FIGURE 1

Single‐cell transcriptomic profile of zebrafish intestinal cells in wildtype and DEAB‐treated zebrafish. (A) Representative images of ENS phenotypes in tg(phox2bb:GFP) zebrafish treated with increasing concentrations of DEAB, showing varying degrees of aganglionosis. Scalebar indicated 100 μm (B) Quantification of ENS phenotypes across tg(phox2bb:GFP) zebrafish treated with increasing DEAB concentrations (n = 20 per condition). The graph illustrates the distribution of phenotypes in percentage, including normal, short‐segment, and total colonic aganglionosis. (C) Uniform manifold approximation and projection (UMAP) analysis of scRNA‐seq showing 9 distinct cell types. UMAP atlas was separated between wildtype and DEAB‐treated zebrafish cells. (D) Marker gene expression profile and cell type characterization. This heatmap illustrates scaled expression levels (z‐score) of significant marker genes defining each cell type in (C). Clustering trees (left panel) reveal relationships among marker genes in each cell type. Cell type indicator genes are listed adjacent to the trees. Expression levels within the heatmap are color‐coded (Red: Up‐regulated; Blue: Down‐regulated, compared to the rest other cell types). Colored squares with numbers (right side of the heatmap) indicate the number of marker genes identified for each cell type. Colored text boxes (right panel) display the top 5 (ordered by p value) enriched Gene Ontology Biological Pathways (GO:BP) derived from the marker genes of each cell type. (E) Bar plot (top panel) and stacked bar plot (bottom panel) showing cell numbers contributing to each cluster for all cell types in wildtype (light‐blue) and DEAB‐treated zebrafish (light‐purple) samples. (F) Cluster size comparison of each cell type between wildtype and DEAB‐treated zebrafish cells. Significantly differentially distributed clusters are represented in orange. Significance was determined by a threshold of the false discovery rate (FDR) < 0.05 and absolute log₂ fold change > 0.58. The dotted line indicates the threshold of log₂ fold change. (G) Expression pattern of ret in the UMAP atlas of wildtype and DEAB‐treated zebrafish samples. Navy‐blue indicates low expression levels, while yellow indicates high expression levels. Gray shows non‐ret expressing cells. (H) Violin plot showing ret expression levels across different cell types in wildtype (blue) and DEAB‐treated (light‐purple) zebrafish.