Figure 2. Spatial transcriptomic analysis revealed altered secretory lineage progenitor signature in the DDX5^ΔIEC mice.

A. Left: H&E staining of the small intestinal swiss rolls from one pair of female WT^IEC and DDX5^ΔIEC littermates used for the spatial transcriptomic analysis (Visium, 10X Genomic). Right: Loupe browser displaying Atoh1^high or Sox4^high spots on each tissue section. Scale bar: 2mm.

B. Heatmap indicating the average expression of IEC lineage-specifying genes in Atoh1^high or Sox4^high spots indicated in A.

C. Schematic of establishing intestinal crypt organoid cultures.

D. Left: Representative bright field images of organoids established from small intestinal crypts of WT^IEC and DDX5^ΔIEC mice following the protocol outlined in⁵⁰. Scale bar, 100μm. Right: Normalized mRNA expressions of Pou2f3 and Vil1 from WT^IEC (n=2) and DDX5^ΔIEC (n=2) small intestinal organoids cultured over 4–7 passages. Each dot represents the result from an independent organoid experiment. Results were averaged from two independent biological experiments. * p<0.05 (unpaired t-test).

E. Left: Representative bright field images of organoids established from colonic crypts of WT^IEC and DDX5^ΔIEC mice following the protocol outlined in⁵⁰. Scale bar, 100μm. Right: Normalized mRNA expressions of Pou2f3 and Vil1 from WT^IEC and DDX5^ΔIEC colonic organoids cultured over 4–7 passages. Each dot represents the result from an independent organoid experiment. Results were averaged from two independent biological experiments. * p<0.05 (unpaired t-test).

F. Left: HOMER de novo analysis identified the top 5 motifs present at the promoters (defined as −1 kb to +500 bp from the transcription start site) of all ileal IEC DDX5-dependent genes identified in Figure 1A³⁹. Promoters from all other IEC expressed genes were used as background. Right: The presence (red) or absence (black) of the motifs indicated on the left at the tuft cell DDX5-dependent gene promoters.

G. Average log₂ fold change of the transcript abundance of tuft cell-expressed genes harboring SIX, HMX, POU2F, NFY motifs, or None of the above motifs at their promoters in ileal IECs from two independent pairs of WT^IEC and DDX5^ΔIEC littermates. Each dot represents one gene.