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Science 327 (5962): 210-213
Copyright © 2010 by the American Association for the Advancement of Science
A Transient Niche Regulates the Specification of Drosophila Intestinal Stem Cells
Divya Mathur1,
Alyssa Bost1,
Ian Driver2, and
Benjamin Ohlstein1,*
1 Department of Genetics and Development, Columbia University Medical Center, New York, NY 10032, USA.
2 The Integrated Program in Cellular, Molecular, Structural and Genetic Studies, Columbia University Medical Center, New York, NY 10032, USA.
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Fig. 1. Characterization of AMP islands during larval development. (A) During late L1, AMPs exist as single, Dl-positive cells (arrowheads). Larval enterocytes are polyploid (arrow), and enteroendocrine cells are Prospero-positive (asterisk). (B) By late L2, an AMP island contains one Dl-positive AMP (arrowhead), and another cell that is Gbe+Su(H)lacZ-positive (arrow). (C) The Gbe+Su(H)lacZ-positive cell (asterisk) extends processes (arrows) around AMPs, throughout mid L3 and (D) late L3, and islands contain multiple Delta-positive AMPs (arrowheads). (A to D) DAPI, nuclear blue; Dl, cytoplasmic red; Prospero, nuclear red; and β-galactosidase, green. (E and F) Notch mutant MARCM clones (green, asterisk) lack a discernible PC [(inset in (E)]. Sometimes, mutant clones merge with each other (dashed line). They also have increased Dl staining at the membranes [inset in (F)], whereas WT islands (arrow) have predominantly vesicular Dl. (G) PswitchAMP UAS-GFP (green), expressed in all cells of an AMP island (late L3 shown) used to (H) ectopically express Nact at early L1, results in differentiation into a PC-like cell (green) with long extensions (arrows) when analyzed at late L3. (E, G, and H) DAPI, nuclear blue; Dl, cytoplasmic red; Prospero, nuclear red; and GFP, green. Scale bars, 10 µm.
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Fig. 2. Developmental fate of AMPs and PCs during metamorphosis. (A) PswitchPC UAS-GFP labels PCs (arrows) surrounding Dl-positive AMPs (arrowheads) during late L3 and (B) 0 hours APF. (C and D) Between 2 and 3 hours APF, PC (asterisk) extensions appear to spread out (arrows), and most AMPs (stars) express GFP and Pdm1, indicating differentiation into enterocytes. A smaller AMP population (arrowheads) remains Pdm1-negative, whereas some cells are Delta-positive. (E) Stat92E-GFP is expressed only in PCs (arrows) at 0 hours APF. (F and G) At 2 hours APF, PC (asterisk) processes break apart (arrow) and AMPs express Pdm1 (arrowheads). (H) PCs stain positive for active caspase-3 (arrows). (I and J) At 4 hours APF, PswitchPC UAS-GFP expression is diminished in differentiating AMPs. Most Pdm1-positive cells accumulate membrane-bound Dl (arrows). Pdm1-negative cells with vesicular Dl (arrowheads) are present at this stage. (K and L) By 14h APF, Pdm1–positive cells lose GFP and Dl expression (arrows), whereas Pdm1-negative cells are Dl-positive (arrowheads). (A to C, F, I, and K) GFP, green; Dl, cytoplasmic red; Prospero, nuclear red; and DAPI, nuclear blue. (D, G, and L) Pdm1, nuclear grayscale, unmerged. (H) GFP, green; active caspase-3, nuclear red; and DAPI, nuclear blue. Scale bars, 10 µm.
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Fig. 3. The PC acts as a niche that regulates AMP differentiation. (A and B) PswitchPC UAS-GFP–mediated expression of UAS-reaper at early L3 results in differentiation of AMPs into polyploid, GFP-positive, Pdm1-positive, enterocyte-like cells with a large cytoplasm (arrows indicate PDM1-positive, polyploid nuclei; dashed lines denote enterocyte-like cells). DAPI, blue; GFP, green; and Pdm1, red, nuclear. (C and D) (Dl channel) PswitchPC, UAS-GFP–driven expression of UAS-P35 results in prolonged PC survival (arrowhead) and delayed AMP differentiation (arrows). DAPI, nuclear blue; GFP, green; Dl, cytoplasmic red; and Prospero, nuclear red. Scale bars, 10 µm.
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Fig. 4. PCs signal via the Dpp pathway to prevent AMP differentiation. (A and B) PswitchPC UAS-GFP–driven expression of dpp RNAi results in (A) AMP differentiation into GFP-positive, polyploid, enterocyte-like cells (arrow, dashed line) that dissociate from other AMPs, indicated by diminished Arm staining, as compared with the Arm expression (arrowhead) between GFP-negative, undifferentiated AMPs. (B) Arm is expressed between cells in islands with GFP-UAS-GFP negative AMPs (arrowhead). MARCM clones of (C) tkv and (D) Mad result in differentiation of AMPs into polyploid enterocyte-like cells (arrows). (A to D) GFP, green; DAPI, nuclear blue. (A and B) Arm, red. Scale bars, 10 µm.
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