Df(3L)H99 rescues growth of emc mutant (A-L) Control and mutant clones in eye and wing imaginal disc were induced at the end of first instar and are associated with sibling twin-spot clones marked by two copies of the GFP marker (brighter grey) that serves as internal control for growth. Clones are labeled by the absence of GFP. (M) emc H99 clones induced in the minute background (N) Quantification of the ratio of clone size to twin-spot measured in wing imaginal discs and standard deviations. After log- transformation of clone/twin-spot ratios to ensure normality, one way ANOVA rejected the null hypothesis that these results are the same (p=2.57 x 10-10). The Holm correction for multiple comparison was used to identify significant differences between all pairs of samples. Whereas clone/twin-spot ratios for Df(3L)H99 and dronci29 did not differ significantly from the FRT80 control (p>0.05), that for emc homozygous clones was significantly different (p=4.95 x 10-8). Ratios for both emc Df(3L)H99 and emc dronc clones differed significantly from emc clones (p=2.19 x 10-8 and 4.89 x 10-4, respectively). Clone/twin-spot ratios for emc dronc clones also differed significantly from dronc clones (p=0.044) and from Df(3L)H99 clones (p=0.048). For columns 2-4, *** denotes significant difference from the FRT80 control (p<0.001), NS denotes no significant difference. For columns 5-6, *** denotes significant difference from the emc clones.Genotypes: (A and C) ywhsF;;FRT80/[UbiGFP]FRT80 (B and D) ywhsF;;emcAP6FRT80/[Ubi- GFP]FRT80 (E and G) ywhsF;;dronci29emcAP6emFRT80/[UbiGFP]FRT80 (F and H) ywhsF;;emcAP6 Df(3L)H99 FRT80/[UbiGFP]FRT80 (I and K) ywhsF;;dronci29 FRT80/[UbiGFP]FRT80 (J and L) ywhsF;;Df(3L)H99FRT80/[UbiGFP]FRT80 (M) ywhsF; emcAP6 Df(3L)H99 FRT80/[UbiGFP] M(3)67C FRT80.

Morphogenetic furrow progression is affected by cell death pathways In all panels, the differentiating neurons are marked by Elav (in blue), and mutant cells are identified by the absence of GFP expression (in green). A) The wave of retinal differentiation from posterior to anterior (right to left), marked here by Senseless expression in R8photoreceptor cells (red) is normal in FRT80 control clones. B) emc null cell lacking GFP shows acceleration of retinal differentiation marked by yellow arrow. C) In contrast, retinal differentiation proceeds at the same pace in emc dronc double mutant clones as in nearby wild type regions in 75% of the eye discs. D) emc H99 double mutants show a stronger suppression of the acceleration of retinal differentiation (compare panel B). Genotypes: (A) ywhsF;;FRT80/[UbiGFP] M(3)67C FRT80 (B) ywhsF;;emcAP6FRT80/[Ubi-GFP] M(3)67CFRT80 (C) ywhsF;;dronci29emcAP6 FRT80/[UbiGFP] M(3)67C FRT80 (n=12)(D) ywhsF;;emcAP6 Df(3L)H99 FRT80/[UbiGFP] M(3)67C FRT80.

Diap1 expression and function in emc mutant clones In mosaic eye discs with (A) FRT80 clones, there is no change in Diap1 levels within and outside the clone. However, both (B) emc mutant clones and (C) emc H99 clones show reduced Diap1 levels. (D) In GMR-DIAP1 eye disc mutant for emc furrow progression is normal and is marked by Senseless staining. Similarly, in GMR-p35 eye disc mutant for emc also show normal furrow progression (E). Genotypes: (A) ywhsF;;FRT80/[UbiGFP] M(3)67C FRT80 (B) ywhsF;;emcAP6FRT80/[Ubi-GFP] M(3)67C FRT80 (C) ywhsF;;emcAP6 Df(3L)H99 FRT80/[UbiGFP] M(3)67C FRT80 (D) ywhsF/GMR-DIAP1;;emcAP6FRT80/[Ubi-GFP] M(3)67C FRT80. (E) ywhsF/GMR-p35;;emcAP6FRT80/[Ubi-GFP] M(3)67C FRT80.

