Project description:We used 10X Genomics Chromium system to profile single cells from a pool of Wild-type Ciona robusta embyros at 12 hours post fertilization (hpf)
Project description: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:Dynamic gene expression programs determine multipotent cell states and fate choices during development. Multipotent progenitors for cardiomyocytes and branchiomeric head muscles populate the pharyngeal mesoderm of vertebrate embryos, but the mechanisms underlying cardiopharyngeal multipotency and heart vs. head muscle fate choices remain elusive. The tunicate Ciona emerged as a simple chordate model to study cardiopharyngeal development with unprecedented spatio-temporal resolution. We analyzed the transcriptome of single cardiopharyngeal lineage cells isolated at successive time points encompassing the transitions from multipotent progenitors to distinct first and second heart, and pharyngeal muscle precursors. We reconstructed the three cardiopharyngeal developmental trajectories, and characterized gene expression dynamics and regulatory states underlying each fate choice. Experimental perturbations and bulk transcriptome analyses revealed that ongoing FGF/MAPK signaling maintains cardiopharyngeal multipotency and promotes the pharyngeal muscle fate, whereas signal termination permits the deployment of a full pan-cardiac program and heart fate specification. We identified the Dach1/2 homolog as a novel evolutionarily conserved second-heart-field-specific factor and demonstrate, through lineage tracing and CRISPR/Cas9 perturbations, that it operates downstream of Tbx1/10 to actively suppress the first heart lineage program. This data indicates that the regulatory state of multipotent cardiopharyngeal progenitors determines the first vs. second heart lineage choice, and that Tbx1/10 acts as a bona fide regulator of cardiopharyngeal multi potency. We performed bulk RNAseq to profile the FACS purified Ciona Robusta Truck Ventral Cells (TVCs) with FGF-MAPK perturbation conditions to address the question- What is the role of FGF signaling pathway during early cardiopharyngeal specification. we performed bulk RNA sequencing of FACS-purified cardiopharyngeal lineage cells isolated from embryos and larvae expressing either a dominant negative form the fibroblast growth factor receptor (dnFGFR), or a constitutively active form of M-Ras (caM-Ras), the sole Ras homolog in Ciona, under the control of TVC-specific enhancers.