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. Author manuscript; available in PMC: 2007 Oct 23.
Published in final edited form as: Cell Stem Cell. 2007 Aug 16;1(2):191–203. doi: 10.1016/j.stem.2007.07.003

The Adult Drosophila Malpighian Tubules Are Maintained by Pluripotent Stem Cells

Shree Ram Singh 1, Wei Liu 1, Steven X Hou 1,*
PMCID: PMC2040309  NIHMSID: NIHMS29270  PMID: 18371350

Summary

All animals must excrete the waste products of metabolism. Excretion is performed by the kidney in vertebrates and by the Malpighian tubules in Drosophila. The mammalian kidney has an inherent ability for recovery and regeneration following ischemic injury. Stem cells and progenitor cells have been proposed to be responsible for repair and regeneration of injured renal tissue. In Drosophila, the Malpighian tubules are thought to be very stable, and no stem cells have been identified. We have identified pluripotent stem cells in the region of lower tubules and ureters of the Malpighian tubules. Using lineage tracing and molecular marker labeling, we demonstrated that several differentiated cells in the Malpighian tubules arise from the stem cells and an autocrine JAK-STAT signaling regulates the stem cells' self-renewal. Identifying adult kidney stem cells in Drosophila may provide important clues for understanding mammalian kidney repair and regeneration during injury.

Keywords: Drosophila, adult kidney stem cell, signal transduction, JAK-STAT

Introduction

Human or mammalian kidneys develop from two sources: ureteric bud (metanephric diverticulum) and the metanephric mesoderm (Saxen, 1987; Moore, 1988; Sorokin et al., 1995). A mature kidney consists of two parts: a nephron, derived from the metanephric mesoderm, and a collecting tubule, derived from the ureteric bud. Tissue culture studies have shown that the mutual induction of ureteric bud and metanephric mesoderm direct the kidney formation. The branching of the ureteric bud is dependent upon induction by the metanephric mesoderm and the differentiation of the nephrons relies on the induction by the collecting tubules (Moore, 1988; Saxen, 1987; Sorokin et al., 1995). The ureteric bud and its branched derivatives first invade the metanephric mesoderm, induce the mesenchymal cells undergoing a structural transition, and become a second polarized epithelium. The two neighboring cell layers then fuse to form a single epithelial structure, the kidney. The epithelium originating from the ureteric bud will give rise to the ureter and the collecting ducts, while the metanephric mesoderm-derived portion will form the nephrons and glomerulli, the blood-filtering and urine-producing tubular units of the mature kidney.

The Drosophila kidneys (Malpighian tubules or MTs) also develop from two sources: hindgut primordium (ectodermal epithelia) and the visceral mesoderm (Denholm et al., 2003; Jung et al., 2005; Schejter and Shilo, 2003). Early in embryogenesis, interaction between the midgut and the hindgut anlagen refines the expression domain of the transcription factor Krüppel (Kr). Kr further regulates the expression of another transcription factor, Cut. As a consequence of Kr and Cut expression, the cells change shape and the MT primordia evert from the hindgut. In parallel to Kr and Cut, the wingless (Wg) also plays a role in MT cell allocation. Within the tubule primordia, a cell called the tip cell is specified via the Notch pathway. The tip cell expresses Kr and also secretes epidermal growth factor (EGF). The EGF then stimulates mitosis in neighboring cells and the growth of tubules by addition of new cells. The tubules then enter the vicinity of the caudal mesoderm and interact with a subpopulation of the caudal mesoderm to induce the mesoderm cells to undergo a mesenchymal-to-epithelial transition. The mesoderm-derived, polarized epithelium then progressively incorporates into the tubule epithelium as stellate cells (SCs), while the ectoderm-derived epithelium becomes the principal cells (PCs) and the ureter.

The above information illustrates that common principles, such as the interaction of two distinct cell populations, are used in the development of the mammalian kidney and Drosophila MTs. Even the pathway (Wnt) or molecules (Kr-Glis2/Klf-6, Cut-Cux-1) that are involved in the induction of tubulogenesis or cell differentiation (Hibris-Nephron) seem to have been conserved through evolution (Denholm et al., 2003; Jung et al., 2005).

