(A and B) Phospho-histone H3 (Ser10) (PH3) staining was done to indicate mitoses in esg-Gal4ts > RFP (A) and esg-Gal4ts > RFP+UAS-upd1 (B) Malpighian tubules. After 6 days of expression of Upd1, mitoses were readily present. Inset: enlarged image of the dashed box showing PH3+ cells are esg >RFP+ RSCs. (C) Quantification of RSCs proliferation induced by expression of UAS-upd1. Data represent means ± SD, *** denotes Student’s t test p<0.001. (D) Representative image of EdU labeling after 2d in wild type Malpighian tubules. Yellow arrowheads denote esg >RFP+ RSCs labeled with EdU. Insets are enlarged images of dashed boxes. (E) Quantified percentage of EdU-positive RSCs after continuous EdU labeling for 2 days and 4 days, n > 10 female flies. (F) Cell cycle phase distribution of RSCs as assessed by FUCCI. (G–J) MARCM clones induced by different heat shock dose. Compared to no heat shock control (G), there was a significant increase in the GFP marked clones in the midgut but not Malpighian tubules after 2X heat shocks (H); the yellow asterisk denotes the autofluorescence from fecal contents in the rectum. (I) Induced clones were seen in the SCZ with 6X heat shocks. As expected, labeling was restricted to the ureter and lower tubules, where RSCs are located. (G’ and I’) Enlarged DAPI channels of the SCZ (dashed red box) corresponding to G and I, respectively. (J) Representative image showing both single cell transient clones (outlined in dashes; the single cell is denoted by yellow arrowhead) and multi-cell RSC clones (outlined in dashes), containing a single Dl+ RSC (denoted by red triangle). No Dl+ cells were detected in the single-cell transient clones. (K) Representative image of GFP marked clones in the lower tubules showing the differential expression levels of Cut in different cell types in RSC clones (outlined by yellow dashes). Cut is expressed at a low level in a single 2 C cell in each clone that is presumably an RSC (white arrows). Downstream cells that are often of higher ploidy show higher Cut expression. For (C), (E) and (F), see also Figure 6—source data 1.