RESUMO
The Ciona larva has served as a unique model for understanding the development of dopaminergic cells at single-cell resolution owing to the exceptionally small number of neurons in its brain and its fixed cell lineage during embryogenesis. A recent study suggested that the transcription factors Fer2 and Meis directly regulate the dopamine synthesis genes in Ciona, but the dopaminergic cell lineage and the gene regulatory networks that control the development of dopaminergic cells have not been fully elucidated. Here, we reveal that the dopaminergic cells in Ciona are derived from a bilateral pair of cells called a9.37 cells at the center of the neural plate. The a9.37 cells divide along the anterior-posterior axis, and all of the descendants of the posterior daughter cells differentiate into the dopaminergic cells. We show that the MAPK pathway and the transcription factor Otx are required for the expression of Fer2 in the dopaminergic cell lineage. Our findings establish the cellular and molecular framework for fully understanding the commitment to dopaminergic cells in the simple chordate brain.
Assuntos
Encéfalo/citologia , Encéfalo/metabolismo , Diferenciação Celular/genética , Ciona/genética , Neurônios Dopaminérgicos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Fatores de Transcrição Otx/genética , Animais , Biomarcadores , Linhagem da Célula/genética , Ciona/citologia , Neurônios Dopaminérgicos/citologia , Imunofluorescência , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Placa Neural/citologia , Placa Neural/metabolismo , Fatores de Transcrição Otx/metabolismo , Transdução de SinaisRESUMO
Cell morphology and tissue integrity are essential for embryogenesis. Caveolins are membrane proteins that induce the formation of surface pits called caveolae that serve as membrane reservoirs for cell and tissue protection during development. In vertebrates, caveolin 1 (Cav1) and caveolin 3 (Cav3) are required for caveola formation. However, the formation of caveola and the function of caveolins in invertebrates are largely unknown. In this study, three caveolins, Cav-a, Cav-b, and CavY, are identified in the genome of the invertebrate chordate Ciona spp. Based on phylogenetic analysis, Cav-a is found to be closely related to the vertebrate Cav1 and Cav3. In situ hybridization shows that Cav-a is expressed in Ciona embryonic notochord and muscle. Cell-free experiments, model cell culture systems, and in vivo experiments demonstrate that Ciona Cav-a has the ability to induce membrane curvature at the plasma membrane. Knockdown of Cav-a in Ciona embryos causes loss of invaginations in the plasma membrane and results in the failure of notochord elongation and lumenogenesis. Expression of a dominant-negative Cav-a point mutation causes cells to change shape and become displaced from the muscle and notochord to disrupt tissue integrity. Furthermore, we demonstrate that Cav-a vesicles show polarized trafficking and localize at the luminal membrane during notochord lumenogenesis. Taken together, these results show that the invertebrate chordate caveolin from Ciona plays crucial roles in tissue integrity and morphology by inducing membrane curvature and intracellular vesicle trafficking during embryogenesis.
Assuntos
Caveolinas/metabolismo , Membrana Celular/metabolismo , Ciona/embriologia , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário , Modelos Biológicos , Animais , Transporte Biológico Ativo , Caveolinas/genética , Membrana Celular/genética , Ciona/citologia , Embrião não Mamífero/citologiaRESUMO
The tailbud stage is part of the organogenesis period-an evolutionarily conserved developmental period among chordates that is essential for determining the characteristics of the chordate body plan. When the volume of the egg is artificially decreased by cutting, ascidians produce a normal-looking but miniature (dwarf) tailbud embryo. Although cell lineages during ascidian embryogenesis are invariant, the number of cell divisions in the dwarf embryo is altered by a different mechanism in each tissue (Yamada and Nishida, 1999). Here, to elucidate the size-regulation strategies of the Ciona robusta dwarf tailbud embryo, we compared anatomical structure, developmental speed, and cell number/volume in each tissue between dwarf and wild type (WT) embryos. To do this, we constructed a 3D virtual mid-tailbud embryo (Nakamura et al., 2012). We could make a Ciona dwarf tailbud embryo from eggs with a diameter over 108 âµm (correspond to â> â40% of the wild type egg volume). The timings of cleavage (~St. 12) and subsequent morphogenesis were nearly the same but blastomeres of animal hemisphere slightly delayed the timing of mitosis in the early cleavage period. Intriguingly, the tissue-to-tissue volume ratios of dwarf tailbud embryos were similar to those of wild type embryos suggesting that the ratio of tissue volumes is essential for maintaining the proper shape of the tailbud embryo. The number of cells in the epidermis, nervous system, and mesenchyme was significantly reduced in the dwarf embryos whereas the cell volume distribution of these tissues was similar in the dwarf and wild type. In contrast, the number of cells in the notochord, muscle, heart, and endoderm were maintained in the dwarf embryos; cell volumes were significantly reduced. Neither parameter changed in germline precursors. These results indicate that each tissue uses different scaling strategies to coordinate cell number and cell volume in accordance with the embryo size.