The adult Drosophila has four MTs, a longer anterior, and a short posterior pair, which converge through common ureters onto the alimentary canal (Pugatcheva and Mamon, 2003; Sözen et al., 1997; Wessing and Eichelberg, 1978; Figure 1A). Each tubule of the longer anterior pair can be divided in four compartments: initial, transitional, main (secretary), and proximal (reabsorptive) (Figure 1A). The latter can be further divided into lower tubule and ureter. The first three compartments of each tubule consist of two cell types and ∼ 150 cells. Type I cells, also known as principal or primary cells, through which cations and organic solutes are transported, are the major tubule cell type (∼80%); type II cells, also known as secondary or stellate cells, through which water and chloride ion flow (Jung et al., 2005; Pugatcheva and Mamon, 2003), are interspersed at regular intervals with the type I cells. The type I cells express the transcription factor Cut and the type II cells express the transcription factor Teashirt (TSH) (Jung et al., 2005). The cut-positive type I cells are found in the initial, transitional, and main segments, and the region of lower tubules and ureters, while the type II cells are located in the initial, transitional, and main segments of the tubules, but were not found in either lower tubules or ureters (Pugatcheva and Mamon, 2003; Sözen et al., 1997; Figure 1B of this study). A previous study (Pugatcheva and Mamon, 2003; Sözen et al., 1997) also identified so-called “tiny” cells in the proximal compartment (including lower tubules and ureters). The tiny cells were proposed to be homologues of myoendocrine cells of the ants Formica, which collect the urine in the renal duct and secrete neurohormones in the hemolymph.

Figure 1.

Figure 1

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The Drosophila MTs and Their Cells

(A) A drawing (adapted from Wessing and Eichelberg, 1978) of the Drosophila MTs. Drosophila has four tubules; the anterior pair is longer than the posterior pair (one tubule of each pair is depicted). Each tubule has four distinct morphologic regions: initial, transitional, and main segments and lower tubule. The two tubules in each pair merge together at ureters and connect to the gut at the midgut-hindgut boundary.

(B and F) The MTs from a tsh-lacZ fly stained with DAPI (blue), anti-β-galatosidase (green), and anti-Cut (red). Red arrows point to the Cut-positive cells in ureter, lower tubule, and main segment. Green arrows point to β-galactosidase-positive type II cell in the main segment. White arrowheads point to tiny cells in the region of lower tubules and ureters. Yellow arrow points to posterior midgut that is negative for both Cut and β-galactosidase. β-galactosidase-positive type II cells interspersed with Cut-positive type I cells in the upper tubules (main, transitional, and initial segments). Scale bars: in (B), (C), and (F) represent 10 μm; in (D) and (E), 5 μm.

It is well known that some adult mammal renal tissues that are lost through injury can be repaired or regenerated. Ischemic injury to the kidney causes death of renal cells, followed by tubular regeneration and recovery of renal function. In animal models, the completely lost glomerular structures can be regenerated after immunologic injury (Haller et al., 2005). In amphibians, such as skate and shark (Elger et al., 2003), the adult animal can regenerate new nephrons. However, the source of the proliferating cells that repopulate the injured nephrons or regenerate the lost renal tissues is not clear. In a recent report, Oliver et al. discovered that the renal papilla contains large numbers of slowly cycling cells and may be a niche for adult kidney stem cells. These slow-cycling cells quickly enter the cell cycle and may participate in renal regeneration after ischemic injury (Oliver et al., 2004). In the little skate, Leucoraja erinacea, a nephrogenic zone has been identified in the adult kidney (Elger et al., 2003). The zone contains stem cell-like mesenchymal cells. These pioneer works strongly suggested the existence of adult kidney stem cells and identified their proximate location in the kidney. However, additional studies are needed to identify these cells and confirm their “stemness,” such as self-renewal and the ability to generate more than one terminally differentiated cell type.

