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First published online January 13, 2009
doi: 10.1242/10.1242/dev.026955

Development 136, 483-493 (2009)
Published by The Company of Biologists 2009
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EGFR signaling regulates the proliferation of Drosophila adult midgut progenitors

Huaqi Jiang and Bruce A. Edgar*

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N., Seattle, WA 98109, USA.


Figure 1
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  		Fig. 1. Development of Drosophila adult midgut progenitors (AMPs). AMPs were marked by GFP expression (green) driven by esgGal4NP7397. The numbers of GFP-positive AMPs, AMP clusters or adult intestinal stem cells (ISCs) are indicated in the appropriate panels. DNA is stained with DAPI (blue). (A) First instar larval midgut (24 hours AED). GFP was detected in the AMPs as individual diploid cells (arrows). (B) Early third instar larval midgut (72 hours AED). Larval enterocytes (ECs) undergo several rounds of endoreplication, enlarging the larval midgut. AMPs remain diploid and their numbers increase during the first two larval stages. However, they remain mostly dispersed as individual cells. Inset shows GFP expression that is overexposed to show cell contacts between two neighboring AMPs. (C) Mid-third instar larval midgut (96 hours AED). AMPs form distinctive 2- to 3-cell clusters. (D) Late third instar larval midgut (120 hours AED). AMPs continue to proliferate and enlarge the clusters. (E) White prepupa stage (0 hours APF, ~130 hours AED). The size of the AMP clusters has increased further. (F) Prepupa stage (4 hours APF). The AMP clusters fuse to form a new midgut epithelium. Larval ECs (out of focal plane) are sloughed into the lumen and histolyze. (G,H) Early pupa stage (8 and 12 hours APF). The majority of the cells in the new midgut epithelium gradually lose GFP expression, except for a few scattered cells that maintain strong GFP expression. (I,J) Pupa stage (24 hours APF). The future adult ISCs are clearly identifiable by strong GFP expression (asterisks in I) and basal localization in the epithelium (asterisks in J, cross-sectional view). GFP expression is lost in the rest of the cells in the new epithelium. Scale bars: 20 µm.

 

Figure 2
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  		Fig. 2. Lineage analysis of the AMPs. (A-B'') Drosophila AMP clones induced using the MARCM system. Clones were induced at either first instar (24 hours AED, A-A'') or third instar (96 hours AED, B-B'') and analyzed at the wandering L3 stage (120 hours AED). When induced at first instar, the clones appear as multiple marked clusters with all cells labeled (A-A''), whereas clones induced late (at 96 hours AED) were all confined to a single cluster that is mosaic for GFP (B-B''). (C-E'') Pupal or adult AMP clones induced using the Flp/Gal4 system. Flp/Gal4 AMP clones were induced at first instar larval stage (24 hours AED) and analyzed at 24 hours APF (C-C'') or from newly eclosed adults (D-E''). At 24 hours APF, each AMP clone contains 0-2 esg-positive cells (C, arrows); the asterisk marks the histolyzing larval midgut. In newly eclosed adults, the midgut contains enteroendocrine cells and ISCs; arrows indicate cells within the clone that are positive for Prospero (D) and Delta (E).

 

Figure 3
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  		Fig. 3. Activated Ras (RasV12) stimulates AMP proliferation. UAS-transgenes were induced in the Drosophila AMPs using the esgGal4ts system. Larvae were shifted to 29°C at the indicated times and dissected at 96 hours AED. (A) GFP (24-96 hours AED, control). (B) RasV12 (72-96 hours AED). (C) RasV12 (48-96 hours AED). (D) RasV12 (24-96 hours AED). (E,F) Cross-sections of posterior midguts from wandering L3 larvae expressing ectopic GFP (E, wild type, WT) or RasV12 (F) throughout larval development (24-120 hours AED). The control AMP clusters are basally localized in the epithelium (E, arrows). PM, peritrophic membrane. The samples in E and F were stained with Toluidine Blue.

