De novo apical domain formation inside the Drosophila adult midgut epithelium
- The Gurdon Institute, University of Cambridge, United Kingdom
Figures
Figure 1 with 1 supplement
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 …
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Figure 1—source data 1
Source data for the graph as Figure 1E.
- https://cdn.elifesciences.org/articles/76366/elife-76366-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Most ISCs and enteroblasts lie beneath tricellular junctions.
(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 …
Figure 2 with 3 supplements
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 …
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 …
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Figure 2—figure supplement 1—source data 1
Source data for graph as Figure 2—figure supplement 1A.
- https://cdn.elifesciences.org/articles/76366/elife-76366-fig2-figsupp1-data1-v2.xlsx
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Figure 2—figure supplement 1—source data 2
Source data for graph as Figure 2—figure supplement 1B.
- https://cdn.elifesciences.org/articles/76366/elife-76366-fig2-figsupp1-data2-v2.xlsx
Figure 2—video 1
Stack images for Figure 2G, showing the ‘closed’ lumen above the PAC.
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.
The cells express the actin marker, Sqh::UtrABD-GFP (green), and are stained for Coracle (red) and Canoe (greyscale).
Figure 3 with 2 supplements
Integrating enteroblasts form an AMIS before forming a PAC.
(A) When enteroblasts reach the level of the septate junction between the adjacent ECs, Canoe (red) and Cora (greyscale) localise to the apical side of the enteroblast, whereas actin is still …
Figure 3—figure supplement 1
Actin and septate junction protein localisation during AMIS and PAC formation.
(A–B) Fim-GFP (green) marking actin. The enteroblasts in A and B are at similar stages to those shown in in Figure 3A and C. Canoe (red) and Cora (greyscale). (C–D) Fim-GFP (green; actin), Mesh …
Figure 3—video 1
Stack images for Figure 3D, showing the ‘closed’ lumen above the PAC.
The cells express the actin marker, Sqh::UtrABD-GFP (green), and are stained for Coracle (greyscale) and Canoe (red).
Figure 4 with 1 supplement
AJs are lost from the enteroblast apical membrane during AMIS formation.
(A) E-cadherin (green) localises all around the plasma membrane of integrating enteroblasts that have not yet reached the septate junction between the overlying enterocytes. Cora (greyscale) is not …
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Figure 4—source data 1
Source data for the fluorescence intensity plot in Figure 4A–D.
- https://cdn.elifesciences.org/articles/76366/elife-76366-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
The localisation of junctional proteins during PAC formation.
(A) Tsp2a (red) localises to the septate junctions that develop between the pre-enterocyte and the neighbouring enterocytes as the PAC forms. Canoe is shown in green and α-spectrin in greyscale. (B) …
Figure 5 with 1 supplement
Sox21a levels fall as the PAC forms during integration.
(A–B) Sox21a (red) is present at high levels in the nuclei of enteroblasts in which actin (Utr-ABD-GFP; green) is not yet polarised apically (arrow in A), is lower in the nuclei of enteroblasts with …
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Figure 5—source data 1
Source data for the graph as Figure 5D.
- https://cdn.elifesciences.org/articles/76366/elife-76366-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Activated enteroblasts take 1-2 days to become enterocytes.
(A) The Sox21a antiserum non-specifically stains the septate junctions. In the left hand panel, a wild-type Sqh::UtrABD-GFP gut stained for Sox21a shows nuclear staining in the two enteroblasts, as …
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Figure 5—figure supplement 1—source data 1
Source data for the graph as Figure 5—figure supplement 1C.
- https://cdn.elifesciences.org/articles/76366/elife-76366-fig5-figsupp1-data1-v2.xlsx
Figure 6
Diagram of the steps in enteroblast integration.
(A) Diagram shows the side view of the steps in enteroblast integration. ‘Unpolarised’ in the second panel of this figure indicates that the enteroblast has not formed a distinct apical domain. At …
Figure 7 with 1 supplement
Neither Canoe or Coracle is required for PAC formation or enterocyte polarity.
(A) Canoe (red) localises to the enterocyte membranes (yellow arrows) that face the lumen above an integrating pre-enterocyte, and to the marginal zone above the newly formed septate junctions …
Figure 7—figure supplement 1
Canoe is not required for PAC formation.
(A) A PAC still forms when both the pre-enterocyte and one of the neighbouring enterocytes are homozygous for canoeR10 (green). Canoe in red and α-spectrin in greyscale. (B) Canoe staining is lost …
Figure 8 with 2 supplements
mesh mutants fail to form a PAC or form an internal PAC.
(A) A meshR2 homozygous MARCM clone marked by GFP (green). The mutant cells lack Mesh staining (red), fail to make septate junctions and do not reach the gut lumen. (B–C) meshR2 (B) and meshf04955 (C…
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Figure 8—source data 1
Source data for the graph as Figure 8J.
- https://cdn.elifesciences.org/articles/76366/elife-76366-fig8-data1-v2.xlsx
Figure 8—figure supplement 1
Phenotypes of other septate junction mutants.
(A–C) The localisations of septate junction proteins are interdependent. Tsp2A (red) is lost from meshR2 mutant cells (green; A) and Mesh is not localised in Tsp2A1-2 (B) and ssk2 mutant cells (C). …
Figure 8—figure supplement 2
mesh and Tsp2a mutants disrupt the apical localisation on Canoe in integrating enteroblasts.
(A–C) Canoe (red) is polarised in early stage Tsp2A1-2 (A) and meshR2 mutant enteroblasts (C). As Tsp2A1-2 mutant cells differentiate and integrate, Canoe becomes diffuse and no longer forms a clear …
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Figure 8—figure supplement 2—source data 1
Source data for the graphs as Figure 8—figure supplement 2D and E.
- https://cdn.elifesciences.org/articles/76366/elife-76366-fig8-figsupp2-data1-v2.xlsx
Author response image 1
