Figure 4(A) Old and new H3T3A distribution in prophase ISC, showing separable domains between H3T3A-GFP (old) and H3T3A-mKO (new) (esg-Gal4>UASp-FRT-H3T3A-eGFP-FRT-H3T3A-mCherry). Data information: Scale bar in (A), (C), (E), (F): 5 µm,(B) Quantification of colocalization between H3T3A-GFP (old) and H3T3A-mKO (new) in prophase and prometaphase ISCs using Pearson's correlation coefficients. The H3 and H2A data are from Figure 2C for direct comparison. Old and new H3T3A distribution is more similar to that of H3 (Pearson's correlation coefficient for H3T3A= 0.49 + 0.02, n = 55 ISCs). Individual data points and mean values are shown. Error bars represent SEM. **** p < 0.0001, * p < 0.05; unpaired t test to compare two individual datasets to each other.(E) In a post-mitotic pair of cells with comparable Dl-nLacZ, both H3T3A-GFP (old) and H3T3A-mKO (new) are symmetrically distributed between the two cells. (F) In a post-mitotic pair of cells with asymmetric Dl-nLacZ expression, H3T3A-GFP (old) is enriched in the Delta-high cell, while H3T3A-mKO (new) is more toward the Delta-low cell. Data information: Scale bar in (E), (F): 5 µm, asterisk, ISC side.(H) Distribution of the Dl-nLacZ-positive (magenta) cells in an H3-expressing midgut, which are well spaced and interspersed within the intestinal epithelium. (I) Distribution of the Dl-nLacZ-positive (magenta) cells in an H3T3A-expressing midgut, which are unevenly distributed in clusters with two or more Delta-positive cells. DAPI: white in (H-I). Data information: Scale bar in (H) and (I): 50 µm(K) Percentages of clusters with 1-, 2-, or >3-Delta-positive cell clusters for H3-expressing midguts (avg. percentage of 1-Dl+ cells = 87.7% + 1.1%, avg. percentage of 2-Dl+ cells = 11.9% + 0.9%, avg. percentage of >3-Dl+ cells = 0.4% + 0.2%, N = 4 midguts, n = 1,482 clusters) and H3T3A-expressing midguts (avg. percentage of 1-Dl+ cells = 68.2% + 1.2%, avg. percentage of 2-Dl+ cells = 28.5% + 0.9%, avg. percentage of >3-Dl+ cells = 3.3% + 0.4%, N = 5 midguts, n = 1,862 clusters). Bars represent mean proportions of cell clusters with 1, 2, or 3+ Dl+ cells out of all cell clusters counted. Error bars represent SEM. Comparing H3-expressing midguts with H3T3A-expressing midguts, there was a statistically significant difference in the distributions of Delta-positive cells per cluster, determined by a Chi-Square test (X2(2, n = 1862) = 908.75, **** p < 0.0001). As shown, there are significant increases in 2 and >3-Delta-positive cells per cluster in the H3T3A-expressing intestines when compared to the H3-expressing intestines.(L) Percentages of single Delta-positive cells out of total Delta-positive cells for H3-expressing midguts (avg. single Dl+ cells = 77.9% + 1.9%, N = 4 midguts, n = 1,674 Dl+ cells) and H3T3A-expressing midguts (avg. single Dl+ cells = 50.4% + 1.5%, N = 5 midguts, n = 2,522 Dl+ cells). Bars represent mean proportion of single Dl+ ISCs out of all ISCs counted. Error bars represent SEM. There are fewer single Delta-positive cells in H3T3A-expressing midguts compared to H3-expressing midguts (two sample t test, *** p < 0.001).(M) Comparison of the mitotic index in H3-expressing (N = 8 midguts) and H3T3A-expressing (N = 8 midguts) midguts. Total number of mitotic ISCs were quantified per midgut by the presence of H3S10 phosphorylation mark. H3T3A-expressing midguts had a significantly higher mitotic index when compared to H3-expressing midguts (two sample t test, * p < 0.05). Bars represent mean number of mitotic cells per midgut. Error bars represent SEM.