Wingless and Dpp signaling are not caspase targets (A) No reduction in the Wg signaling reporter Fz3-RFP was detectable in emc clones. See Figure 4 Figure Supplement 1 for Fz3-RFP expression in the wild type. (B) Retinal differentiation is accelerated in emc nkd3 double mutant clones like in emc clones. Senseless expression in R8 photoreceptor cells and Elav staining in differentiating photoreceptors are shown. (C) p-Mad accumulates around the morphogenetic furrow in eye discs containing control clones(Vrailas & Moses, 2006; Firth, Bhattacharya, & Baker, 2010) (E) Except for the advanced progression, p-Mad levels were unchanged in emc clones. Genotypes: (A) Fz3-RFP/+; emcAP6FRT80/[Ubi-GFP] M(3)67C FRT80 (B) ywhsF;;emcAP6nkd3FRT80/[Ubi-GFP] M(3)67C FRT80 (C) ywhsF;;FRT80/[UbiGFP] M(3)67C FRT80 (D) ywhsF;;emcAP6FRT80/[Ubi-GFP] M(3)67C FRT80.

Ci expression and function in emc mutant clones A) Ci is elevated within emc mutant cells (yellow arrows). This was also true posterior to the morphogenetic furrow (orange arrow) B) Higher Ci was also seen in emc dronc mutant cells, even when the morphogenetic furrow progressed normally, as indicated by Elav staining. C) Ci levels were completely normal in emc H99 clones. D) emc and ci doubly-mutant clone lacking GFP shows accelerated retinal differentiation (blue arrow). Genotypes: (A) ywhsF;;emcAP6FRT80/[Ubi-GFP] M(3)67C FRT80 (B) ywhsF;;dronci29emcAP6 FRT80/[UbiGFP] M(3)67C FRT80 (C) ywhsF;;emcAP6 Df(3L)H99 FRT80/[UbiGFP] M(3)67C FRT80 (D) ywhsF;;emcAP6FRT80/[Ubi-GFP] ci+ M(3)67C FRT80;ci[94]/ci[94].

Non-apoptotic caspase signaling targets Notch pathway by promoting Dl expression (A) E(spl), an important N target for lateral inhibition, is elevated in the morphogenetic furrow region of emc clones (yellow arrows). (B) emc dronc clones have normal levels of E(spl) protein.(C) Senseless staining shows a neurogenic phenotype in psn mutant clones, due to reduced Notch signaling. Morphogenetic furrow progression is unaffected. (D) A neurogenic phenotype was also observed in emc psn clones, along with normal furrow progression. (E) Su(H) mutant clones identified by absence of GFP labeling show a strong neurogenic phenotype as well as advanced retinal differentiation (orange arrow) (Li & Baker, 2001). The cell-autonomous effect results in a discontinuity at the borders of Su(H) clones, where differentiation outside the clones lags that within clones (orange arrow) Genotypes: (A) ywhsF;;emcAP6 FRT80/[Ubi-GFP] M(3)67C FRT80 (B) ywhsF;; dronci29emcAP6 FRT80/[UbiGFP] M(3)67C FRT80 (C) ywhsF/+;;psnV1 FRT80/[Ubi-GFP]M(3)67CFRT80 (D) ywhsF/+;; emcAP6 psnV1 FRT80/[Ubi- GFP]M(3)67CFRT80 ((E) ywhsF; Su(H)D47FRT40/FRT40[UbiGFP].

Caspase regulation of Delta levels (A) Normal levels of Delta protein are seen in FRT80 clones as compared to elevated Delta levels in (B) emc clones whereas that phenotype is reversed in (D) emc H99 clones. However, as seen in (C) dronc emc clones show high Delta levels. Genotypes: (A) ywhsF;;FRT80/[UbiGFP] M(3)67C FRT80 (B) ywhsF;;emcAP6FRT80/[Ubi-GFP] M(3)67C FRT80 (C) ywhsF;;dronci29emcAP6 FRT80/[UbiGFP] M(3)67C FRT80 (D) ywhsF;;emcAP6 Df(3L)H99 FRT80/[UbiGFP]FRT80.

