Single-cell transcriptome profiling of the Ciona larval brain
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ABSTRACT: The tadpole-type larva of Ciona has emerged as an intriguing model system for the study of neurodevelopment. The Ciona intestinalis connectome has been recently mapped, revealing the smallest central nervous system (CNS) known in any chordate, with only 177 neurons. This minimal CNS is highly reminiscent of larger CNS of vertebrates, sharing many conserved developmental processes, anatomical compartments, neuron subtypes, and even specific neural circuits. Thus, the Ciona tadpole offers a unique opportunity to understand the development and wiring of a chordate CNS at single-cell resolution. Here we report the use of single-cell RNAseq to profile the transcriptomes of single cells isolated by fluorescence-activated cell sorting (FACS) from the whole brain of Ciona robusta (formerly intestinalis Type A) larvae. We have also compared these profiles to bulk RNAseq data from specific subsets of brain cells isolated by FACS using cell type-specific reporter plasmid expression. Taken together, these datasets have begun to reveal the compartment- and cell-specific gene expression patterns that define the organization of the Ciona larval brain.
Project description:Among urochordates (tunicates)âthe closest living relatives of vertebratesâCiona intestinalis is increasingly being used as a model organism in the field of developmental biology. Ciona intestinalis is the seventh animal which genome published; the ~120-Mbp euchromatin region is estimated to contain ~16,000 protein-coding genes. In addition, analyses of more than one million ESTs have provided the foundation for gene models and associated transcriptomes. The fertilized Ciona intestinalis egg develops into a tadpole larva with a simplified chordate body plan, and then it metamorphose into adult sea squirt of sessile filter feeder. One of interests in the field of developmental biology is to understand what kind of genes are expressed in the body and how spatially and/or temporally coordinated expression of genes is controlled. In this study, we investigated the entire gene expression of 11 organs of adult Ciona; the neural complex, branchial sac, esophagus, stomach, endostyle, intestine, body-wall muscle, heart, blood cells, ovary, and testis. Our data would provides basic information of transcriptome in each organ and help to understand gene expression control of organ specific genes. Gene expressions in 11 organs of adult Ciona intestinalis; blood cells, branchial sac, digestive grand, endostyle, esophagus, heart, body-wall muscle, neural complex, ovary, stomach and testis. Three independent experiments were performed at each tissue using different individuals for each experiment.
Project description:Among urochordates (tunicates)—the closest living relatives of vertebrates—Ciona intestinalis is increasingly being used as a model organism in the field of developmental biology. Ciona intestinalis is the seventh animal which genome published; the ~120-Mbp euchromatin region is estimated to contain ~16,000 protein-coding genes. In addition, analyses of more than one million ESTs have provided the foundation for gene models and associated transcriptomes. The fertilized Ciona intestinalis egg develops into a tadpole larva with a simplified chordate body plan, and then it metamorphose into adult sea squirt of sessile filter feeder. One of interests in the field of developmental biology is to understand what kind of genes are expressed in the body and how spatially and/or temporally coordinated expression of genes is controlled. In this study, we investigated the entire gene expression of 11 organs of adult Ciona; the neural complex, branchial sac, esophagus, stomach, endostyle, intestine, body-wall muscle, heart, blood cells, ovary, and testis. Our data would provides basic information of transcriptome in each organ and help to understand gene expression control of organ specific genes.
Project description:We report the comprehensive sequencing of small RNA libraries created from different developmental stages (larva and gastrula) of the basal chordate, Ciona intestinalis. These libraries were used for the identification of microRNAs in this organism. Sequencing of small RNA libraries from 2 stages of Ciona intestinalis.
Project description:DNA binding profiles of three maternal factors (Tcf7, Gata.a and Zic-r.a) in chordate 16-cell stage embryo. 16-cell stage ciona intestinalis embryos were collected from multiple batches and the extracted chromatin was immunoprecipitated by the antibodies which specifically recognize ciona Gata.a, Tcf7 and Zic-r.a, respectively.
