Intestinal epithelium-derived BMP controls stem cell self-renewal in Drosophila adult midgut
- University of Texas Southwestern Medical Center at Dallas, United States
Figures
Figure 1
BMP signaling is required for midgut regeneration.
(A) Left: an ISC lineage in Drosophila adult midguts. ISC: intestinal stem cell; EB: enteroblast; EC: enterocyte; EE: enteroendocrine cell. ISC and EB are collectively called precursor cells. Dl and …
Figure 2 with 2 supplements
BMP signaling is required for ISC self-renewal.
(A–F″) 3- to 5-day-old adult females expressing esgts>GFP (A, D–D″) or expressing esgts>GFP together with UAS-Tkv-RNAi (B, E–E″) or UAS-Put-RNAi (C, F–F″) were shifted to 29°C for 8 days, followed …
Figure 2—figure supplement 1
Blocking apoptosis does not rescue ISC loss caused by inactivation of BMP signaling.
(A–D) 3–5-day-old adult females expressing esgts>GFP (Con; A), esgts>Diap1 (B), esgts>Put-RNAi (C), or esgts>Put-RNAi + Diap1 (D) were shifted to 29°C for 8 days, followed by immunostaining for DI …
Figure 2—figure supplement 2
RFP/GFP two-color twin spot clonal analysis.
(A) Confocal images of a posterior midgut containing FRT82 ubi-GFP (red) and FRT82 ubi-RFP (red) and immunostained with the nuclear marker DRAQ5 (blue) prior to clonal induction. Both ubi-GFP and ubi…
Figure 3 with 3 supplements
Characterization of midgut phenotypes caused by differential inactivation of BMP pathway components.
(A–H) Midguts containing the control clones (A, C, E, G) or putP clones (B, D, F, H) were immunostained for DI (red in A, B, G, H) or Pdm1 (red in C–F), GFP (green), and Su(H)-lacZ (blue in A, B, G, …
Figure 3—figure supplement 1
Tkv and Sax act redundantly in the regulation of ISC self-renewal.
(A) Schematic drawing of Tkv and Sax coding regions with numbers indicating the nucleotide positions. The red lines indicate the regions targeted by individual RNAi lines. The red bars indicate the …
Figure 3—figure supplement 2
Both tkv8 and mad1–2 mutant clones caused non-cell autonomous ISC overproliferation.
Posterior midguts carrying control (A), tkv8 (B), mad1–2 (C), or tkv8 mad1–2 (D) clones and immunostained for PH3, GFP, and DRAQ5 at 8 days ACI. Arrows and asterisks indicate PH3+ cells inside and …
Figure 3—figure supplement 3
Inactivation of BMP signaling in ECs caused ISC proliferation.
(A–D) Control midguts (Myo1Ats) or midguts expressing the indicated RNAi lines at 29°C for 8 days were immunostained for PH3 and DRAQ5. Inactivation of BMP signaling in ECs resulted in elevated ISC …
Figure 4 with 1 supplement
Asymmetric BMP signaling regulates ISC self-renewal.
(A–A′, C–E′) High magnification views of wild type adult midguts immunostained for pMad (red in A–A′, D–E′), dad-lacZ (red in C), Dl-lacZ (blue in A′, E′), Su(H)-GFP (green in A′, C, E′) or PH3 …
Figure 4—figure supplement 1
Effect of misexpressing Dpp or Gbb alone in precursor cells on ISC self-renewal.
(A–B′) Adult midguts expressing esgts>Dpp + GFP (A, A′) or esgts>Gbb + GFP (B, B′) at 29°C for 12 days were immunostained for Dl (red), GFP (green) and DRAQ5 (blue). Misexpression of Dpp or Gbb …
Figure 5 with 1 supplement
BMP signaling promotes ISC self-renewal by antagonizing N.
(A–E′) Adult midguts expressing NICD (8d) (A–A′), TkvQ235D (8d) (B–B′), Dpp + Gbb (12d) (C–C′), or the indicated combinations of transgenes (D–E′) under the control of esgts were immunostained for …
Figure 5—figure supplement 1
Integrated pMad levels in control or N knock down precursor cells.
Quantification of pMad signals in pairs of ISC/EB in the posterior control midguts (Con; esgts>GFP) or in clusters of precursor cells expressing esgts>N-RNAi (mean ± SD: n = 20 for control ISC/EB …
Figure 6 with 2 supplements
Both Dpp and Gbb are expressed in ECs.
