Rae1 is required for cycB accumulation, overgrowth, and survival upon loss of Hippo signaling.
(A) Wts loss (MARCM wtsx1 clones) in eye discs increases cycB protein levels (lane 2) compared to control FRT82B discs (lane 1). Concomitant reduction of Rae1 either through RNAi (lane 3) or removing a genomic copy (lane 4) restores cycB levels. (B) Sav loss (MARCM sav4 clones) in eye discs increases cycB protein levels (lane 2) compared to control FRT82B discs (lane 1). Concomitant reduction of Rae1 either through RNAi (lane 3) or removing a genomic copy (lane 4) restores cycB levels. Relative levels of cycB (normalized to Tubulin) are indicated for blots in A, B. (C) Control wing (nub>dcr). (D) RNAi to hpo in the wing using nubgal4 (nub>dcr, hpoIRT) causes wing overgrowth. (E-F) Reducing Rae1 levels slightly by removing one copy suppresses tissue overgrowth (E, nub>dcr, hpoIRT, Rae1ex28/+) while greater reduction in Rae1 levels by RNAi both suppresses overgrowth and causes tissue lethality (F, nub>dcr, hpoIRT, Rae1IRV). (G) Control wing showing overgrowth due to hpo RNAi using the wing driver ms1096gal4 (ms1096>hpoIRT) at 25°C. (H-H”) Removing one copy of Rae1 using Rae1ex28 (ms1096>hpoIRT, Rae1ex28/+) suppresses the overgrowth (H). The gal4/UAS system is temperature responsive. Increasing the temperature leads to increased expression of inverted repeats and increased knockdown of target genes. Increasing the temperature to 27°C (H’) and 30°C (H”) further decreases Hippo signaling by increased RNAi to hpo. Blue overlaid tracings show control ms1096>hpoIRT wing outlines from the indicated temperatures; wings are more overgrown but do not lie flat; overlaid traced images of these wings are smaller than actual wing size. Removing one copy of Rae1 (which maintained the Rae1 gene dosage to that in H) causes more dramatic tissue loss with further increasing the temperature to 27°C (H’) and 30°C (H”). (I) Control en>dcr wing at 18°C. (J) RNAi to hpo (en>dcr, hpoIRT) in the posterior wing at 18°C; wings are so enlarged that flattened, mounted wings appear smaller than they are; see S12C and S12D Fig for examples of overgrown wings still attached to flies corresponding to the wings in J, K). (K-L) Reducing Rae1 by concurrent Rae1 RNAi (en>dcr, hpoIRT, Rae1IRV, K), or introducing Rae1ex28 (L) suppresses overgrowth and causes overgrowing wings to blacken and shrivel (tracing of a control en>dcr, hpoIRT wing is overlaid to highlight growth suppression). (M-P) RNAi to Rae1 causes growth suppression and tissue disruption upon concurrent RNAi to Mer (en>dcr, merIRN, Rae1IRV, N) compared to RNAi to Mer alone (en>dcr, merIRN, Rae1IRV, M, green tracing in N) or concurrent RNAi to ex (en>dcr, exIRT, Rae1IRV, P) compared to RNAi to ex alone (en>dcr, exIRT, O, purple tracing in P). Overgrowth due to wts RNAi with engal4 was so extensive wings were too overgrown to mount for comparison. (Q) Control wing (c5gal4/+). (R) RNAi to wts in the wing using c5gal4 (c5>wtsIRT) causes wing overgrowth (purple tracing in T, U, and V). (S) RNAi to yki (c5>wtsIRT, yikIRN, yellow tracing in T) suppresses tissue overgrowth, restoring wing size to that of a control wing (black tracing). (U-V) In contrast to yki RNAi in S-T, removing one copy of Rae1 using Rae1ex28 not only suppresses overgrowth but promotes significant tissue loss (c5>wtsIRT, Rae1ex28/+, U, pink tracing in V). The resulting wings are significantly smaller than either control wings (Q) or wings suppressed by yki RNAi (S); this is highlighted by an overlay of tracings in (V). Increasing the temperature to 27°C (U’) and 30°C (U”) further decreases Hippo signaling by increased RNAi to wts. Blue overlaid tracings show control c5>wtsIRT wing outlines from the indicated temperatures; as noted earlier, overgrowth in these context is quite dramatic such that wings no longer lie flat; mounted wings thus wrongly appear smaller once they are flattened to be photographed. Under these conditions, removing one copy of Rae1 (which maintained the Rae1 gene dosage to that in U) causes even more dramatic loss of tissue.