Figure 3.

Sox100B Is Required for Sox21a Expression in ISCs and EBs

(A and B) Representative confocal images of 9-day-old MARCM clones showing that Sox21a expression is reduced in Sox100B^d1 homozygous mutant clones. Expression level of Sox21a is quantified based on Sox21a antibody staining and relative Sox21a intensity is calculated by comparing Sox21a intensity within Sox100B^d1 MARCM clones to that of the nearest Sox21a-positive cell outside clones, and quantification is shown in (B). In (A), Delta and Prospero staining are separated by cellular localization (Delta, membrane staining; Prospero, nuclear staining).

(C and D) Representative images of Sox21a antibody staining in wild-type- and Sox100B^RNAi- expressing posterior midguts, illustrating the reduced Sox21a expression after 7day Sox100B knockdown. Category scoring of endogenous Sox21a expression is performed and quantification is shown in (D).

(E and F) Sox21a intensity quantification in individual cells specifically in ISCs (E) and EBs (F) confirms that Sox100B is required for Sox21a expression in both ISCs and EBs.

(G) qRT-PCR data demonstrates that Sox21a mRNA expression is strongly reduced in the intestine, while escargot and Delta mRNA expression are slightly induced after ISC/EB-specific Sox100B knockdown for 7 days. All mRNA expression is normalized to actin5c expression. Four independent biological replicates were analyzed per condition.

In (D), p values are calculated using Fisher's exact test (p < 10⁻⁵ versus control); the number of guts scored for each condition is indicated above the bar. In (B) and (E)–(G), values are presented as averages ± SEM; p values are calculated using unpaired two-tailed Student's t test; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001.