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.

Subcluster analysis of enteric nervous system (ENS) cells in wildtype and DEAB‐treated zebrafish. (A) Uniform manifold approximation and projection (UMAP) analysis of ENS cells, showing 12 subclusters. UMAP atlas was separated between wildtype and DEAB‐treated zebrafish cells. (B) Marker gene expression profile and ENS subcluster characterization. This heatmap illustrates scaled expression levels (z‐score) of significant marker genes defining each ENS subcluster in (A). Clustering trees (left panel) reveal relationships among marker genes in each ENS subcluster. ENS subcluster indicator genes are listed adjacent to the trees. Expression levels within the heatmap are color‐coded (Red: Up‐regulated; Blue: Down‐regulated in each ENS subcluster, compared to the rest other ENS subclusters). Colored squares with numbers (right side of the heatmap) indicate the number of marker genes identified for each ENS subcluster. 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 ENS subcluster. (C) Bar plot (top panel) and stacked bar plot (bottom panel) showing the subcluster size number of ENS cells contributing to each subcluster in wildtype (light‐blue) and DAEB‐treated zebrafish (light‐purple) samples. (D) Cluster size comparison of each ENS subcluster between wildtype and DEAB‐treated zebrafish cells. Significantly differentially distributed subclusters 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. Dotted line indicates the threshold of log₂ fold change. (E) Gene set enrichment analysis (GSEA) of hallmark gene signatures representing different functionalities of ENS subclusters [45]. Normalized enrichment scores within the heatmap are color‐coded; blue indicates downregulated pathways, and red indicates up‐regulated pathways in DEAB‐treated zebrafish ENS. The significance threshold of FDR adjusted p value (Adjusted p) < 0.05 was applied.

Increased epithelial subcluster populations in DEAB‐treated zebrafish. (A) Uniform manifold approximation and projection (UMAP) analysis of epithelium showing all nine subclusters. UMAP atlas was separated between wildtype and DEAB‐treated zebrafish cells. (B) Marker gene expression profile and epithelial subcluster characterization. This heatmap illustrates scaled expression levels (z‐score) of significant marker genes defining each epithelial subcluster in (A). Clustering trees (left panel) reveal relationships among marker genes in each epithelial subcluster. Epithelial subcluster indicator genes are listed adjacent to the trees. Expression levels within the heatmap are color‐coded (Red: Up‐regulated; Blue: Downregulated in each epithelial subcluster, compared to the rest other epithelial subclusters). Colored squares with numbers (right side of the heatmap) indicate the number of marker genes identified for each epithelial subcluster. 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 epithelial subcluster. (C) Bar plot (top panel) and stacked bar plot (bottom panel) showing cell numbers contributing to each subcluster of epithelial cells in wildtype (light‐blue) and DEAB‐treated zebrafish (light‐purple) samples. (D) Cluster size comparison of each epithelium subcluster between wildtype and DEAB‐treated zebrafish cells. Significantly differentially distributed subclusters are represented in orange. Significance was determined by the threshold of the false discovery rate (FDR) < 0.05 and absolute log₂ fold change > 0.58. The dotted line indicated the threshold of log₂ fold change. (E) Gene set enrichment analysis (GSEA) of hallmark gene signatures representing different functionalities of epithelium subclusters [45]. Normalized enrichment scores within the heatmap are color‐coded; blue indicates downregulated pathways and red indicates up‐regulated pathways in ret mutant zebrafish epithelium. The significance threshold of FDR adjusted p value (Adjusted p) < 0.05 was applied.

