Figure 4

Epithelial subcluster identification and functional pathway shifts in ret mutant zebrafish

(A) Uniform manifold approximation and projection (UMAP) analysis of the epithelium shows all 9 subclusters. UMAP atlas was separated between wildtype and ret mutant zebrafish cells.

(B) Feature dot plot represents the expression level of the top 5 transcripts (ordered by average log2 fold change) per subcluster of epithelium between wild-type and ret mutant zebrafish cells. The colored dot on the top indicates subclusters, as shown in (A), and features were separated according to subclusters.

(C) Cluster size comparison of each epithelium subcluster between wildtype and ret mutant zebrafish cells. Significantly differentially distributed subclusters are represented in orange. The significance of differences 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.

(D) Bar plot (top) and stacked bar plot (bottom) show the cell numbers contributing to each subcluster of all epithelial cells in wildtype (light-blue) and ret mutant zebrafish (light-purple) samples.

(E) Gene set enrichment analysis (GSEA) of hallmark gene signatures represents different functionalities of epithelium subclusters.³⁵ Blue indicates down-regulated pathways in ret mutant zebrafish epithelium subclusters, 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. NES: Normalized enrichment score. Due to only 1 cell being contained in the ret mutant enterochromaffin cells cluster, differentially expressed genes could not be detected, resulting in the enterochromaffin cells cluster being excluded from the GSEA approach.

(F) Heatmap of differentially expressed genes, which were involved in significantly altered hallmark pathways between wildtype and ret mutant epithelium subclusters. Green indicates down-regulated genes in ret mutant epithelium subclusters, while pink indicates up-regulated genes in ret mutant epithelium subclusters.

(G) Pseudotime trajectories (left) show the developmental track of the epithelium in wildtype and ret mutant zebrafish intestines. Dark-blue indicates lower pseudotime scores, light-blue indicates higher pseudotime scores, and the number within trajectories indicates the branch node. Ridge chart (right) shows the distribution of epithelium along with pseudotime trajectory (left), separating between wildtype and ret mutant zebrafish. Box chart shows the statistical pseudotime score analysis between wildtype and ret mutant epithelium. t test was used to calculate p value.

(H) Subcluster-based individual ridge charts show the distribution of each epithelium subcluster along with pseudotime trajectory, as shown in (G) right, separating between wildtype and ret mutant samples. Box chart shows the statistical pseudotime score analysis between wildtype and ret mutant epithelium. t test was used to calculate p value. NS.: Not significant; ∗∗: p < 0.01 and ∗∗∗: p < 0.001.