DEV026955 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 - Adobe PDF

• Supplemental Figure S2 - Adobe PDF

• Supplemental Figure S1 -

  Fig. S1. Pupal development of the midgut. The esg-positive adult intestinal progenitor cells were marked by esgGal4-driven GFP expression, and the numbers of esg-positive cells were counted in the newly formed adult midgut epithelium and are indicated. (A-A′′′) At 24 hours APF, only the esg-positive cells resume proliferation in the newly formed adult midgut (arrows). The asterisk marks the histolyzing larval midgut. (B-B′′′) At 48 hours APF, the esg-positive cells continue to proliferate and their numbers increase (PH3-positive cells marked by an arrow). (C-C′′′) At 72 hours APF, the esg-positive cells continue to proliferate (arrows) and some of them differentiate into enteroendocrine cells (Prospero-positive staining marked by arrowheads). Throughout pupal development, no Delta staining was observed in these esg-positive cells (data not shown).

• Supplemental Figure S2 -

  Fig. S2. Downregulation of EGFR signaling impairs AMP proliferation during Drosophila larval development. Activity of the EGFR signaling pathway was inhibited in the AMPs by ectopic expression of either gene-specific inverted repeats (IR, RNAi) targeting multiple crucial genes in the pathway, or dominant-negative Raf (Raf^DN) and Mkp3. Expression of transgenes was induced using the esgGal4^ts system in first instar larvae (24 hours AED), which were dissected at white pupae formation (0 hours APF). (A) GFP. (B) Egfr RNAi. (C) Ras RNAi. (D) Raf RNAi. (E) Raf^DN. (F) Ras^V12 + Raf^DN. (G) Mkp3. (H) Mkp3 + Ras^V12. AMPs are marked by GFP (green) and ECs by DNA (blue, large nuclei).

• Supplemental Figure S3 -

  Fig. S3. Flp/Gal4 cell lineage analysis of the AMPs in vn mutants. AMP clones were induced using the Flp/Gal4 system. To induce the clones, first instar larvae (24 hours AED) were heat shocked for 20 minutes at 37°C and then dissected at the wandering L3 stage. (A-A′′) AMP Flp/Gal4 clone in the control midgut (vn^P1749/+). (B-B′′) AMP Flp/Gal4 clone in vn mutant midgut (vn^γ7/P1749). Whereas the control clones contain multiple clusters (A-A′′, ∼15 clusters/clone), the clones in vn mutant midgut appear as a single cluster (B-B′′ arrow, 1 cluster/clone). Genotypes: A-A′′, y w hsflp; tub>CD2>Gal4 UAS-CD8-GFP/+; vn^P1749/+; B-B′′, y w hsflp; tub>CD2>Gal4 UAS-CD8-GFP/+; vn^P1749/γ7.

• Supplemental Figure S4 -

  Fig. S4. vn knockdown experiment. UAS-Vn RNAi was induced in AMPs and ECs using the esgGal4^ts (A-A′′) and MyoIAGal4^ts (B-B′′) drivers throughout larval development (24-120 hours AED). Induction of Vn RNAi in the AMPs did not effect their development (A). The same was true when Vn RNAi was induced in the larval ECs (B). Compare to control in Fig. 1D.

• Supplemental Figure S5 -

  Fig. S5. Local induction of UAS-Vn rescued the vn mutant phenotype in the larval midgut. UAS-Vn1.2 was induced in vn^P1749 mutants throughout larval development (24-120 hours AED) in AMPs and ECs using the esgGal4^ts (A-A′′) and MyoIAGal4^ts (B-B′′) drivers. The loss of AMP clusters in vn mutants were rescued by ectopic induction of UAS-Vn expression in the AMPs (A). UAS-Vn induction in the larval ECs promoted ectopic proliferation of AMPs (B). The AMP hyperplasia is likely to be the result of higher Vn expression induced by endoreplicated ECs (more copies of UAS-Vn transgenes) and ensuing higher EGFR activation in the AMPs. Genotypes: A, w; esgGal4 tubGal80^ts UAS-GFP/+; vn^P1749/P1749 UAS-Vn1.2; B, w; MyoIAGal4 tubGal80^ts UAS-GFP/+; vn^P1749/P1749 UAS-Vn1.2.