GSE99844
A single cell transcriptional roadmap for cardiopharyngeal fate diversification [scRNA-seq]
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. 1823 FACS purified Ciona cardiopharyngeal progenitor cells at successive developmental stages (12, 14, 16, 18, 20 hpfs) have been sequenced in this research, encompassing the developmental spectrum from single multipotent progenitors to diverse fate-restricted progenitor cells.  1796 out of 1823 cells have reads successfully mapped to Ciona genome (i.e only 1796 samples have FPKM data in the *txt processed data files). We adopted multiple quality control criteria to filter out low quality single cell transcriptomes, the contaminating subpopulations and the doublets. Eventually, 848 high-quality cells were retained for further analysis. Based on previously identified cell type specific markers and the well established lineage tree, we identified all five cardiopharyngeal progenitor subtypes (TVC, STVC, ASM, FHP, SHP) and in silico reconstructed three unidirectional trajectories corresponding to the specification of pharyngeal and cardiac fate. Our study enabled us to characterize the global gene expression patterns of heterogeneous cardiopharyngeal progenitors, and interrogate the spatial-temporal dynamics of cardiopharyngeal specification. Overall design: Single cell RNA-seq of FACS purified cardiopharyngeal progenitors (Mesp>tagRFP/Handr-r>tagBFP/MyoD905>EGFP) in Ciona Robusta at 12, 14, 16, 18, 20 hours post fertilization (hpf). Please note that multiple quality control criteria have been applied to filter out low quality single cell transcriptomes, the contaminating subpopulations and the doublets. [1] only single cells that had more than 2,000 and less than 6,000 expressed genes, and genes that were detected in more than 3 cells were retained. [2] Cells with mapping rates less than 30% and total reads more than 2-million were removed. [3] Contaminating non-cardiopharyngeal lineage cells were removed [4] Cells that expressed noticeable degree of both cardiac and ASM markers were considered as doublets and  removed.Eventually, 848 high quality cells were retained for further analysis. However, all samples and their associated raw data files have been included in the records for other researchers who might want to apply their own criteria to pre-process the raw data, although they are not all described in the associated manuscript.