Assuntos
Ciona/embriologia , Embrião não Mamífero/embriologia , Morfogênese , Análise de Célula Única , Animais , Ciona/citologia , Ciona/genética , Embrião não Mamífero/citologiaRESUMO
Integrated analyses of regulated effector genes, cellular processes, and extrinsic signals are required to understand how transcriptional networks coordinate fate specification and cell behavior during embryogenesis. Ciona cardiopharyngeal progenitors, the trunk ventral cells (TVCs), polarize as leader and trailer cells that migrate between the ventral epidermis and trunk endoderm. We show that the TVC-specific collagen-binding Discoidin-domain receptor (Ddr) cooperates with Integrin-ß1 to promote cell-matrix adhesion. We find that endodermal cells secrete a collagen, Col9-a1, that is deposited in the basal epidermal matrix and promotes Ddr activation at the ventral membrane of migrating TVCs. A functional antagonism between Ddr/Intß1-mediated cell-matrix adhesion and Vegfr signaling appears to modulate the position of cardiopharyngeal progenitors between the endoderm and epidermis. We show that Ddr promotes leader-trailer-polarized BMP-Smad signaling independently of its role in cell-matrix adhesion. We propose that dual functions of Ddr integrate transcriptional inputs to coordinate subcellular processes underlying collective polarity and migration.
Assuntos
Movimento Celular , Polaridade Celular , Ciona/citologia , Receptores com Domínio Discoidina/fisiologia , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas Morfogenéticas Ósseas/fisiologia , Diferenciação Celular , Linhagem da Célula , Junções Célula-Matriz , Colágeno/metabolismo , Receptores com Domínio Discoidina/metabolismo , Desenvolvimento Embrionário , Integrina beta1/metabolismo , Transdução de Sinais , Proteínas Smad/metabolismo , Proteínas Smad/fisiologiaRESUMO
Complex gene regulatory networks are at the heart of cell fate specification and differentiation. The simple chordate Ciona has remarkable advantages for the dissection of these regulatory networks, including a stereotypically chordate but extremely small and simple embryo, a streamlined and compact genome, and highly efficient transgenesis by electroporation. Here we use the Ciona notochord as an example of how these characteristics can be exploited to understand both the early network controlling cell fate as well as the tissue-specific network controlling notochord differentiation and morphogenesis.
Assuntos
Ciona/genética , Redes Reguladoras de Genes , Notocorda/metabolismo , Animais , Ciona/citologia , Ciona/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Notocorda/embriologiaRESUMO
Placodes and neural crests represent defining features of vertebrates, yet their relationship remains unclear despite extensive investigation1-3. Here we use a combination of lineage tracing, gene disruption and single-cell RNA-sequencing assays to explore the properties of the lateral plate ectoderm of the proto-vertebrate, Ciona intestinalis. There are notable parallels between the patterning of the lateral plate in Ciona and the compartmentalization of the neural plate ectoderm in vertebrates4. Both systems exhibit sequential patterns of Six1/2, Pax3/7 and Msxb expression that depend on a network of interlocking regulatory interactions4. In Ciona, this compartmentalization network produces distinct but related types of sensory cells that share similarities with derivatives of both cranial placodes and the neural crest in vertebrates. Simple genetic disruptions result in the conversion of one sensory cell type into another. We focused on bipolar tail neurons, because they arise from the tail regions of the lateral plate and possess properties of the dorsal root ganglia, a derivative of the neural crest in vertebrates5. Notably, bipolar tail neurons were readily transformed into palp sensory cells, a proto-placodal sensory cell type that arises from the anterior-most regions of the lateral plate in the Ciona tadpole6. Proof of transformation was confirmed by whole-embryo single-cell RNA-sequencing assays. These findings suggest that compartmentalization of the lateral plate ectoderm preceded the advent of vertebrates, and served as a common source for the evolution of both cranial placodes and neural crest3,4.
Assuntos
Evolução Biológica , Ciona/citologia , Ciona/embriologia , Ectoderma/citologia , Crista Neural/citologia , Vertebrados/embriologia , Animais , Sequência de Bases , Linhagem da Célula , Ciona/crescimento & desenvolvimento , Ectoderma/embriologia , Hormônio Liberador de Gonadotropina/metabolismo , Larva , Crista Neural/embriologia , Placa Neural/citologia , Placa Neural/embriologia , Análise de Célula Única , XenopusRESUMO
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.
Assuntos
Núcleo Celular/metabolismo , Ciona , Mitocôndrias/metabolismo , Aglutinina de Amendoim/química , Ouriços-do-Mar , Animais , Ciona/citologia , Ciona/metabolismo , Masculino , Camundongos , Ouriços-do-Mar/citologia , Ouriços-do-Mar/metabolismoRESUMO
Physical and chemical cues from the environment are used to direct animal behavior through a complex network of connections originating in exteroceptors. In chordates, mechanosensory and chemosensory neurons of the peripheral nervous system (PNS) must signal to the motor circuits of the central nervous system (CNS) through a series of pathways that integrate and regulate the output to motor neurons (MN); ultimately these drive contraction of the tail and limb muscles. We used serial-section electron microscopy to reconstruct PNS neurons and their hitherto unknown synaptic networks in the tadpole larva of a sibling chordate, the ascidian, Ciona intestinalis. The larva has groups of neurons in its apical papillae, epidermal neurons in the rostral and apical trunk, caudal neurons in the dorsal and ventral epidermis, and a single tail tip neuron. The connectome reveals that the PNS input arises from scattered groups of these epidermal neurons, 54 in total, and has three main centers of integration in the CNS: in the anterior brain vesicle (which additionally receives input from photoreceptors of the ocellus), the motor ganglion (which contains five pairs of MN), and the tail, all of which in turn are themselves interconnected through important functional relay neurons. Some neurons have long collaterals that form autapses. Our study reveals interconnections with other sensory systems, and the exact inputs to the motor system required to regulate contractions in the tail that underlie larval swimming, or to the CNS to regulate substrate preference prior to the induction of larval settlement and metamorphosis.