Fig. 7. Reversibility of DAPT treatment. (A-D) Whole-mount double staining of metanephroi with anti-Wt1 and DBA; (E-M) sections through metanephroi cultured under conditions similar to the wholemounts. The antigens detected are indicated on the right in the appropriate colors. In all sections, bars at the bottom reflect days in DMSO (white bars) and/or in DAPT (blue bars). Note that NICD and Jag1(J) reside in the same cells (insets and frames in J). Expression of both is inhibited by DAPT (K) but recovers after DAPT removal (frames in L,M). After a 2-day inhibition, Wt1^HIGH cells are again detected throughout the metanephroi (C,G). After 3.5 days, despite recovery of NICD and Jag1 (K,L), no Wt1^HIGH cells appear at the periphery, and only Wt1^LOW; Pax2-positive (not shown) cells increase in abundance, and branching resumes (D,H,I). We conclude that a crucial period exists between day 2 and 3 in DAPT, after which restoration of Notch signaling fails to rescue podocyte differentiation. This is summarized in I.