The adult Drosophila MTs have been thought to be very stable (Skaer, 1993). The developmental program of the tubules is mostly completed during embryogenesis. During metamorphosis, the other parts of the gut are entirely remodeled, the larval gut is degenerated, and the adult gut is formed. The MTs are an exception; they did not remodel during metamorphosis and remain almost unmodified in the adult. No adult stem cells have been described in the MTs. In this report, we show that the tiny cells in the region of lower tubules and ureters function as pluripotent adult stem cells and that an autocrine JAK-STAT signaling regulates the stem cells' self-renewal.

Results

The Proximal Region of MTs Contains Unique Cell Types

We re-examined the cell types in the MTs using an antibody to Cut and a tsh-lacZ enhancer trap line. Consistent with previous reports, we found that the Cut-positive type I cells are in the whole MTs (including initial, transitional, main segments, lower tubules, and ureters; Figures 1C, 1E, and 1F), while the β-Gal-positive type II cells are only found in the upper tubules (including initial, transitional, and main segments; Figures 1C and 1E). The nuclei of the type I cells are larger than those of the type II cells (Figures 1B and 1D). The β-Gal-positive type II cells have “stellate” morphology in the main segments and have “bar-shaped” morphology in the initial and transitional segments. The posterior midgut expresses neither Cut nor tsh-lacZ (Figure 1F, yellow arrow).

The lower tubules and ureters are composed of three types of cells based on their nuclear sizes (Figures 2A and 2B). The first type has a small nucleus (white arrowhead in Figure 2A) and mostly lies close to the tubular walls (Figure 2B). Cells of the second type are Cut-positive (Figures 1C and 1F), have large and oval nuclei (white arrow in Figure 2A), and are distant from the tubular walls (Figure 2B). The small cells may correspond to the previously reported tiny cells in the lower tubules and ureters (Sözen et al., 1997). Cells of the third type have intermediate nuclear size (green arrowhead in Figure 2A) and may be the transition type cells between the small and large nuclear cells.

Figure 2.

Figure 2

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The Small Nuclear Cells are Proliferating and Express Unique Molecular Markers in the Region of Lower Tubules and Ureters of Adult Drosophila MTs

(A and B) Cells with three different-sized nuclei occupy distinct positions in the region of lower tubules and ureters (phalloidin, red; DAPI, blue). White arrow, green arrowhead, and white arrowhead in (A) point to the large, intermediate, and small nuclear cells, respectively. The cells with small nuclei lie primarily in close proximity to the tubular walls, while the cells with large and oval nuclei are distant from the tubular walls (B).

(C) BrdU labels the small (white arrows), intermediate (yellow arrow), and large (green arrow) nuclear cells (anti-BrdU, red; DAPI, blue).

(D and E) The phospho-histone-H3 labels only a small nuclear cell (yellow arrows; anti-phospho-histone H3, red; anti-Arm, green; DAPI, blue).

(F) Anti-Arm staining outlines the small nuclear cells (white arrows; anti-Arm, red; DAPI, blue).

(G) esg-Gal4/UAS-GFP is specifically expressed in the small nuclear cells (white arrows in G and I) and (white arrows; anti-GFP, green; anti-Arm, red; DAPI, blue)

(H) kr-Gal4/UAS-GFP is specifically expressed in the small nuclear cells (white arrows anti-GFP, green; anti-Arm, red; DAPI, blue).

(I and J) Both esg (I) and kr (J) sometimes label a pair of phospho-histone-H3 positive dividing cells (white arrows in I and J; anti-GFP, green; anti-phospho-histone H3, red; DAPI, blue). Scale bars: in (G) and (H) represent 10 μm; in (A), (C–F), and (I–J), 5 μm; in (B), 2 μm.

