Identification of major cell types across five developmental stages in S. clava.

(A) Representative sampling stages and corresponding larval and juvenile morphologies. (B) Cell type annotations derived from scRNA-seq data, aligned with the respective developmental stages. Hpf, hours post-fertilization; Dpf, days post-fertilization. (C) Inferred corresponding relationships between cell types across successive stages. Each cell type at a later stage (y-axis) is matched to its most similar cell type from the previous stage (x-axis) based on transcriptomic similarity calculated via K-nearest neighbor (KNN) analysis. A similarity score > 0.7 is used as the criterion for inferring the corresponding relationship. Cell types are color-coded by their germ layer of origin: ectoderm (blue), mesoderm (yellow), and endoderm (red). (D) UMAP plot integrating scRNA-seq data from all five stages, with cells colored by developmental stage, illustrating the cellular state transitions before and after metamorphosis. Cells from pre-metamorphic stages are outlined by red dashed lines, while post-metamorphic cells are enclosed by green dashed lines.

Germ layer-based cell type composition and dynamics during metamorphosis of S. clava.

(A) UMAP plot of all 26,099 cells integrated across five developmental stages, showing the distribution of cell types. Cluster labels represent a combination of developmental stage and cell type identity. (B) Dot plot displaying the expression of representative germ layer-specific marker genes. Dot color indicates the average expression level within each cluster, and dot size reflects the proportion of cells expressing the gene. (C) UMAP plot highlighting cells derived from the three germ layers, showing the distribution of ectoderm-, mesoderm-, and endoderm-derived populations before and after metamorphosis. (D) Bar plot illustrating the proportional changes in cell populations derived from each germ layer across the five developmental stages.

Developmental trajectories of endoderm lineage cells.

(A) UMAP plot showing sub-clustering of endodermal lineage cells and their distribution during metamorphosis. (B) Box plot summarizing the predicted differentiation levels of endodermal lineage cells based on CytoTRACE scores. (C) UMAP overlaid with CytoTRACE differentiation scores of endodermal lineage cells, where red indicates higher differentiation potential states and blue indicates more differentiated states. (D) UMAP plot of endodermal lineage cells colored by developmental stages of trj and mj. (E) Dot plot showing expression levels of representative marker genes distinguishing the two major endodermal lineages. Dot color reflects average expression within each cluster, and dot size indicates the proportion expressing cells. (F) Pseudotime trajectory analysis visualized on the UMAP plot. Cells are colored by pseudotime score. (G) RNA velocity streamlines overlaid on the UMAP plot, indicating the predicted directions of cell state transition within the endodermal lineage. (H) Schematic illustration summarizing the two major endodermal lineages and their respective differentiation fates. (I) (Top) Heatmap showing cross-species transcriptomic similarity between ascidian and mouse endodermal lineages. AUROC scores were calculated using expression of one-to-one orthologous genes. (Bottom) Schematic diagram of mouse definitive endoderm and visceral endoderm lineages. PrE, primitive endoderm; ParE, parietal endoderm; VE, visceral endoderm; emVE, embryonic visceral endoderm; exVE, extra-embryonic visceral endoderm; TE, trophectoderm; ExE, extra-embryonic ectoderm; EPI, epiblast; DE, definitive endoderm.

Characterization of stomach and intestine progenitor cells.

(A) UMAP plot showing sub-clustering of digestive tract cells at the metamorphic juvenile stage. Cells are colored according to CytoTRACE-inferred differentiation scores, with red indicating higher differentiation potential. (B) Dot plot showing enriched Gene Ontology (GO) terms for the top 100 differentially expressed genes in digestive cells compared to all other cells at a certain stage. (C) Heatmap displaying the expression of representative marker genes in the major digestive tract cell types. Each column represents a cell type, and each row represents a marker gene. (D) Schematic diagram of the adult digestive tract (stomach and intestine), with dotted lines indicating the anterior-posterior levels of transverse sections (left). Violin plots showing the expression levels of marker genes (CD166 and AK1A1) in stomach progenitor cells and maker genes (UROM and HMCN1) in intestinal progenitor cells (middle). In situ hybridization results validating marker gene expression in ascidian digestive tract (right). Red and blue arrows indicate signals in stomach and intestinal regions, respectively.

Lineage-specific signaling and cross-species comparison of stomach and intestine progenitor cells

(A) Developmental trajectory of digestive progenitor cells, colored by digestive cell types. Arrowheads indicate inferred directions of differentiation. The inset (right) shows the same trajectory colored by pseudo-time scores, where darker shades indicate closer to the root cells. (B) Heatmap showing gene expressions along the differentiation trajectories of intestinal and stomach progenitors. Lineage-specific transcription factors (TFs) are labeled. The expression dynamics of representative TFs (red) are displayed along the corresponding trajectories. (C) Network showing inferred signaling interactions between digestive progenitor cells and other cell types at the same developmental stage. Edge thickness reflects the strength of inferred communication. (D) Dot plot showing the expression levels of ligand–receptor pairs between digestive tract cell types and interacting cell types. Dot color indicates communication probability, while dot size represents statistical significance (p-value from one-sided permutation test). (E) Heatmap of cross-species transcriptomic similarity between ascidian and mouse digestive progenitor cells. AUROC scores were calculated based on the expression of one-to-one orthologous gene pairs. (F) Sankey diagram showing homologous relationships between ascidian and mouse digestive tract progenitor cells (AUROC > 0.7). Small_int, small intestine.