FIG 5 .

Impact of microbiota on cell identity of the gut. (A) The mitotic activity of the guts of conventionally reared flies is significantly higher than that of axenically reared flies, as measured by immunostaining with anti-Ph3 antibody. The impacts of microbiota on mitotic activity are more pronounced in the posterior region of the gut. Mean values from four experiments (n = 10 guts each) ± SE are shown; anterior, P = 0.04; middle, P = 0.39; posterior, P = 0.0019. Results from Oregon^R are shown; similar results were obtained with Canton^S. (B) There is no significant difference in the number of midgut enterocytes in axenic and conventionally reared flies (mean values from 10 guts each ± SE; P = 0.4995). (C to F) Composition of cell types (eb, enteroblasts; ee, enteroendocrine cells) is altered in the guts of axenic versus conventionally reared flies. Quantitative measurements (C) of the ratio of cell types per region are shown. The expression of cell identity markers (D to F) in the guts of axenic or conventionally reared flies was compared. Expression of green fluorescent protein (GFP) under the control of progenitor cell (D) (stem cell and enteroblast; esg-Gal4^TS; UAS-nlsGFP) or enteroblast (E) [Su(H)GBE-Gal4; UAS-mcd8GFP] reporter genes was monitored. (C and F) Enteroendocrine cells were quantified using anti-Prospero antibody. Representative images of GFP or anti-Prospero antibody signal from the guts of 4- to 7-day-old flies are shown. Guts were stained with DAPI and examined by fluorescence microscopy at magnification ×20, and images were taken of the anterior (R2) and posterior (R5) regions of the gut. Quantitative measurements were made by counting the number of GFP⁺ or anti-Prospero cells and total number of DAPI-positive cells per field of 10 individual guts and replicated at least three times (for panel D, anterior, P < 0.0001; posterior, P = 0.0003; for panel E, anterior, P = 0.106; posterior, P = 0.005; for panel F, anterior, P = 0.0002; posterior, P = 0.0017).