The Tiny Cells Are Proliferating and Express Unique Molecular Markers

To further characterize the proliferating cells in the MTs, we performed 5-bromodeoxyuridine (BrdU) incorporation experiments. We found that BrdU labels all three cell types in the region of lower tubules and ureters (Figure 2C). No BrdU-labeled cells were detected in the upper tubules (data not shown). In both larvae and adult tissues, many cells undergo endoreplication (Edgar and Orr-Weaver, 2001). Both endoreplicating and dividing cells synthesize new DNAs and can be labeled by BrdU. We further stained the tissue for phospho-histone-H3 to distinguish endoreplicating from dividing cells in the MTs. While BrdU labels all three cell types in the region of the lower tubules and ureters, the phospho-histone-H3 staining was only detected among the population of cells with small nuclei (Figures 2D and 2E, yellow arrows; Figures 2I and 2J, white arrows). These data suggest that the cells with small nuclei are dividing, and the cells with intermediate and large nuclei are undergoing endoreplication. We also stained the tissue for Armadillo (Arm, the β-Catenin homolog). The Arm staining clearly outlined the small nuclear cells in the region of lower tubules and ureters (Figure 2F).

To characterize the tiny cells further, we searched for additional cell-specific molecular markers in the MTs. We found that the transcription factors escargot (esg; Figures 2G and 2I) and kr (Figures 2H and 2J) are specifically expressed in the small nuclear cells (arrows) in the region of the lower tubules and ureters. We further found that both esg and kr occasionally label a pair of phospho-histone-H3 positive dividing cells (Figures 2I and 2J, arrows). The function of the esg gene is to maintain cells as diploid in Drosophila imaginal cells (Fuse et al., 1994). Therefore, the esg-positive small nuclear cells in MTs are likely diploid cells. In the Drosophila digestive system, the region of lower tubules and ureters is anatomically next to the posterior midgut, a transcriptional reporter of the Notch signaling pathway [Su(H)GBE-lacZ], and the transcription factor Prospero (Pros) were reported to be expressed in the small cells in the posterior midgut (Micchelli and Perrimon, 2005; Ohlstein and Spradling, 2005). We checked the Su(H)GBE-lacZ and Pros in the MTs and found that neither Su(H)GBE-lacZ nor Pros is expressed in the region of lower tubules and ureters (data not shown). Therefore, the MTs and posterior midgut express different molecular markers; Cut and tsh-lacZ are expressed only in the MTs and not in the posterior midgut (Figure 1F), while Su(H)GBE-lacZ and Pros are only expressed in the posterior midgut and not in the MTs. Therefore, the MTs and posterior midgut are composed of distinct cell types.

The Small Nuclear Cells in the Region of Lower Tubules and Ureters Are Pluripotent Stem Cells

To find out whether the MTs are maintained by stem cells, we first used a positively marked mosaic lineage (PMML) labeling technique (Kirilly et al., 2005) to label and trace cells that undergo mitotic divisions. The production of GFP-marked clones is dependent on mitotic recombination. Therefore, the technique allowed us to directly detect proliferating cells. The adult flies bearing transgenes of a heat-shock-inducible flipase (hs-FLP), a reporterless actin5C promoter, and a promoterless Gal4 with a UAS-EGFP were heat-shocked to induce FLP-FRT-mediated mitotic recombination and generate an active actin5C-Gal4 for driving UAS-EGFP expression. Following heat shock in 2-, 5-, or 10-day-old wild-type adult flies, GFP-marked clones were detected only in the region of the lower tubules and ureters, and not in the upper tubules of the MTs (Figure 3A). No GFP-marked clones were detected in control animals that had identical genotypes but did not undergo heat shock treatment (data not shown). Two days after clone induction (ACI), the GFP mostly labeled the primarily the small nuclear cells (white arrows in Figures 3A and 3B). Only one large nuclear cell clone (white arrowhead in Figure 3A) was usually detected in each MT. As described below, the marked small nuclear cell is a stem clone, and the marked, large nuclear cell is a non-stem cell, ‘transient’ clone. Since the non-stem cell clones are very infrequent, these cells must have very limited proliferation potential.

Figure 3.

Figure 3

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The Tiny Cells in the Regions of Lower Tubules and Ureters are Pluripotent Stem Cells.

GFP (green) label cells derived from PMML clones.

(A) GFP marks primarily the small nuclear cells (white arrow) in the region of lower tubules and ureters but not the TSH-positive (red, green arrow) type II cells in the upper tubules of the MTs (two days ACI). One transient large nuclear cell GFP clone (white arrowhead) was also detected in the MTs. DAPI staining in blue.

