DEV035196 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. The standard quantitative method used in analyzing mosaic midguts. (A) Diagram of the adult Drosophila gastrointestinal tract (see Miller, 1950). The adult midgut extends the length between the cardia and the pylorus (heavy black outline). Data were collected from two defined regions of each midgut analyzed: the middle of the anterior midgut and the middle of the posterior midgut. To reproducibly determine the middle of both the anterior and posterior midgut, each segment was first independently divided into a series of non-overlapping 40× fields of view or ‘frames’ using the confocal microscope (green and red asterisks, respectively). The anterior segment extends from the narrowest region following the cardia to the anterior limit of the copper cells; the anterior most-frame we designated a1. The posterior segment extends from the posterior limit of the copper cells to the anterior limit of the pylorus where the Malpighian tubules enter the tract; the anterior-most frame of the posterior midgut we designated p1. Under our experimental conditions, we found that the anterior and posterior segments of the midgut can each be covered in the span of 5 (or 4) frames (e.g. a1-a5, p1-p4). (B) In a 10-frame midgut, the middle of the anterior is therefore centered at a3 (green frame), and the middle of the posterior is centered at p3 (red frame). (C) In an 8-frame midgut the middle of the anterior is centered at a2.5, and the middle of the posterior is centered at p2.5. Midguts were measured twice before selecting the final frames used for analysis.

• Supplemental Figure S2 -

  Fig. S2. Apc loss leads to increased Dl levels. (A,B) The MARCM system was used to positively identify ISC lineages with GFP 5 days after induction (anti-GFP, green; anti-Pros and anti-Dl, red; DAPI, blue). Note that anti-Delta and anti-Pros are both shown in red; Dl staining appears punctate and membrane localised, whereas Pros expression is saturated and nuclear. (A′,B′) Clone boundary indicated by green outline, anti-Delta and anti-Pros are both shown in white. (A,A′) Wild-type ISC lineages. (B,B′) ISC lineages lacking Apc. Note that, compared with wild type, Dl appears at higher levels in some ISC/EB pairs both within and outside of marked Apc lineages. Scale bar: 50 µm.

• Supplemental Figure S3 -

  Fig. S3. Loss of Apc in ISCs enhances hyperplasia and multilayering of the N^RNAi phenotype. (A,B) The MARCM system was used to positively identify ISC lineages with GFP 10 days after induction (anti-GFP, green; anti-Pros, red; DAPI, blue). A series of progressively deeper optical sections from the surface of the midgut is shown at 10 µm, 30 µm and 50 µm from the basement membrane. (A-A′′) ISC lineages expressing N^RNAi. (B-B′′) ISC lineages expressing N^RNAi but lacking Apc. Hyperplasia, multilayering and disruption of midgut organization are evident. Scale bar: 50 µm.

• Supplemental Figure S4 -

  Fig. S4. Quantification of clone size and mitotic index in N^RNAi, Apc lineages. (A-D) The MARCM system was used to positively identify ISC lineages with GFP 5 days after induction (anti-GFP, green; DAPI, blue). (A′-D′) Clone boundary indicated by green outline. (A′,B′) Quantification of the number of cells per clone. To quantify the phenotype only esg⁺ pros⁻ cells were scored (esg-lacZ, red; anti-Pros; blue). (A′) N^RNAi; (B′) N^RNAi, Apc. (C′,D′) Quantification of mitotic index. To quantify the phenotype, only small pHH3⁺ pros⁻ cells were scored (anti-Pros, red; anti-phospho-histone H3, white; DAPI, blue). (C′) N^RNAi; (D′) N^RNAi, Apc. Scale bar: 50 µm.

• Supplemental Figure S5 -

  Fig. S5. Wnt activation in the ISC lineage leads to an increase in clone size. (A-D) The MARCM system was used to positively identify posterior ISC lineages with GFP 5 days after induction (anti-GFP, green; DAPI, blue). (A) Wild-type ISC lineages. (B) ISC lineages expressing arm^S10. (C) ISC lineages lacking Apc. (D) Quantitation of the number of cells per clone in arm^S10 and Apc mutant lineages. arm^S10 expression leads to a significant increase in clone size compared with wild type, although not as great as that caused by Apc loss. (wild type, n=41; arm^S10, n=144; Apc, n=157). Error bars dentote s.e.m. Scale bar: 50 µm.