Caspases contribute to R7 photoreceptor and cone cell defects in emc mutants In all panels emc mutant cells are marked by the absence of GFP expression (in green) and photoreceptor neurons are marked by Elav in blue. (A) Runt (in red) is expressed in R7 and R8 (yellow arrowhead) photoreceptor cells inside and outside the clone in FRT80 controls. (B) Inside emc clone, Runt expression is lost from R7 cells, while expression in R8 cells remains unaffected (red arrow). (C) However, inside the emc H99 clones, Runt is expressed in both R7 and R8 cells. (D) Cut (in red) is expressed in cone cells in FRT80 controls. (E) Inside emc clone, cut is delayed. (F) However, inside the emc H99 clones, cut staining is not delayed. Genotypes: (A, D) ywhsF;;FRT80/[UbiGFP] M(3)67C FRT80 (B, E) ywhsF;;emcAP6FRT80/[Ubi-GFP] M(3)67C FRT80 (C, F) ywhsF;;emcAP6 Df(3L)H99 FRT80/[UbiGFP] M(3)67C FRT80.

Model of emc effects on Drosophila eye development.

A) Loss of emc allows Da protein to form heterodimers and activate ex transcription, increasing SHW pathway activity. SHW activity reduces DIAP1 expression, thereby derepressing caspase activity. In the eye, non- apoptotic caspase activity increases expression of the Notch ligand Delta. Elevated Delta expression accelerates morphogenetic furrow progression, while cis-inhibiting Notch signaling posterior to the morphogenetic furrow, inhibiting R7 cell specification and cone-cell differentiation. B) Like many proteins, Delta has multiple potential caspase cleavage sites, here predicted suing Cascleave 2.0 (Wang et al., 2014). None are high-confidence predictions and only one is in the intracellular domain where caspase access is plausible (position 675, predicted score 0.647). The intracellular domain is required for Delta signaling. Cleavage at position 675 would remove the main ubiquitylation site required for signaling, as well as the binding site for mindbomb, so is not anticipated to enhance signaling activity (Daskalaki et al., 2011).

Morphogenetic furrow progression in emc mutant cells In eye imaginal discs, normal furrow progression was observed in (A) dronc -/- and (B) Df(3L)H99 homozygous clones, indicated by Senseless and Elav staining. Homozygous mutant clones of emc-/- (C) lacking GFP expression, have no Emc staining (yellow arrowhead) and higher Da (red arrowhead). At the morphogenetic furrow as shown by yellow arrow, Emc expression goes down and Da goes up. Similar results were observed in (D) dronc-/- emc-/- clones and (E) emc-/- Df(3L) H99 -/- clones confirm that these are emc null clones. Genotypes: (A) ywhsF;;dronci29FRT80/[UbiGFP] M(3)67C FRT80 (B) ywhsF;; Df(3L)H99 FRT80/[UbiGFP] M(3)67C FRT80. (C) ywhsF;;emcAP6FRT80/[Ubi-GFP] M(3)67C FRT80 (D) ywhsF;;dronci29emcAP6 FRT80/[UbiGFP] M(3)67C FRT80 (E) ywhsF;;emcAP6 Df(3L)H99 FRT80/[UbiGFP] M(3)67C FRT80.

Apoptosis and furrow progression

Representative images of eye imaginal discs stained for TUNEL (A) ywhsF;;FRT80/[UbiGFP] M(3)67C FRT80 (B) ywhsF;;emcAP6FRT80/[Ubi-GFP] M(3)67C FRT80. In (C) pieEB3 homozygous clones, furrow progression occurs at normal speed marked by Senseless staining in R8 photoreceptors. (D) In GMR-p35 eye disc mutant for emc also show lack of Dcp1staining. Genotypes: (A) ywhsF;;FRT80/[UbiGFP] M(3)67C FRT80 (B) ywhsF;;emcAP6FRT80/[Ubi-GFP] M(3)67CFRT80 (C) ywhsF/+; pieEB3FRT40/FRT40Alz. (D) ywhsF/GMR-p35;;emcAP6FRT80/[Ubi-GFP] M(3)67C FRT80.

Fz3-RFP/+ eye disc showing RFP and Elav staining.

Ci155 levels when cell death pathways are blocked

Representative image of eye imaginal disc in emc H99 clones in the non-Minute background, showing similar areas inside and outside clones. Genotype: ywhsF;; emcAP6 Df(3L)H99 FRT80/[UbiGFP]FRT80

Patched staining in emc mutants (A) ywhsF;;FRT80/[UbiGFP] M(3)67C FRT80 (B) ywhsF;;emcAP6FRT80/[Ubi-GFP] M(3)67C FRT80 (C) ywhsF;;emcAP6 Df(3L)H99 FRT80/[UbiGFP] M(3)67C FRT8.