(B) An enlarged view of GFP-marked clones (two days ACI) showing that the GFP marks primarily the small nuclear cells (anti-Arm, red; anti-GFP, green; DAPI, blue).

(C-F) 4 days ACI. The GFP marks clones of cluster cells with small, intermediate, and large nuclei. Two examples of clones show the basal RNSC (arrowheads), RB (arrows) and apical direction of growth (dashed arrows; anti-GFP, green; DAPI, blue).

(G and H) 6 days ACI. The GFP marks cluster cells with small, intermediate, and large nuclei in the region of the lower tubules and ureters (anti-Arm, red; anti-GFP, green; DAPI, blue).

(I) 4 days ACI. Some of the GFP-marked small nuclear cells (arrows) migrated to the main segment (anti-Arm, red; anti-GFP, green; DAPI, blue).

(J-N) 10 days ACI. In the region of the lower tubules and ureters, the GFP marks cell clusters (one cluster of cells is highlighted in [K]). In the upper tubule, the GFP labels both Cut-positive (red in [M] and [N]) type I cells (yellow arrowheads) and TSH-positive (red in [L]) type II cells (yellow arrows in [L–N]). White dashed arrows in (A) point from the ureter to upper tubules. Scale bars: in (A) represents 20 μm; in (G) and (J), 10 μm; in (B–F), (H–I), and (K–N), 5 μm.

We further traced the GFP-marked clones for 4, 6, and 10 days ACI to follow the fates of the marked cells. At 4 and 6 days ACI, the GFP clones were restricted primarily to the region of the lower tubules and ureters (Figures 3C–3H). In this region, the GFP marked individual small nuclear cells (yellow arrows in Figure 3G) and cluster cells with small, intermediate, and large nuclei (two examples are shown in large views in Figures 3C-3F). By observing the clonal expansion from 4 to 6 days ACI, we found that most basal diploid cells (white arrowheads in Figures 3C and 3E) function as stem cells, which we term the renal and nephric stem cells (RNSCs). The RNSCs contacted their immature diploid daughters (white arrows in Figures 3C and 3E), which we term the renalblast (RB). Following additional RNSC divisions, the former RB began to expand in size and DNA content, becoming intermediate and large nuclear cells (Figures 3C–3F). The intermediate and large nuclear cells in the region of the lower tubules and ureters expressed Cut but may function differently from the Cut-positive type I cells in the upper tubules because the upper and lower tubules of MTs have distinguishable functions in excreting the waste products. We term the intermediate and large nuclear cells the early and late renalcyte (RC)

At 4 and 6 days ACI, the GFP also marked some diploid small nuclear cells in the main segments (Figure 3I). The GFP-marked small nuclear cells in the main segments likely came from the lower tubules because they were not in the main segments 2 days ACI. Further, these main segment small nuclear cells did not proliferate and never formed clusters of cells; they moved up and differentiated into both TSH-positive and Cut-positive cells in the initial and transition segments at 10 days ACI (Figures 3L–3N).

At 10 days ACI, the GFP labeled the entire MT. In the region of lower tubules and ureters, the GFP marked individual small nuclear cells and cluster cells with small, intermediate, and large nuclei (Figures 3J and 3K); in the main segments, the GFP labeled small nuclear cells (similar to Figure 3I); in the initial and transition segments, the GFP labeled both TSH-positive type II and Cut-positive type I cells (Figures 3L–3N). The ratio of TSH-positive/Cut-positive cells was about 4:1, suggesting that the type II cells turn over faster than type I cells in the upper tubules.

We also used another sensitive lineage labeling method that did not rely on Gal4 (Harrison and Perrimon, 1993). In these experiments, clones expressing a nuclear-targeted β-galactosidase (β-gal) protein were generated after the adult flies bearing transgenes of a hs-FLP, a reporterless tubulin promoter, and a promoterless lacZ were heat-shocked to induce FLP-FRT-mediated mitotic recombination and generate an active tubulin promoter-lacZ gene. Similar results were observed using the second clone marking technique (data not shown).

