Fig. S1. Characterisation of a novel null hpo allele, hpo^5.1. (A) Schematic representation of the hpo locus. The coding sequence in shown black; 5′- and 3′- UTRs are shown in white. Two kinase-dead mutations for hpo (hpo^42-47 and hpo^JM1) are shown. Two null hpo mutations, hpo^5.1 and hpo^BF33, are shown in red. hpo^5.1 was generated by excision of the P-element G3315 (triangle). The peptide recognised by the Hpo66 antibody is shown in blue. (B-E′) XY sections of wing imaginal discs containing heat-shock induced clones of hpo^5.1 (B,B′), hpo^JM1 (C,C′), hpo^BF33 (D,D′) or hpo^42-47 (E,E′). (B,C,D,E) Stainings for Hpo using the Hpo66 antibody, recognising the C-terminal part of Hpo kinase domain. (B′,C′,D′,E′) Merged images of B,C,D,E with GFP (green). Scale bar: 10 m. (F) Graph presenting the survival rate of flies containing clones of different alleles of hpo, induced with the eyFLP driver. In theory, 50% of the flies should have the genotype to allow formation of clones. In practice, depending on the allele used, only 2.1-36.4% of the hatched flies carried clones. The kinase-dead alleles hpo^JM1 and hpo^42-47 have a stronger effect on lethality than the null alleles hpo^5.1 and hpo^BF33. (G) Schematic of the different apicobasal complexes of a wing epithelial cell. Genotypes: (B,B′) yw hsFLP/w; FRT42D GFP/FRT42D hpo^5.1. (C,C′) yw hsFLP/w; FRT42D GFP/FRT42D hpo^JM1. (D,D′) yw hsFLP; FRT42D GFP/FRT42D hpo^BF33. (E,E′) yw hsFLP; FRT42D GFP/FRT42D hpo^42-47. (F) yw eyFLP; FRT42D GFP/FRT42D hpo^5.1, yw eyFLP; FRT42D GFP/FRT42D hpo^JM1, yw eyFLP; FRT42D GFP/FRT42D hpo^BF33, yw eyFLP; FRT42D GFP/FRT42D hpo^42-47.

Fig. S2. Elevated levels of apical polarity determinants in hpo clones. (A-G′) Clones of hpo mutant cells (marked by absence of GFP) in third instar wing imaginal discs. (A,A′,D,D′) hpo^JM1 clones; (B-C′,E-G′) hpo^5.1 clones. (A-F′) XY sections on the apical side of wing imaginal discs. (A′-F′) Merged images of A-F with GFP (green). Scale bar: 10 m. (G,G′) Highly magnified transverse section of wing imaginal cells. Apical is to the top. (G′) Merged image of G (in red) with GFP (green). Scale bar: 5 m. The arrowhead points to the subapical region of the columnar epithelial cells of the disc. The yellow bracket indicates the position of the peripodial membrane. In XY sections, as in transverse sections, the accumulation of the apical proteins aPKC (A,A′,G,G′), Crb (C,C′), P-Moe (D,D′), DE-cad (E,E′) and Arm (F,F′) can be seen in hpo mutant cells, whereas Dlg levels are unchanged (B,B′). The increase in aPKC is seen in columnar epithelial but not peripodial cells (G,G′). Genotypes: (A,A′,D,D′) yw hsFLP/w; FRT42D GFP/FRT42D hpo^JM1. (B-C′,E-G′) yw hsFLP/w; FRT42D GFP/FRT42D hpo^5.1.

Fig. S3. Notch on the surface of loss and gain-of-function clones for the Hpo pathway. (A-F′) Transverse sections of wing imaginal discs. Apical is to the top. (A′-F′) Merged images of A-F with GFP (green). In grey or red: stainings for NICD. Scale bar: 10 m. Cells mutant for Mer/ex (A,A′), for hpo (B,B′) or for wts (C,C′), all marked by absence of GFP, have an increase in Notch at their apical surface. Conversely, cells overexpressing hpo (marked by GFP in F,F′) possess less apically localised Notch. The same is true for cells mutant for crb (E,E′) and for cells mutant for crb/wts (D,D′), both marked by absence of GFP. Genotypes: (A,A′) yw mer4; FRT40A pw⁺ mer⁺ UbiGFP/FRT40A ex^e1; MKRS hsFLP/+. (B,B′) yw hsFLP/w; FRT42D GFP/FRT42D hpo^JM1. (C,C′) yw hsFLP/w; FRT82B GFP/FRT82B wts^latsX1. (D,D′) yw hsFLP/w; FRT82B GFP/FRT82B crb^11a22 wts^latsX1. (E,E′) yw hsFLP/w; FRT82B GFP/FRT82B crb^11a22. (F,F′) yw hsFLP/w; UAS::hpo/Act>CD2>Gal4, UAS::GFP, UAS::p35.

