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. 2022 Sep 28;11:e76366. doi: 10.7554/eLife.76366

Figure 1. ISCs/early enteroblasts are polarised and reside underneath tri-cellular junctions.

(A) Par-6 localises apically in ISCs and early enteroblasts. Su(H)GBE >mCD8 GFP expression (arrow) marks an early enteroblast, while the GFP-negative cell (arrowhead) is an ISC. Nuclear Prospero staining (white) marks an entero-endocrine cell (*) and cytoplasmic Dlg (greyscale) labels the septate junctions. (B) An ISC/early enteroblast expressing Par-6-GFP (green), which localises apically. Mesh (red) marks the septate junctions and basal labyrinth. (C) Canoe-YFP (green) localises to the marginal zone above the septate junctions (Tsp2a; red) in mature enterocytes. Although ISC/early enteroblasts do not form septate junctions, Canoe-YFP localises apically. (D) Adherens junctions (DE-cadherin-GFP; green) form throughout the cell-cell contacts between ISC/early enteroblasts and enterocytes. Coracle (red) marks the septate junctions; Canoe; white. (E) A graph showing the proportion of ISCs/early enteroblasts (ISC/EB) or entero-endocrine cells (ee) that localise beneath tri-cellular junctions (@TCJ) and bicellular junctions (@BCJ). Most ISCs/EBs localise beneath tricellular junctions where three enterocytes meet, whereas entero-endocrine cells mainly lie beneath bicellular junctions. DE-Cad-GFP expressing midguts were stained for GFP to mark cell contacts and Prospero to mark the ees. Cells with a low DNA content (~2 n) were counted by imaging from the basal side. 390 ISC/early enteroblasts and 141 ee were scored in 6 guts. (F) An example of an ISC/EB beneath a tricellular junction marked by Anakonda-GFP (Aka-GFP; green). Scale bars = 5 µm.

Figure 1—source data 1. Source data for the graph as Figure 1E.

Figure 1.

Figure 1—figure supplement 1. Most ISCs and enteroblasts lie beneath tricellular junctions.

Figure 1—figure supplement 1.

(A) Related to Figure 1C. A DECad-GFP expressing midgut at homeostasis stained for GFP (green) and Prospero (red). The arrows indicate the Prospero+ entero-endocrine cells in the magnified images on each side. (B) Par-6 (red) is localised apically in ISCs (Deltats >GFP positive cells; arrow) and early enteroblasts (Deltats >GFP negative cell; arrowhead). (C–D) Par-6 (red; C) localises apically in esgts >GFP positive ISC/early enteroblasts, whereas F-actin (red; D) is not apically-enriched. Dlg (greyscale; C) marks the septate junctions. Scale bars = 5 µm.