The above data suggest that the small nuclear cells in the regions of the lower tubules and ureters function as pluripotent stem cells in the renal tubules. An RNSC can produce one RB through asymmetric division. The RB can have two fates. In the region of lower tubules and ureters, the RB can become a mature RC in about 5 days through endoreplication. The RB can also move toward the distal upper tubules and finally differentiate into a type I or II cell in the transitional and initial segments. It takes about 10 days for a RB to move up and finally differentiated into a type I or II cell in the transitional and initial segments.

An Autocrine JAK-STAT Signaling Operates in the small nuclear cells

In Drosophila testis and ovary, germline stem cells are anchored to a group non-dividing somatic cells. These somatic cells reside in a fixed location and function as stem cell niches. In the MTs, the RNSCs do not attach to any particular cell types and are scattered over the region of the lower tubules and ureters. We wondered how the RNSCs are regulated in such an arrangement. To identify the signaling that regulates the RNSC self-renewal, we examined the expression of the components of the JAK-STAT signal transduction pathway in MTs because the signaling regulates stem cell self-renewal in several other stem cell systems (Decotto and Spradling, 2005; Kiger et al., 2001; Tulina and Matunis, 2001). The Drosophila JAK/STAT signal transduction pathway has five major components (Arbouzova and Zeidler, 2006). A transmembrane protein Domeless (Dome, also called Master of Marrelle (Mom); Brown and Hombria, 2001; Chen et al., 2002) functions as a receptor of the signal transduction pathway. A secreted glycoprotein Unpaired (Upd) is a ligand of the Dome receptor (Harrison et al., 1998). The signal is trasduced through the only Drosophila JAK kinase homolog, Hopscotch (Hop; Binari and Perrimon, 1994), to the only Drosophila STAT homolog, Stat92E (Hou et al., 1996; Yan et al., 1996). The pathway is also regulated by a negative regulator Socs36E (Callus, B. Mathey-Prevot, 2002). The activated Stat92E then enters the nucleus to activate its target genes' transcription (Arbouzova and Zeidler, 2006).

We first examined the receptor dome expression in the MTs using the dome-Gal4/UAS-GFP flies. The dome-Gal4/UAS-GFP was expressed in a mosaic pattern in the MTs (Figure 4A). In the region of the lower tubules and ureters, it was expressed in both small and large nuclear cells (Figure 4B). We also examined upd expression in the upd-Gal4/UAS-GFP flies and found that the upd-Gal4/UAS-GFP was only expressed in the single small nuclear cells in the region of lower tubules and ureters (Figures 4C and 4D, arrows). Some of the upd-Gal4/UAS-GFP labeled cells were also phospho-histone-H3 positive (Figure 4E, arrow). However, unlike the esg-Gal4/UAS-GFP (Figure 2I) and the kr-Gal4/UAS-GFP (Figure 2J), the upd-Gal4/UAS-GFP only labeled a single phospho-histone-H3 positive cell (Figure 4E) and never labeled a pair of phospho-histone-H3 positive dividing cells (compare Figure 4E with Figures 2I and 2J). It is likely that upd is only expressed in RNSCs and esg and kr are expressed in both RNSCs and RBs. One pair of anterior MTs had an average of 97 upd-expressed cells (n=12), 157 esg-expressed cells (n=7), and 174 kr-expressed cells (n=8). If we consider that half of the esg-expressed cells or the kr-expressed cells are RNSCs, the total number of RNSCs in one pair of anterior MTs should be ∼79-87. However, we observed that some RNSCs were quiescent cells and were not asymmetrically dividing into one RNSC and one RB. Therefore, the actual number of RBs should be less than RNSCs. We estimate that the number of RNSCs in one pair of anterior MTs should close to the number of upd-expressed cells (∼97).

Figure 4.

Figure 4

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The JAK-STAT Signaling is Operating in RNSCs.

(A) dome-Gal4/UAS-GFP is expressed in the whole MT (anti-GFP, green; DAPI, blue).

(B) In the region of lower tubules and ureters, dome-Gal4/UAS-GFP is expressed in both the small nuclear cells (arrows) and the large nuclear cells (arrowheads; anti-Arm, red; anti-GFP, green; DAPI, blue).

