A transient niche regulates the specification of Drosophila intestinal stem cells

Abstract

Stem cell niches are locations where stem cells reside and self-renew. Although studies have shown how niches maintain stem cell fate during tissue homeostasis, less is known about their roles in establishing stem cells. The adult Drosophila midgut is maintained by intestinal stem cells (ISCs); however, how they are established is unknown. Here, we show that an ISC progenitor generates a niche cell via Notch signaling. This niche uses the bone morphogenetic protein 2/4 homolog, decapentaplegic, to allow progenitors to divide in an undifferentiated state and subsequently breaks down and dies, resulting in the specification of ISCs in the adult midgut. Our results demonstrate a paradigm for stem cell-niche biology, where progenitors generate transient niches that determine stem cell fate and may give insights into stem cell specification in other tissues.

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) Pswitch^AMP UAS-GFP (green), expressed in all cells of an AMP island (late L3 shown) used to (H) ectopically express N^act 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.

Fig. 2

Developmental fate of AMPs and PCs during metamorphosis. (A) Pswitch^PC 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, Pswitch^PC 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.

Fig. 3

The PC acts as a niche that regulates AMP differentiation. (A and B) Pswitch^PC 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) Pswitch^PC, 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.

Fig. 4

PCs signal via the Dpp pathway to prevent AMP differentiation. (A and B) Pswitch^PC 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.