSCs). Recently an important role in post-transcription regulation has been attributed to two type of ncRNAs: microRNAs and pseudogenes. Ciona robusta, due to its philogenetic position close to vertebrates, is an excellent model to investigate mechanisms of post-transcriptional regulation evolutionarily highly conserved in immune homeostasis. The combined use of NGS and bioinformatic analyses suggests that in the pharynx, the hematopoietic organ of Ciona robusta, the Tgf-ß, Wnt, Hedgehog and FoxO pathways are involved in tissue homeostasis, as they are in human. Furthermore, ceRNA network interactions and 3'UTR elements analyses of Tgf-ß, Wnt, Hedgehog and FoxO pathways genes suggest that different miRNAs conserved (cin-let-7d, cin-mir-92c, cin-mir-153), species-specific (cin-mir-4187, cin-mir-4011a, cin-mir-4056, cin-mir-4150, cin-mir-4189, cin-mir-4053, cin-mir-4016, cin-mir-4075), pseudogenes (ENSCING00000011392, ENSCING00000018651, ENSCING00000007698) and mRNA 3'UTR elements are involved in post-transcriptional regulation in an integrated way in C. robusta.
Assuntos
Ciona/metabolismo , Proteína Forkhead Box O1/metabolismo , Regulação da Expressão Gênica , Fator de Crescimento Transformador beta/metabolismo , Proteínas Wnt/metabolismo , Regiões 3' não Traduzidas , Animais , Linhagem da Célula , Biologia Computacional , Proteínas Hedgehog/metabolismo , Hematopoese , Sequenciamento de Nucleotídeos em Larga Escala , Homeostase , Sistema Imunitário , MicroRNAs/metabolismo , Faringe/metabolismo , Mapeamento de Interação de Proteínas , RNA-SeqRESUMO
Recent studies suggest that transcriptional activators and components of the pre-initiation complex (PIC) form higher order associations-clusters or condensates-at active loci. Considerably less is known about the distribution of repressor proteins responsible for gene silencing. Here, we develop an expression assay in living Ciona embryos that captures the liquid behavior of individual nucleoli undergoing dynamic fusion events. The assay is used to visualize puncta of Hes repressors, along with the Groucho/TLE corepressor. We observe that Hes.a/Gro puncta have the properties of viscous liquid droplets that undergo limited fusion events due to association with DNA. Hes.a mutants that are unable to bind DNA display hallmarks of liquid-liquid phase separation, including dynamic fusions of individual condensates to produce large droplets. We propose that the DNA template serves as a scaffold for the formation of Hes condensates, but limits the spread of transcriptional repressors to unwanted regions of the genome.
Assuntos
Ciona/metabolismo , Embrião não Mamífero/metabolismo , Animais , Ciona/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/genética , Transcrição Gênica/fisiologiaRESUMO
The notochord is a defining feature of the chordates. The transcription factor Brachyury (Bra) is a key regulator of notochord fate but here we show that it is not a unitary master regulator in the model chordate Ciona Ectopic Bra expression only partially reprograms other cell types to a notochord-like transcriptional profile and a subset of notochord-enriched genes is unaffected by CRISPR Bra disruption. We identify Foxa.a and Mnx as potential co-regulators, and find that combinatorial cocktails are more effective at reprogramming other cell types than Bra alone. We reassess the network relationships between Bra, Foxa.a and other components of the notochord gene regulatory network, and find that Foxa.a expression in the notochord is regulated by vegetal FGF signaling. It is a direct activator of Bra expression and has a binding motif that is significantly enriched in the regulatory regions of notochord-enriched genes. These and other results indicate that Bra and Foxa.a act together in a regulatory network dominated by positive feed-forward interactions, with neither being a classically defined master regulator.
Assuntos
Ciona/genética , Ciona/metabolismo , Proteínas Fetais/genética , Proteínas Fetais/metabolismo , Notocorda/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Animais , Ciona intestinalis/genética , Ciona intestinalis/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Notocorda/crescimento & desenvolvimento , Transativadores , Fatores de Transcrição/metabolismoRESUMO
Spatial reorganization of cytoplasm in zygotic cells is critically important for establishing the body plans of many animal species. In ascidian zygotes, maternal determinants (mRNAs) are first transported to the vegetal pole a few minutes after fertilization and then to the future posterior side of the zygotes in a later phase of cytoplasmic reorganization, before the first cell division. Here, by using a novel fluorescence polarization microscope that reports the position and the orientation of fluorescently labeled proteins in living cells, we mapped the local alignments and the time-dependent changes of cortical actin networks in Ciona eggs. The initial cytoplasmic reorganization started with the contraction of vegetal hemisphere approximately 20 s after the fertilization-induced [Ca2+] increase. Timing of the vegetal contraction was consistent with the emergence of highly aligned actin filaments at the cell cortex of the vegetal hemisphere, which ran perpendicular to the animal-vegetal axis. We propose that the cytoplasmic reorganization is initiated by the local contraction of laterally aligned cortical actomyosin in the vegetal hemisphere, which in turn generates the directional movement of cytoplasm within the whole egg.