 

Figure 4
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  		Fig. 4. EGFR signaling mutant AMPs fail to proliferate. Egfr-/- or Ras-/- Drosophila AMP clones were generated using the MARCM system, which positively marks mutant cells with GFP expression. (A) FRT42D only (control). (B) FRT42D Egfr[CO]. (C) FRT82B Ras{Delta}c40b. The boxed regions in A-C are shown to the right as GFP (A'-C'), DNA (A''-C'') and merged (A'''-C''') images. Unlike in the control (A-A'''), where GFP-positive clones form multiple clusters, clones of Egfr[CO] and Ras{Delta}c40b AMPs (B-B'''; C-C''') appear as single GFP-positive cells. Arrowheads indicate the positions of Egfr-/- or Ras-/- AMPs. The asterisk in C indicates one larval EC with non-specific GFP expression from the MARCM system (FRT82B).

 

Figure 5
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  		Fig. 5. Expression and activity of the EGFR ligands spitz, Keren and vein in the larval midgut. (A-A'') MAPK activity (dpERK staining) in the midgut of late third instar Drosophila larvae. (B-B'') The expression of UAS-GFP driven by spiGal4NP0261 in the midgut of L3 wandering larvae. (C-C'') vnlacZ reporter expression pattern in the midgut. Large arrows indicate the circular visceral muscle cells, which form four distinct rows (two are shown). Small arrows indicate the longitudinal visceral muscle cells. (D,D') Krn RNA in situ hybridization in L3 wandering larval midgut. (E,E') spi RNA in situ hybridization in L3 wandering larval midgut. Arrowheads indicate the positions of the AMP clusters in all panels.

 

Figure 6
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  		Fig. 6. Expression of sSpi, Krn or sKrn in the AMPs induces their proliferation. The ligands were induced in the Drosophila AMPs using the esgGal4ts system starting at 24 hours AED, and larvae were dissected at 96 hours AED. (A,A') GFP (control). (B,B') Activated (secreted) Spi (sSpi). (C,C') Activated (secreted) Krn (sKrn). (D,D')Krn. (A-D) GFP marks the AMP clusters. (A'-D') Merged images of GFP (green) and DNA (DAPI, blue). (E) The ectopic expression of strong EGF ligands in the AMPs dramatically reduces the total number of AMP clusters in the midgut. WT, wild-type.

 

Figure 7
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  		Fig. 7. Vein is required for AMP proliferation. (A-C) Posterior midguts from white prepupa (0 hours APF) of wild-type (WT) Drosophila contain multiple AMP clusters (A), which are missing from the midguts of vn mutants (B, vnP1749; C, vn{gamma}7). Midguts are outlined with dashed lines. (D-E') Posterior midguts from wandering L3 larvae in which vn was specifically knocked down in the visceral muscle cells throughout larval development (24-120 hours AED, D) or only during late larval development (72-120 hours AED, E). Most of the remaining small cells in B-D are visceral muscle cells. Arrows in D point to the few AMP clusters in the midgut. (F,F') Induction of UAS-Vn expression throughout larval development (24-120 hours AED) using the muscle-specific driver howGal4ts rescued the vn mutant phenotype. D'-F' show merged images of DNA (DAPI, blue) and howGal4ts-driven GFP expression (green) in the visceral muscle and trachea (asterisks in D',F') cells. (G) Knockdown of vn mRNA in the midgut by vn RNAi. Relative levels of vn mRNA in the larval midgut were quantified by qRT-PCR. Only UAS-Vn RNAi expression driven by the muscle-specific Gal4 driver, howGal4ts, knocked down vn significantly in the larval midgut.

 

Figure 8
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  		Fig. 8. Postembryonic development of the Drosophila midgut epithelium. AMPs (green) proliferate in two phases and several EGFR ligands are involved in each phase. Also note the specification of future adult intestinal stem cells during early metamorphosis and the reappearance of enteroendocrine cells (red) at a late stage of metamorphosis (72 hours APF). See text for details.

 

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© The Company of Biologists Ltd 2009