Figure 1. Role of cell competition after irradiation.
Panels A-G and I-L show third instar wing imaginal discs labeled to detect the activated Dcp1 caspase in apoptotic cells at the indicated times post-irradiation. (A) Extensive cell death follows within 4 hr after gamma-irradiation (4000 Rads). (B) Little change is seen in the rpS12G97D mutant. (C) Most of the acute cell death is p53-dependent, consistent with a DNA damage response. (D) A small amount of cell death persists in the rpS12G97D p53- double mutant. (E) Cell death is reduced to more quantifiable levels 4 hr after a lower radiation dose (500 Rad). (F) This DNA damage-induced cell death is not significantly affected by the rpS12G97D mutation. (G) Most cell death 4 hr after irradiation is p53-dependent. (H) Quantification of cell death (numbers of cells per wing pouch) 4 hr following irradiation with 500 Rad. ns – difference not statistically significant (p>0.05). ** - difference highly significant (p<0.01). N = 6 for each genotype. (I) Cell death 24 hr after irradiation (4000 Rad). (J) Comparable levels of cell death in rpS12G97D mutants. (K) Although cell death appears reduced in p53 mutant wing discs 24 hr after irradiation compared to the wild type control (4000 Rad), this p53-independent cell death is substantially increased compared to 4 hr after irradiation (compare panel C). (L) p53-independent cell death is reduced in the rpS12G97D p53- double mutant compared to the p53- mutant. (M) Cell death quantification in p53 and rpS12G97D p53- wing discs, and in wing discs from rpS12G97D p53- larvae carrying genomic transgenes encoding either wild type rpS12 or rpS12G97D. The results show that between 66% and 86% of p53-independent cell death was RpS12-dependent. We did not quantify cell death in wild-type and rpS12G97D wing discs because of the large number and aggregation of dead cells. ns – difference not statistically significant (p>0.05). ** - difference highly significant (p<0.01). N: 10 (p53-); 13 (rpS12G97D p53-); 9 (P{rpS12+} rpS12G97D p53-); 8 (P{rpS12G97D} rpS12G97D p53-). (N) Twenty-four hr after irradiation, p53 mutant wing discs exhibit cells expressing nuclear Xrp1 in a pattern similar to that of dying cells. We were unable to double-label with anti-Xrp1 and anti-active Dcp1 simultaneously because both are rabbit antisera. n: 33 (p53-); 26 (rpS12G97D p53-). (O) Fewer Xrp1-expressing cells were seen in rpS12G97D p53- double mutant wing discs. (P) Quantification of Xrp1 expression. Most (58%) of the p53-independent Xrp1 expression 24 hr post-irradiation was RpS12-dependent. ** - difference highly significant (p<0.01). (Q) Irradiated adult flies with a short, thin scutellar bristle (white arrow, compare normal contralateral bristle – black arrow) like those typical of Rp+/- mutant flies. (R) The frequency of sporadic Rp-like bristles increases 3.33x and 4x on the thoraces of rpS12G97D and rpS12G97D p53- flies (respectively) where cell competition is inhibited. ns – difference not statistically significant (p>0.05). ** - difference highly significant (p<0.01). N = 3 sets of 100 flies for each genotype. (S) Frequencies of y bristles found on the thoraces of ~2000 y/+; rpS12G97D female and +/Y; rpS12G97D male flies following irradiation (1000 rad) in the mid-third larval instar. The preponderance of y bristles in females suggests that induced y mutations typically affect other genes including essential genes. The occurrence of phenotypically y M bristles in females is consistent with deletions extending at least from the y locus to the nearest Rp locus, RpL36. Statistics: Significance was assessed using t-test (panel P) or one-way ANOVA with the Holm procedure for multiple comparisons (panels H,M,R). ** = p<0.01. ns = not significant. Genotypes: A, (E, I) w11-18 B, (F, J) rpS12G97D (C,G,K,N) p535A-1-4 D, (L, O) rpS12G97D p535A-1-4.