Fig. S4. The apical hypertrophy is specific to the Hpo pathway loss of function. (A-D′) Transverse sections of wing imaginal discs stained for aPKC. (A′-D′) Merges images of A-D with GFP (green). Apical is to the top. Scale bar: 10 m. Cells overexpressing yki under the ptc-Gal4 promoter have an increased apical localisation of aPKC (A,A′; marked by GFP) whereas cells overexpressing the Hpo pathway targets cyclinE (cycE), DIAP1 and the miRNA bantam under the same promoter do not (B,B′; marked by GFP). Cells overexpressing activated ras (ras^v12) (C,C′; marked by GFP) as well as cells overexpressing cyclinD (cycD) and ckd4 (D,D′; marked by GFP) also do not show such an increase. Genotypes: (A,A′) w; ptc>Gal4,UAS::GFP/+; UAS::yki/+. (B,B′) UAS::diap1/w; ptc>Gal4, UAS::GFP/+; UAS::cycE, ban^EP3622 FRT80B/+. (C,C′) yw hsFLP/w; Act>CD2>Gal4, UAS::GFP, UAS::p35/UAS::ras^v12-HA. (D,D′) yw hsFLP/w; Act>CD2>Gal4, UAS::GFP/UAS::cycD,UAS::cdk4.

Fig. S5. DE-cad and apical protein accumulation. (A-B′′) Transverse sections of wing imaginal discs containing hpo clones (marked by absence of β-gal). Apical is to the top. (A′′,B′′) Merged images of A,A′ and B,B′ with β-gal (blue). Scale bar: 5 m. Arm (A) and DE-cad (A′) stainings increase in hpo mutant cells, but their colocalisation remains unchanged (A′′). In wild type as in hpo mutant cells, DE-cad staining is mainly junctional (B), and DE-cad and the early endosome marker Rab5 (B′), are rarely found to colocalise (B′′). (C-D′) Transverse sections of wing imaginal discs overexpressing shg, arm and gfp under the patched promoter. Apical is to the top. Scale bar: 10 m. (C′,D′) Merged images of C,D with GFP (green). In grey or red: stainings for aPKC. Overexpressing shg and arm is not sufficient to induce accumulation of apical determinants as visualised by aPKC staining (C,C′). The phenotype is clearly different from loss of Hpo signalling as shg- and arm-expressing cells occasionally delaminate and lose their apicobasal polarity, resulting in a circular staining for aPKC (D,D′). Genotypes: (A-B′′) yw hsFLP/w; FRT42D GFP/FRT42D hpo^5.1. Merged images of C-D′) w; ptc>Gal4,UAS::GFP,UAS::p35/UAS::shg,UAS::arm.

Fig. S6. Apical constriction occurs in Hpo pathway loss-of-function cells but cannot account for the apical determinant accumulation. (A-C′′) Apical XY sections of third instar wing imaginal discs containing GFP-labelled clones (green) mutated for hpo (A-A′′), wts (B-B′′) and crb/wts (C-C′′) and stained for Arm (A-C) and F-actin (A′-C′). (A′′,B′′,C′′) merged images of A,B, A′,B′ and of GFP. (D) Graph presenting, for each genotype, the apical cell surface ratio (r_ACS) between mutant tissue and surrounding wild-type tissue. P-values from Mann-Whitney tests are shown on the graph. (A-A′′) yw tubGAL4 hsFLP 122 UAS-nucGFPmyc/w; FRT42D hpo^5.1/FRT42D tubG80. (6B-B′′) yw tubGAL4 hsFLP 122 UAS-nucGFPmyc/w; FRT82B wts^latsX1/FRT82B CD21 y+ tubG80.LL3.(C-C′′) yw tubGAL4 hsFLP 122 UAS-nucGFPmyc/w; FRT82B crb^11a22 wts^latsX1/FRT82B CD21 y+ tubG80.LL3. (D; Table 4: yw tubGAL4 hsFLP 122 UAS-nucGFPmyc/w; FRT42D hpo^5.1/FRT42D tubG80, yw tubGAL4 hsFLP 122 UAS-nucGFPmyc/w; FRT82B wts^latsX1/FRT82B CD21 y+ tubG80.LL3, yw tubGAL4 hsFLP 122 UAS-nucGFPmyc/w; FRT82B crb^11a22 wts^latsX1/FRT82B CD21 y+ tubG80.LL3.