Figure 2.

Sox100B Is Required for Proper ISC Differentiation

(A and B) Representative confocal images of 7-day-old MARCM clones in the adult posterior midgut showing that Sox100B^d1 homozygous mutant ISCs maintain Delta expression––indicated by arrowheads in (A)––and the ability to form multicellular clones. Note that absence of Sox100B antibody staining in the Sox100B^d1 homozygous mutant clones further validates the mutant allele. Number of cells per clone is plotted in (B) showing that Sox100B is not essential for ISC proliferation. In (A) Delta and Prospero staining are separated by cellular localization (Delta, membrane staining; Prospero, nuclear staining).

(C) Cell size distribution in Sox100B^d1 homozygous mutant clones is affected compared with control clones. Cell shape within clones is outlined by membrane Arm staining and sizes of individual cell represented by pixels in the outlined area is plotted.

(D–G) Clonal analysis of Sox100B^d1 mutant 7-day-old MARCM clones using differentiation markers shows that proper ISC differentiation to ECs and EEs is impaired. Representative confocal images are shown in (D). Numbers of Pdm1-positive ECs and Prospero-positive EEs are counted in individual clones and the ratios to total cell number in each clone are plotted in (E) and (G), respectively. Distributions of total clones categorized by whether containing Pdm1-positive ECs in control and Sox100B^d1 mutant clones is shown in (F).

(H–K) Sox100B^RNAi-expressing 5-day-old MARCM clones shows normal clone size (I) but impaired EC differentiation (J), confirming that Sox100B is not required for ISC proliferation but is required for EC differentiation. EE differentiation appears to be not affected in Sox100B^RNAi-expressing MARCM clones (K).

In (A), (D), and (H), GFP expression labels MARCM clones. Scale bars, 25 μm. In (B), (C), (E), (G), and (I)–(K), values are presented as averages ± SEM; all p values are calculated using unpaired two-tailed Student's t test; ^∗p < 0.05, ^∗∗∗∗p < 0.0001. In (F), the p value is calculated using the chi-squared test (p < 10⁻¹⁶).