Decreased immune cell populations and suppressed inflammatory responses in DEAB‐treated zebrafish. (A) Uniform manifold approximation and projection (UMAP) analysis of immune cells showing all 6 subclusters. UMAP atlas was separated between wild type and DEAB‐treated zebrafish cells. (B) Marker gene expression profile and immune subcluster characterization. This heatmap illustrates scaled expression levels (z‐score) of significant marker genes defining each immune subcluster in (A). Clustering trees (left panel) reveal relationships among marker genes in each immune subcluster. Immune subcluster indicator genes are listed adjacent to the trees. Expression levels within the heatmap are color‐coded (Red: Up‐regulated; Blue: Downregulated in each immune subcluster, compared to the rest other immune subclusters). Colored squares with numbers (right side of the heatmap) indicate the number of marker genes identified for each immune subcluster. 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 immune subcluster. (C) Bar plot (top panel) and stacked bar plot (bottom panel) showing cell numbers contributing to each subcluster of immune cells in wildtype (light‐blue) and DEAB‐treated zebrafish (light‐purple) samples. (D) Cluster size comparison of each immune cell subcluster between wildtype and DEAB‐treated zebrafish. Significantly differentially distributed subclusters 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 indicated the threshold of log₂ fold change. (E) Gene set enrichment analysis (GSEA) of hallmark gene signatures representing different functionalities of immune subclusters [45]. Normalized enrichment scores within the heatmap are color‐coded; blue indicates downregulated pathways and red indicates up‐regulated pathways in DEAB‐treated zebrafish immune cells. The significance threshold of FDR adjusted p value (Adjusted p) < 0.05 was applied. NES: Normalized enrichment score.

Increased myofibroblast populations and altered ECM dynamics in DEAB‐treated zebrafish. (A) Uniform manifold approximation and projection (UMAP) analysis of extracellular matrix (ECM) showing all 11 subclusters. UMAP atlas was separated between wildtype and DEAB‐treated zebrafish cells. (B) Marker gene expression profile and ECM subcluster characterization. This heatmap illustrates scaled expression levels (z‐score) of significant marker genes defining each ECM subcluster in (A). Clustering trees (left panel) reveal relationships among marker genes in each ECM subcluster. ECM subcluster indicator genes are listed adjacent to the trees. Expression levels within the heatmap are color‐coded (Red: Up‐regulated; Blue: Downregulated in each ECM subcluster, compared to the rest other ECM subclusters). Colored squares with numbers (right side of the heatmap) indicate the number of marker genes identified for each ECM subcluster. 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 ECM subcluster. (C) Bar plot (top panel) and stacked bar plot (bottom panel) showing the cell number contributing to each subcluster of all ECM cells in wildtype (light‐blue) and DEAB‐treated zebrafish (light‐purple) samples. (D) Cluster size comparison of each ECM subcluster between wildtype and DEAB‐treated zebrafish cells. Significantly differentially distributed subclusters are represented in orange. Significance of differences was determined by the threshold of t false discovery rate (FDR) < 0.05 and absolute log₂ fold change > 0.58. The dotted line indicated the threshold of log₂ fold change. (E) GSEA of hallmark gene signatures representing different functionalities of ECM subclusters [45]. Normalized enrichment scores within the heatmap are color‐coded; blue indicates the downregulated pathways in DEAB‐treated zebrafish ECM subclusters, and red indicates the up‐regulated pathways in DEAB‐treated zebrafish ECM. The significance threshold of FDR adjusted p value (Adjusted p) < 0.05 was applied. (F) Violin plot showing collagen scores across ECM subclusters fibroblasts‐1 and myofibroblasts‐1 in wildtype (blue) and DEAB‐treated (light‐purple) zebrafish. p‐value was calculated by t test. ***p < 0.001.

Single‐cell transcriptomic analysis of intestinal cell populations in wildtype, ret mutant, and DEAB‐treated zebrafish. (A) Uniform manifold approximation and projection (UMAP) analysis of scRNA‐seq showing nine distinct cell types. UMAP atlas was separated between wildtype, ret mutant, and DEAB‐treated zebrafish cells. (B) Bar plot (top panel) and stacked bar plot (bottom panel) showing the cell number contributing to each subcluster of all intestinal cells in wildtype (light‐blue), ret mutant (light‐purple) and DEAB‐treated zebrafish (pink) samples. (C) Feature dot plot representing expression levels of selected genes per subcluster between wildtype, ret mutant, and DEAB‐treated zebrafish cells. Expression levels within the dot plot are color‐coded (Pink: Up‐regulated; green: Downregulated, in each cell type compared to the rest other cell types). Gene expressed percentages in each cell type among wildtype, ret mutant, and DEAB‐treated zebrafish are indicated by dot sizes.