Assuntos
Actinas/metabolismo , Ciona/metabolismo , Zigoto/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/fisiologia , Animais , Ciona/fisiologia , Citoplasma/metabolismo , Feminino , Fertilização/fisiologia , Células Germinativas/metabolismo , Oócitos/metabolismo , Urocordados/metabolismo , Zigoto/fisiologiaRESUMO
The 5' and 3' RACE is a method to obtain full-length 5' and 3' ends of cDNA using known cDNA sequences from expressed sequence tags (ESTs), subtracted cDNA, differential display, or library screening. Here is described the identification of full-length 5' and 3' ends of Ciona robusta Mif1 and Mif2 cDNA by using 5' and 3' RACE method.
Assuntos
Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Ciona/genética , DNA Complementar , Fatores Inibidores da Migração de Macrófagos/genética , Animais , Ciona/metabolismo , Genoma , Fatores Inibidores da Migração de Macrófagos/metabolismo , Capuzes de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The technique of in situ hybridization can be used to visualize the spatial and temporal pattern of gene expression during development. Ascidians are invertebrate chordates that develop with a fixed cell cleavage pattern into a tadpole larvae. The knowledge of the cell lineage allows the earliest steps of cell fate specification to be followed at a single cell resolution. This protocol describes preparation of Ciona intestinalis embryos, classical in situ hybridization protocol coupled with nuclear staining, and a guide to identify gene expression in specific precursors of the developing brain at neural plate stages of development.
Assuntos
Ciona/embriologia , Ciona/metabolismo , Hibridização In Situ/métodos , Placa Neural/embriologia , Placa Neural/metabolismo , Urocordados/embriologia , Urocordados/metabolismo , AnimaisRESUMO
The solute carrier 6 (SLC6) gene family, functioning as neurotransmitter transporters, plays the crucial roles in neurotransmission, cellular and organismal homeostasis. In this study, we found an expansion of SLC6 family gene in the genome of chordate invertebrate Ciona savignyi. A total of 40 candidate genes including 29 complete and 11 putative genes were identified as SLC6 family gene homologs. Phylogenetic analysis revealed that most of these Ciona SLC6 genes were highly conserved with the vertebrate ones, although gene duplication and gene losses did exist. Four genes were selected from SLC6 subfamilies to be further investigated for their functional characteristics on cell growth and migration through overexpression approach in cultured cell lines. The results showed both SLC6A7 and SLC6A17 from amino acid transporters AA1 and AA2 sub-families, respectively, significantly suppressed the cell proliferation and migration. While SLC6A1 and SLC6A4, which were classified into GABA and monoamine transporters, respectively, did not affect the cell proliferation and migration in HEK293T, HeLa, and MCF7 cells. The whole set of C. savignyi SLC6 genes identified in this study provides an important genomic resource for future biochemical, physiological, and phylogenetic studies on SLC6 gene family. Our experimental data demonstrated that Ciona amino acid transporters, such as SLC6A7 and SLC6A17, were essential for cell physiology and behaviors, indicating their crucially potential roles in the control of cell proliferation and migration during ascidian embryogenesis.
Assuntos
Ciona/metabolismo , Clonagem Molecular/métodos , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Animais , Movimento Celular , Proliferação de Células , Ciona/genética , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Família Multigênica , FilogeniaRESUMO
Peanut agglutinin (PNA) is an established marker of the mammalian acrosome. However, we observed that PNA specifically binds to a unique intracellular structure alongside the nucleus in ascidian sperm. Here, we characterize the PNA-binding structure in sperm of marine invertebrates. PNA bound to the region between the mitochondrion and nucleus in spermatozoa of ascidians, sea urchins, and an appendicularian. However, PNA-binding substances were not exposed by the calcium ionophore ionomycin in three ascidian species, indicating that it is a distinct structure from the acrosome. Instead, the ascidian PNA-binding region was shed with the mitochondrion from the sperm head via an ionomycin-induced sperm reaction. The ascidian PNA-binding substance appeared to be solubilized with SDS, but not Triton X-100, describing its detergent resistance. Lectins, PHA-L4 , SSA, and MAL-I were detected at an area similar to the PNA-binding region, suggesting that it contains a variety of glycans. The location and some of the components of the PNA-binding region were similar to known endoplasmic reticulum (ER)-derived structures, although the ER marker concanavalin A accumulated at an area adjacent to but not overlapping the PNA-binding region. Therefore, we conclude that ascidian sperm possess a non-acrosomal, Triton-resistant, glycan-rich intracellular structure that may play a general role in reproduction of tunicates and sea urchins given its presence across a wide taxonomic range.