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. 2015 Jun 15;34(15):2025–2041. doi: 10.15252/embj.201591517

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© 2015 The Authors. Published under the terms of the CC BY NC ND 4.0 license

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

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ReDDM-based analysis unveils hitherto unrecognized plasticity of progenitor cells

A Representative confocal image of an esg^Re^DDM adult midgut 7 days after the temperature shift. The large esg⁺ cell clusters (arrows) reflect substantial production by the individual ISCs in the absence of cell turnover in their local surroundings. Not-yet renewed cells (old mature cells) are detected by DAPI (not shown in the image) and outlined by the adherens junction protein marker Dlg-1 (grey).

B, C A high magnification image (ISC, intestinal stem cell; EB, enteroblast, EC, enterocyte) in which the lower EB represents a middle stage, and the upper EB a typical late stage (enterocyte-committed, polyploid enteroblast). (C) Co-staining with GBE-Su(H)-lacZ (white) identified the enteroblasts from their mother ISCs. Note the stronger esg > GFP signal and larger size of Su(H)⁺ (EB) cells.

D–H Confocal images of esg⁺ cells in unfixed wild-type adult midguts in homeostatic conditions. Enteroblasts are distinguished by their brighter GFP signal and larger size (see B). Cells are co-labelled by GFP (detecting esg > mCD8-GFP, green), RFP-actin (D), RFP-moesin (E) and RFP-tubulin (F–H). The image shows a single channel of RFP-tubulin (G) or mCD8-GFP (H).

I Magnification from (A) showing the intestinal epithelial cells outlined by Dlg-1 labelling (grey) and the interstitial position of the enteroblasts (which lacks the epithelial marker Dlg-1). Enteroblasts extend long protrusions along the EC borders.

J Magnification from (A) showing EBs in non-regenerated area.

K–N Representative confocal images showing Flybow (FB2.0) clones 14 days ACI (after clone induction). (K) Clones are varied in size and elongated, round or irregular in shape revealing plastic patterns of tissue turnover. Neighbouring clones can have intercalated cells (K–M, arrows). (N) Scheme of the Flybow MARCM method (Lee & Luo, 2001; Hadjieconomou et al, 2011) of expected outcome (a multicellular clone or a single cell) depending on whether the labelled daughter cell after induction of FLP to activate FRT-mediated mitotic recombination is an ISC or a EB.

O, P Images illustrate multicellular clones (O) and single-cell clone (P). The two neighbouring clones are composed of newly differentiated (hexagonal cells, EC) and undifferentiated (EB) cells, which tend to be strongly polarized. (P) A single-cell clone with long protrusion and large size typical of undifferentiated enteroblast surrounded by not-yet renewed cells. The old intestinal cells are colourless and visualized by DAPI nuclear counterstaining (white).