BMP signaling inhibits Dl-independent N pathway activity to promote ISC self-renewal

(A) A scheme for the ISC lineage in Drosophila midgut. (B-E’’) Representative images of Control guts (B-B’’), midguts expressing UAS-Put-RNAi (C-C’’), UAS-Dl-RNAi (D-D’’), or UAS-Put-RNAi + UAS-Dl-RNAi (E-E’’) with esg-Gal4ts, UAS-GFP at 29°C for 10 days and immunostained for Su(H)-lacZ (grey or red) and GFP (green). Su(H)-lacZ is used as a marker for EB. DAPI (blue) staining indicates nuclei. Compared with control guts (B-B’’), Put knockdown (C-C’’) in precursor cells (green) caused an increase of EB pairs. Dl knockdown induced stem cell-like tumor. Put and Dl double knockdown induced a dramatic increase of EBs. Scale bar (20 μm) is shown in B.

Numb is important for ISC maintenance when BMP pathway activity is attenuated

(A-D’’) Representative images of adult midguts expressing UAS-mCherry-RNAi (Control) (A-A’’), UAS-Mad-RNAi (B-B’’), UAS-Numb-RNAi (C-C’’) and UAS-Mad-RNAi + UAS-Numb-RNAi (D-D’’) with esg-Gal4ts, UAS-GFP at 30°C for 14 days and immunostained for Dl-lacZ (red), E(spl)mβ-CD2 (cytoplasmic magenta) and Pros (nuclear magenta), which are markers for ISC, EB and EE, respectively. DAPI (blue) staining indicates nuclei. Yellow arrows indicate ISCs (Dl-lacZ+ E(spl)mβ-CD2- Pros-) and white arrowheads indicate EBs (E(spl)mβ-CD2+) in Control, Mad, or Numb single knockdown guts. Red arrow indicated a Dl-lacZ+, E(spl) mβ-CD2+ cells in Mad and Numb double knockdown guts. Scale bar (20 μm) is presented in (A). (E-G) Quantification of number of precursor cells (E), percentage of ISC cells (F) and percentage of EB cells (G) of each genotype. Data are mean ± SD from three independent experiments. ****, p < 0.0001.

Loss of ISC in Numb and Mad depleted guts is due to ISC-to-EB differentiation

(A-H’) Representative images of adult midguts containing MARCM clones (green) of FRT40 (Control) (A, A’, E, E’), mad1-2 (B, B’, F, F’), numb4 (C, C’, G, G’) and mad1-2, numb4 (D, D’, H, H’) and immunostained for GFP (green) and Dl (red and grey in A-D’) or E(spl)mβ-CD2 and Pros (red in A-D’ and grey in E-H’) at 14 days (grown at 18°C) after clone induction. GFP marks the clones. DAPI (blue) staining indicates nuclei. ISCs inside and outside the clones are indicated by yellow and white arrows, respectively.

(I) Representative images of adult midguts containing MARCM clones (green) of control (I, I’) or mad1-2, numb4 (J, J’) immunostained for GFP (green), E(spl)mβ-CD2 and Pros (red), and Pdm1 (magenta and grey). Scale bar (20 μm) is presented in (A).

(K) Quantification of clone size for the indicated genotypes 14 days after clone induction.

(L) Quantification of numbers of clones with or without ISCs. Data are mean ± SD from three independent experiments. **, p < 0.01, ****, p < 0.0001.

Depletion of both Numb and Mad leads to more EB/EB division

(A) Scheme of an ISC division that produces differentially labeled daughter cells (RFP+ GFP- and RFP- GFP+) through FRT-mediated mitotic recombination. Adapted from Tian and Jiang 2014.

(B) Scheme of differentially labeled twin clones generated by FLP/FRT-mediated mitotic recombination of dividing ISCs. Adapted from (Tian and Jiang 2014).

(C) Scheme of twin-spot experiments. 3∼5-day-old adult flies of indicated genotype are grown at 29°C for 14 days before heat shock to induce clones. After one-day recovery at 29°C, the flies are raised at 18°C for 4 days prior to analysis.

(D-O) Representative images of twin-spot clones from adult midguts of the indicated genotypes. Scale bar 20 μm is shown in (D).

(P) Quantification of twin spots of different classes from guts of the indicated genotypes.

Data are mean ± SD from three independent experiments. *, p < 0.05, **, p < 0.01.

numb mutant clones exhibit weak stem cell loss phenotype

(A-C’) Representative images of adult midguts containing MARCM clone (green) of FRT40 (Control) (A, A’), numb4 (B, B’), and numb15 (C, C’) and immunostained for Dl (red), GFP (green) and DAPI (blue) at 14 days after clone induction. GFP marks the clones. ISCs inside and outside the clones are indicated by yellow and white arrows, respectively. Scale bar (20 μm) is shown in (A).

(D) Quantification of clone size distribution for the indicated genotypes at 14 days after clone induction.

(E) Quantification of numbers of clones with or without ISC.

Data are mean ± SD from three independent experiments.*, p < 0.05, **, p < 0.01.

Numb is required for EE fate determination.

(A-C’) ISC MARCM clone (green) of control (A, A’), numb4 (B, B’), and numb15 (C, C’) are stained for Pros (red) at 14 days after clone induction. Representative clone in control guts (A, A’) contains EE cells (Pros positive), as indicated with yellow arrows. Representative clones in numb4 (B, B’) and numb15 (C, C’) guts do not contain any EE cells. Scale bar (20 μm) is presented in (A).

Numb is critical for ISC maintenance during regeneration

(A-I’) Adult flies of indicated genotype were treated with sucrose, bleomycin or DSS for 24h at 14 days after clone induction and recovered for another 4 days before dissection. Guts containing MARCM clones of the indicated genotype were stained for GFP (green) and Dl (red and white). GFP marks the clones. DAPI (blue) staining indicates the nuclei. Stem cells inside and outside the clones are indicated by yellow and white arrows, respectively. Scale bar (20 μm) is shown in (A).

(J) Quantification of the percentage of clones with or without ISCs.

(K) Quantification of clone size distribution for the indicated genotypes.

Data are mean ± SD from three independent experiments.*, p < 0.05, **, p < 0.01 ****, p < 0.0001.

Model for Numb and BMP signaling in ISC/EB fate decision.

(A) During asymmetric ISC division, the basal ISC daughter transduces higher level of BMP signaling and inherits higher level of Numb activity than the apical one. Inhibition of N by BMP signaling and Numb promotes ISC fate.

(B) In numb mutant background, differential BMP signaling between the basal and apical ISC daughters is sufficient to generate differential N pathway activities to drive asymmetric fate decision.

(C) In mad mutant background, the shallow BMB activity gradient acts in conjunction with the asymmetric Numb activity to generate differential N pathway activities between the basal and apical ISC daughters to drive asymmetric fate decision.

(D) In numb mad double mutant background or in guts containing numb mutant clones and injured by bleomycin (Bleo) feeding, the shallow BMB activity gradient is often insufficient to generate asymmetric N pathway activation, leading to precocious ISC-to-EB differentiation.

BM: basement membrane. Bleo: Bleomycin; BM: basement membrane; thin line and dashed line indicate weak inhibition.