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. Author manuscript; available in PMC: 2020 Sep 15.
Published in final edited form as: Nat Cell Biol. 2019 Jun 3;21(6):674–686. doi: 10.1038/s41556-019-0336-z

Figure 1∣. Cell clustering and cell-type-specific markers.

Figure 1∣

(a) Early cardiopharyngeal development in Ciona, and sampling stages and established lineage tree. Cardiopharyngeal lineage cells are shown for only one side and known cell-type-specific marker genes are indicated. st., FABA stage55; hpf, hours post-fertilization; TVC, trunk ventral cell; STVC, second trunk ventral cell; FHP, first heart precursor; ASMF, atrial siphon muscle founder cells; SHP, second heart precursor; iASMP, inner atrial siphon muscle precursor; oASMP, outer atrial siphon muscle precursor; LoM, longitudinal muscles; QC, quality control. Dotted line: midline. (b) t-distributed Stochastic Neighbor Embedding (t-SNE) plots of 20 hpf scRNA-seq data (n=288 cells) showing distinct clusters of progenitor subtypes: FHP (red), SHP (orange), iASMP (blue) and oASMP (dark blue). Color-coded marker gene expression levels are shown on corresponding clusters. (c) Expression heatmap of 20 hpf single cell transcriptomes showing top predicted differentially expressed marker genes across different cell types. Blue: previously known ASM and heart markers, red: candidate markers. (d) Violin plots and FISH validations of candidate cell-type-specific markers in St. 28 embryos. mRNAs visualized by whole mount fluorescent in situ hybridization (green). Cardiopharyngeal nuclei marked by Mesp>nls::LacZ revealed by anti beta-galactosidase antibody (red). Mesp>hCD4::mCherry, revealed by anti-mCherry antibody, marks cell membranes (blue). Anterior to the left. Scale bar, 10 μm. Solid arrowheads, ASM; open arrowheads, SHPs; arrows, FHPs; M, midline (dotted line). The numbers of observed embryos and those showing the illustrated gene expression pattern are indicated at the right bottom corner of each image. Violin plots are to visualize the distributions of the expression (log FPKM) of the indicated genes. The wide of the violin indicates the frequency of cells with indicated gene expression level. The number of cells in each cell cluster is summarized in Supplementary Table 6 (Data sheet: cell identity and number).