(A and B) Low magnification views of adult midguts expressing one (a) or two (b) copies of UAS-GFP transgene under the control of dpp-Gal4 were immunostained for GFP and DRAQ5. dpp>GFP is expressed …
Figure 6—figure supplement 1
Characterization of dpp and gbb expression in Drosophila by RNA in situ hybridization.
(A–D) RNA in situ hybridization with dpp (A and B) or gbb (C and D) probe for wild type wing discs (A, C) or wing discs expressing UAS-Dpp (B) or UAS-Gbb (D) with wing disc specific Gal4 driver MS109…
Figure 6—figure supplement 2
dpp and gbb mRNAs are not detected in precursors or VM.
High magnification views of the indicated regions of adult midguts expressing esg>GFP (A–C′, G–I′) or how>GFP (D–F′’, J–L′) and probed for dpp (A–F′) or gbb (G–L′) expression by RNA in situ …
Figure 7 with 4 supplements
EC-derived Dpp and Gbb regulate ISC self-renewal.
(A and B) Control (A) or midguts coexpressing both Dpp and Gbb with Myo1Ats (B) were immunostained for Dl and Su(H)-lacZ. (C–I) Control guts (C, G), guts expressing strong Dpp-RNAi line (D, H), …
Figure 7—figure supplement 1
Characterization of Dpp and Gbb RNAi lines.
(A–E) Adult wing phenotypes associated with Dpp (B and C) or Gbb (D and E) knockdown using a wing specific Gal4 driver, MS1096, to express the indicated RNAi lines. Arrows in D and E indicate …
Figure 7—figure supplement 2
Characterization of Dpp and Gbb knockdown in ECs.
Adult midguts expressing Myo1Ats>GFP (A and A′), Myo1Ats>GFP + Dpp-RNAiS (B and B′), or Myo1Ats>GFP + Gbb-RNAiS (C and C′) at 29°C for 25 days followed by immunostaining for pMad (red), GFP (green), …
Figure 7—figure supplement 3
Partial loss of BMP in ECs stimulates ISC proliferation.
(A and B) Control guts (A) and guts expressing strong Dpp-RNAi line (B) for 10 days were immunostained for PH3 (red) and DRAQ5 (blue). (C) Quantification of PH3+ cells in midguts from adults of the …
Figure 7—figure supplement 4
Characterization of Dpp and Gbb in trachea and VM.
(A) RNA in situ hybridization of midguts expressing Btl>GFP with a dpp probe. dpp mRNA was detected in Btl>GFP+ tracheal cells. (B–D) Adult midguts expressing Btlts-Gal4 (B), Btlts>Dpp (C) or Btlts>D…
Figure 8 with 4 supplements
Regulation of ISC self-renewal by Vkg and BMP activity gradient.
(A–C) Low (A and B) and high (A′, B′, C) magnification sagittal views of adult midguts expressing Myo1Ats>GFP (A–A′) or Myo1Ats>Dpp-GFP (B–C) and immunostained with the indicated antibodies or dyes. …
Figure 8—figure supplement 1
Dpp regulates Gbb subcellular localization.
(A–C′’) Sagittal views of adult midguts expressing Myo1Ats>Gbb-GFP (A and A′), Myo1Ats>Gbb-GFP + Dpp (B and B′), or Myo1Ats>Gbb-GFP + Dpp-HA (C and C′) were immunostained with antibodies against GFP …
Figure 8—figure supplement 2
Dpp physically interacts with Gbb.
Adult midguts expressing Dpp-HA and Gbb-GFP individually or in combination with Myo1Ats at 29°C for 5 days were subjected to immunoprecipitation and western blot analysis with the indicated …
Figure 8—figure supplement 3
Integrated pMad levels in precursor cells of control or vkg mutant midguts.
Quantification of pMad signals in pairs of wild type ISC/EB (Con; esgts>GFP) or in clusters of vkg mutant (vkg00236/01,029) precursor cells in the posterior midguts (mean ± SD: n = 20 for control …
Figure 8—figure supplement 4
Dpp localization at the BM is diminished in vkg mutant guts.
(A–A″) Adult midguts were immunostained for the Drosophila integrin βPS subunit Integrin/Mys (red), Vkg-GFP (green), Phalloidin and DRAQ5 (blue). Integrin/Mys is enriched at the basal membrane (BM) …
Figure 9
A working model for how BMP regulates ISC self-renewal.
Basal/basolateral secretion coupled with basement membrane (BM) trapping sets up an apical-basal BMP activity gradient consisting of Dpp-Gbb heterodimers. Basally localized ISC daughter cells …
