An empty Drosophila stem cell niche reactivates the proliferation of ectopic cells - PubMed (original) (raw)

An empty Drosophila stem cell niche reactivates the proliferation of ectopic cells

Toshie Kai et al. Proc Natl Acad Sci U S A. 2003.

Abstract

Stem cells are thought to reside in regulatory microenvironments ("niches") generated by stable stromal neighbors. To investigate the significance of empty niches vacated by stem cell loss, we studied Drosophila ovarioles, which maintain two to three germ-line stem cells in a niche requiring adhesive stromal cap cells and Decapentaplegic signals. After experimentally emptying the germ-line stem cell niche, cap cell activity persists for several weeks. Initially, somatic inner germarium sheath cells enter the empty niche, respond to Dpp, but fail to divide. Subsequently, follicle cell progenitors, including somatic stem cells enter the niche, respond to Dpp, and proliferate as long as cap cells remain. Proliferation requires the normal hedgehog signal of the somatic stem cells as well as proximity to the niche. Thus, empty niches can persist, signal incoming cells, and support ectopic proliferation. Similar events may underlie some disease states.

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Figures

Figure 1

Figure 1

The GSC niche is associated with local enhanced Dpp signal transduction. (a) A schematic drawing of a Drosophila germarium showing germ-line cells (pink) including the GSC, CB, and developing cysts (note branched fusomes, red). Somatic cells are terminal filament (TF; yellow-green), CpCs (green), IGSs (blue), and prefollicle cells (yellow). (b) A germarium from strain PZ1444-lacZ, an enhancer trap line that marks CpCs (strong green, arrowhead) and IGSs (light green, arrow). Hts staining is shown in red to mark fusomes and cell boundaries. (c) A wild-type germarium stained for pMAD (green) and Hts (red). High levels of pMAD are present only in GSCs (arrowhead). Elevated staining is not observed near the SSCs (arrows). (d) A Dad-lacZ germarium stained for LacZ (green) and Hts (red). LacZ is expressed strongly only in GSCs (arrowhead) and weakly in young CBs. Elevated staining is not seen near the SSCs (arrows). (e and f) Tumorous germaria from a c587-GAL4/UAS-dpp strain (e, see Materials and Methods) or a bam strain (f) stained for pMAD (green) and Hts (red). pMAD is widely distributed in the pattern dictated by the c587 line, indicating the early germ cells are competent to respond to Dpp throughout the germarium. In the bam tumor, pMAD is found only in endogenous GSCs located at the anterior tip, indicating that this location is unique in its ability to up-regulate the Dpp pathway. Insets in e and f show a GSC at higher magnification. (Bar = 10 μm.)

Figure 2

Figure 2

Empty GSC niches interact with two successive populations of ectopic cells. The changing cellular composition of the GSC niche and surroundings is shown 4 (a, a′), 7 (b, b′), 10 (c, c′),14 (d, d′), and 18 (e, e′) days after the ablation of GSCs by using hs-bam. Micrographs (a_–_e) of PZ1444 germaria are shown (anterior is left) stained with anti-β-galactosidase (green) to label CpCs (arrowhead) and IGSs (arrow), and anti-fusome Abs (red) against Hts protein. (f) Enhancer trap line PZ2954 (green) shows the position of follicle cell progenitors 4 days after GSC ablation. Hts staining is shown in red. (g) Enhancer trap line PZ10613 (green) shows the position of IGSs adjacent to the CpCs (unlabeled, arrowhead) 4 days after ablation. Hts staining is shown in red. (h) Staining for ApopTag (green) 4 days after ablation reveals that IGSs die by programmed cell death (PZ1444-lacZ is shown in red). The stability of terminal filament cells (bracket) is revealed by using hh-lacZ at 10 (i) and 18 days (j). CpCs are also stable (a_–_d and i) but disappear by 18 days as shown by staining for PZ1444 (e) and hh-lacZ (j). (k) Lineage analysis of follicle cell behavior after GSC ablation. An SSC clone generated by inducing FLP-mediated recombination at the same time as GSC ablation is shown after staining 7 days later. Follicle progenitor cells (green) lie posterior to an unlabeled group (arrowhead) consisting of CpCs and four to five IGSs. (a′_–_e′) The changing shape and cellular composition of the germarium after GSC ablation is summarized diagrammatically. The locations of CpCs are indicated by an arrowhead. (Bar = 10 μm.)

Figure 3

Figure 3

Somatic cell changes in the vicinity of the GSC niche after stem cell departure. A graph summarizes changes in the number of follicle cell progenitors (yellow), IGSs (blue), CpCs (green), and the number of somatic cells in the vacated GSC niche that label with BrdUrd (red) as a function of time after ablation of the GSC by a heat-induced pulse of Bam protein. Note that two different scales are used. Data are from the experiments of Figs. 2 and 4.

Figure 4

Figure 4

The niche induces ectopic cells to up-regulate Dpp signaling and to divide. (a_–_c) Cells responding to Dpp signaling were assayed by using Dad-lacZ. Germaria 4 (a), 10 (b), and 18 (c) days after GSC ablation are shown. Staining is with anti-β-galactosidase (green) and anti-Hts (red); arrowhead indicates CpCs. (d_–_f) To determine whether cells entered the cell cycle, BrdUrd incorporation was assayed in PZ1444-lacZ females. Germaria were stained for LacZ (red) and BrdUrd (green) at various times after GSC ablation. (d) Seven days, ectopic IGSs in the niche are not labeled; only cells associated with developing follicles have incorporated BrdUrd; SSCs are also negative at this time (arrow). (e) Ten days, ectopic follicle cell progenitors located in the niche adjacent to CpCs (arrowhead) strongly incorporate BrdUrd. (f) Eighteen days, no incorporation is seen after CpC function (arrowhead) is lost. The germarium is outlined. (Bar = 10 μm.)

Figure 5

Figure 5

Ectopic niche cells still require hedgehog. Control flies (PZ1444) and flies containing hs-bam and either a temperature-sensitive allele of dpp (a) or hh (b) that had been grown at the permissive temperature were subjected to a brief heat shock, incubated for 5 more days at the permissive temperature, and then shifted to the nonpermissive temperature for 5 days. BrdUrd incorporation within ectopic follicle progenitor cells in the vacated niche was observed in control flies (see Fig. 4_e_) and when Dpp was reduced (a) but not when Hh was reduced (b). The number of BrdUrd-incorporating cells was counted, and the average values in control (PZ1444) and the experimental genotypes were plotted (c). (d) The effects of ectopic Hh during niche repopulation was determined by measuring BrdUrd incorporation in flies containing both hs-hh and hs-bam transgenes, and that were repeatedly subjected to heat shocks for 18 days before analysis. Under these conditions, BrdUrd incorporation is observed in follicle cell progenitors throughout much of the germarium, not just in the niche. (e) A model showing the stimulation of ectopic SSCs (red) by Hh and possibly by Dpp in the vacated GSC niche. (Bar = 10 μm.)

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