Project description:Tunicates, including ascidians, are recognized as the true “sister group” of vertebrates and are emerging as models to study the development and degeneration of central nervous system (CNS). Ascidian larvae have the typical chordate body plan that includes a dorsal neural tube. During their metamorphosis, a deep tissue reorganization takes place, with some tissues that degenerate while others develop to become functional during the adult life. The larval CNS also degenerates and most neurons disappear, making room to the formation of adult CNS. The genome of the ascidian Ciona intestinalis has been sequenced and annotated, with several CNS specific genes that have been characterized, revealing specification mechanisms shared with humans. These features make ascidian metamorphosis a good model to study the mechanisms underlying physiological CNS degeneration and to compare them to the pathological condition typical of neurodegenerative diseases. In order to shed light on the molecular determinants of C. intestinalis metamorphosis and neurodegeneration, we analyzed its transcriptome at three stages of development: swimming larva (SwL, Hotta stage 28), settled larva (SetL, Hotta stage 32) and metamorphosing larva (MetL, Hotta stage 34). Supported by SoE-SEED-2020 Grant, University of Milan.
Project description:We report the comprehensive sequencing of small RNA libraries created from different developmental stages (larva and gastrula) of the basal chordate, Ciona intestinalis. These libraries were used for the identification of microRNAs in this organism.
Project description:We report the comprehensive sequencing of small RNA libraries created from different developmental stages of the basal chordate, Ciona intestinalis. Surprisingly, half of the identified miRNA loci encode not two, but up to four distinct small RNAs. These additional RNAs, termed M-bM-^@M-^\moRsM-bM-^@M-^], are generated from sequences adjacent to the predicted ~60-nt pre-miRNA. moRs appear to be produced by RNAse-III-like processing, are ~20-nt in length, have 5M-bM-^@M-^Y-monophosphate and 3M-bM-^@M-^Y-hydroxl termini, and like miRNAs, are developmentally regulated. Sequencing of small RNA libraries from 4 stages of Ciona intestinalis. Ciona_LarvaMir309_RNASeq and Dmel_Toll10b_RNASeq are sequencing runs to detect if the expression of these RNA products is specific to Ciona or not. An ectopic transcript of the Drosophila melanogaster miR-309 locus was expressed in Ciona to see if it processed the miRs differently than Drosophila melanogaster. The Dmel sequence is from the endogenous case and is a control for comparison.
Project description:Hox cluster genes play crucial roles in the establishment of the body plan along the antero-posterior axis during animal development. Hox genes are expressed in the chordate endodermal tissues, and unraveling functions there is necessary for elucidating the mechanisms of endoderm specification. In the invertebrate chordate Ciona intestinalis, the endodermal tissues are in the premature state at the larval stage, and they form the differentiated digestive tract during metamorphosis. In this study, we showed that a Hox gene Ci-Hox10 is required for the formation of the intestine during metamorphosis. To know the downstream genes that are controlled by Hox10, microarray analysis of Hox10 knock-down embryos was performed. Gene expressions in the tail region of Hox10 knockdown embryos that were injected with Hox10 morpholino (MO) and control embryos of Ciona intestinalis were examined by comparative analysis with two color detection. Dye-swap analysis was carried out.
Project description:A genome decoded chordate Ciona intestinalis is one of the suitable animal for analyzing genetic controls in early embryogenesis and zygotically expressed genes are especially intensely studied. However maternally expressed genes are less studied than zygotic ones because of their huge numbers. These microarray experiments were carried out to screen rapidly degrading mRNA to identify crucial maternal mRNAs for Ciona embryogenesis in relatively a short period before their degradation. Two samples (unfertilized eggs vs 32-cell stage embryos), two biological replicates, dye Swap design
Project description:Recent whole-genome studies and in-depth expressed sequence tag (EST) analyses have identified most of the developmentally relevant genes in the urochordate, Ciona intestinalis. In this study, we made use of a large-scale oligo-DNA microarray to further investigate and identify genes with specific or correlated expression profiles. This large-scale oligo-DNA microarray for C. intestinalis should facilitate the understanding of global gene expression and gene networks during the development and ageing of a basal chordate. Keywords: gene expression, development, ageing, life cycle