Figure 2. Integrating enteroblasts form an apical domain before reaching the gut lumen.

(A) A transverse section of a Su(H)GBE >mCD8 GFP midgut imaged 1 day after heat shock. Large spherical lumens surrounded by plasma membranes (α-spectrin; greyscale) have formed between the integrating enteroblasts and the overlying enterocytes. One enteroblast is still GFP-positive, indicating that it has recently been activated, whereas the other has lost Su(H)GBE >mCD8 GFP (green) expression. Canoe (red) labels the apical corners of the enteroblasts. (B) The lumen-facing side of the enteroblast is marked by MyoIA-YFP (green) and Myo7a (red), which are markers for the enterocyte brush border and apical cortex respectively. (C) A GFP protein trap line in the transmembrane transporter Picot (green) labels the apical brush border in enterocytes and the lumen-facing membrane in an integrating enteroblast. Note that multiple lumens have formed between the enteroblast and the enterocytes above. Plasma membranes are labelled with α-spectrin (red). (D) Fimbrin-GFP (Fim-GFP; green) marks the apical cortex of an integrating enteroblast and the enterocyte brush border. Note that the enteroblast to the right has not yet formed a lumen but has Fimbrin, Canoe (red) and Coracle (greyscale) localised to its apical surface. (E) The actin binding domain of Utrophin (Sqh::UtrABD-GFP; green) marks the enterocyte brush border and the apical side of an integrating enteroblast. The apical recycling endosome marker, Rab11 (red) also labels the apical region of the enteroblast. (F) A transmission electron micrograph showing that the lumen above an integrating enteroblast is surrounded by brush border microvilli. Scale bar, 2 µm. (G) An integrating enteroblast with a closed pre-apical compartment (PAC) and lumen that lie below the septate junction between the overlying enterocytes. The cells express the actin marker, Sqh::UtrABD-GFP (green), and are stained for Coracle (red) and Canoe (greyscale). (H) An integrating enteroblast stained as in (H) with an open lumen that is continuous with the gut lumen. (I) A model for enteroblast integration in which a ‘closed’ lumen above the PAC precedes an ‘open’ lumen. The ‘closed’ lumen stage represents the pre-EC with a PAC forming underneath the septate junction between the neighbouring enterocytes (purple), creating an isolated, closed lumen inside the epithelial layer. The cap over the ‘closed’ lumen comes from the neighbouring enterocytes. Adherens junctions form between pre-EC (light blue) and neighbouring enterocytes. New septate junctions also form between the pre-EC and the adjacent enterocytes (purple). The ‘open’ lumen represents a fully-developed enterocyte after the lumen has fused with the gut lumen, turning the PAC into the apical domain. To simplify the cartoons in the following figures, we combine the membranes between pre-EC and neighbouring ECs into one line. Scale bars in A-E, G and H, 5 µm.

Figure 2—figure supplement 1. Heatshock increases the number of integrating enteroblasts without inducing a regeneration response.

(A) Graph showing the distribution of enteroblast nuclear volumes in control guts and in guts imaged one day after a 2 hour heat shock. The horizontal lines indicate the median values, which are significantly different by a two-tailed t test (*p<0.05). After heat-shock treatment, the Su(H)GBE >GFP + enteroblasts have larger nuclear volumes on average, indicating that more cells are activated and becoming polyploid. N shows the number of midguts analysed in each experiment and n, the number of nuclei. (B) Graph showing the number of cells in mitosis (phospho-histone H3 (PH3) staining) in control guts and in guts imaged one day after a 2 hr heatshock. The heat shock increases the number of ISC divisions. The horizontal lines indicate the median values, which are significantly different by a two-tailed t test (**p<0.01). N shows the number of midguts analysed in this experiment. (C) Phosphorylated eukaryotic translation initiation factor 4E-binding protein 1 (p-4EBP1; green) staining in a control gut (left panel) and a gut 24 hr after a 2-hr heat shock (right panel). The heat shock induces a modest increase in p-4EBP1 levels. DNA is stained with DAPI (blue). (D) Phosphorylated Extracellular Regulated Kinase (p-ERK; green) staining in a control gut (left panel) and a gut 24 hr after a 2-hr heat shock (right panel). p-ERK levels are not increased after this treatment. (E) Par-6-GFP localisation to the lumen-facing domain of an integrating enteroblast. Canoe is labelled in (red) and the septate junctions are labelled with Coracle (greyscale). (F) β_H-spectrin-GFP (green) localises to the enterocyte apical membrane and marks the lumen-facing membranes of both the integrating enteroblast and the overlying enterocytes. Canoe is labelled in (red) and the septate junctions are labelled with Coracle (greyscale). (G) A GFP protein trap line in the actin crosslinker, Cheerio (Cher; green). Cheerio localises to apical brush border of the enterocytes and to the apical, lumen-facing surface of an integrating enteroblast. Mesh (red) marks the septate junctions and basal labyrinth and α-spectrin (greyscale) marks the cell membranes. (H) Separate channels from the image of an integrating enteroblast with a pre-apical compartment (PAC) beneath a closed lumen shown in Figure 2G. (I) Separate channels from the image of an integrating enteroblast with an open lumen shown in Figure 2H. Scale bar=5μm.

Figure 2—video 1. Stack images for Figure 2G, showing the ‘closed’ lumen above the PAC.

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The cells express the actin marker, Sqh::UtrABD-GFP (green), and are stained for Coracle (red) and Canoe (greyscale).

Figure 2—video 2. Stack images for Figure 2H, showing the ‘open’ lumen with a new enterocyte exposed to the gut lumen, it has a concave shape apical domain which is originated from the PAC.

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The cells express the actin marker, Sqh::UtrABD-GFP (green), and are stained for Coracle (red) and Canoe (greyscale).