TUNEL detection confirmed that there were dying cells in scrib mutant clones expressing aPKCCAAXDN and the ectopic expression of the JNK pathway reporter, msn-lacZ, in the mutant tissue suggested that this was due to a failure to rescue JNK-dependent cell death.
The mean and stdev for the percentage of wild-type or btd mutant clones expressing UAS-pntP1 driven by tub-GAL4 are calculated by bootstrapping.
Consistent with this, scrib mutant clones expressing both aPKCCAAXDN and BskDN still showed occasional scaring at clonal edges suggestive of an impaired cell adhesion.
Furthermore, scrib mutant clones expressing aPKCCAAXDN no longer exhibited ectopic CycE or BrdU incorporation posterior to the MF, although ectopic CycE and BrdU positive cells were still sometimes observed surrounding the mutant clones of tissue (data not shown).
Indeed, we found that some lgl-/ mutant clones expressing Rab5DN in the proximal regions overgrew (13 out of 27 scored, Figure 5C), while clones in the wing pouch never did.
We recovered large baz mutant clones expressing BazS980A-GFP only rarely, and the resulting egg chambers often had large gaps in the follicle cell layer, suggesting that mutant cells fail to integrate into the epithelium.
Elav and phalloidin staining generally revealed a normal regular array of differentiating ommatidial clusters in scrib mutant clones expressing aPKCCAAXDN, although sometimes clonal borders showed separation between the mutant and wild-type tissue resulting in tissue scars (data not shown) and occasional basally retracted mutant photoreceptor nuclei.
Only in 3 out of total 10 btd mutant clones expressing UAS-PntP1, we observed that mature INPs were partially rescued to 9.3 ± 3.2 per lineages, which is still much fewer than the number of mature INPs (20 30 per lineages) in normal type II NB lineages.
Genotypes for making brm mutant clone: hsflp, tub-Gal4, UAS-GFPnls; tubGal80 FRT80B/FTR80B brm 2. brm mutant clone expressing Yki transgene: hsflp, tub-Gal4, UAS-GFPnls;UAS-Yki/+;tubGal80 FRT80B/FTR80B brm 2. Hpo mutant clone expressing Brm transgene: yw UAS-GFP hsflp FRT42D hpo BF33 /FRT42D tub-Gal80; tub-Gal80al4/Brm.
SDS-PAGE Coomassie and Western blot analyses confirmed that the espG/orf3Δ core mutant clone expressed and secreted wild-type levels of the EspA,B,D translocator proteins in the absence of Map, Tir or Intimin with EspF secreted levels reduced due to the absence of its chaperone, CesF (data not shown).