(C) upd-Gal4/UAS-GFP is only expressed in the region of lower tubules and ureters (arrows), but not in the upper tubules (anti-GFP, green; DAPI, blue).

(D) In the region of lower tubules and ureters, upd-Gal4/UAS-GFP is expressed only in the small nuclear cells (arrows; anti-Arm, red; anti-GFP, green; DAPI, blue) and one upd-expressed cell is also phospho-histone-H3 positive (arrow in [E]; anti- phospho-histone-H3, red; anti-GFP, green; DAPI, blue).

(F) Stat92E protein is expressed only in the small nuclear cells (arrow) in the region of lower tubules and ureters (anti-Stat92E, green; anti-Arm, red; DAPI, blue). White dashed arrows in (A–D) point from the ureter to upper tubules. Scale bars: in (A) represent 40 μm; in (C), 20 μm; in (B) and (D), 10 μm; in (E) and (F), 5 μm.

We also examined Stat92E protein expression in wild type flies (Figure 4F). The nuclear Stat92E was detected in both single and pairs of small nuclear cells (possibly in both RNSCs and RBs) in the region of the lower tubules and ureters. Because Stat92E protein level is regulated by the JAK-STAT signaling (Chen et al., 2002) and the Stat92E protein expression is readout of the signaling, the JAK-STAT signaling is activated in both RNSCs and RBs. The RNSCs produced both the ligand Upd and the receptor Dome; therefore, an autocrine JAK-STAT signaling operates in the RNSCs.

The JAK-STAT Signaling Regulates the RNSCs' Self-renewal

We examined the JAK-STAT pathway's function on RNSCs. Using the PMML technique, we first generated GFP-marked clones that also overexpress upd (PMML-UAS-upd; Figure 5). In the PMML-UAS-upd flies, we found that the MT size was enlarged significantly (compare Figures 5B with 5A). The number of GFP-marked clones (Figure 5C) and Stat92E-positive small nuclear cells was dramatically higher (Figure 5E). However, most Stat92E-positive small nuclear cells were GFP-negative, indicating that the secreted-Upd from the GFP-positive clones stimulated cell proliferation neighboring cells. We also counted the total number of cells and the number of GFP-positive cells in both the PMML-wild-type and the PMML-UAS-upd flies, based on DAPI and GFP stainings. One pair of anterior MTs had an average of 497 cells (n=23) in the PMML-wild-type flies and 2645 cells (n=23) in the PMML-UAS-upd flies. 2 days ACI, one pair of anterior MTs had an average of 35 GFP-positive cells (25 RNSCs+RBs and 10 RCs; n=67) in the PMML-wild-type flies and 1350 GFP-positive cells (550 RNSCs+RBs and 800 RCs; n=34) in the PMML-UAS-upd flies. Unlike the mostly single small nuclear cell clones in the PMML- wild-type flies (Figures 3A and 3B), most clones were composed of cluster cells 2 days ACI in the PMML-UAS-upd flies (Figures 5F-5J). Each cluster had one RNSC (arrowhead in Figure 5J), one RB (arrow in Figure 5J), and one RC. The PMML-UAS-upd flies also showed an increase in phospho-histone H3 staining (mitotic index = phospho-histone H3 positive cells/one pair of anterior MTs: mitotic index in the PMML-UAS-upd flies=8, N=17; mitotic index in the PMML-wild-type flies=3, N=14). These data suggest that overexpression of upd makes RNSCs more active, accelerating both their self-renewal and differentiation into RC. We also traced the GFP-marked clones for 4, 6, and 10 days ACI in the PMML-UAS-upd flies and found that the GFP-marked small nuclear cells moved up and differentiate into type I and II cells in the upper tubules, like those in the wild-type control flies. Therefore, the accelerating proliferation and differentiation of RNSCs was confined to the region of lower tubules and ureters, and cell migration and differentiation in the upper tubules in the Upd overexpressed flies was similar to that in the wild type control flies. These findings suggest that factors other than the JAK-STAT signaling may restrict the RNSCs to the region of the lower tubules and ureters.

Figure 5.

Figure 5

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The JAK-STAT signaling regulates MT proliferation.

(A) MTs with PMML wild-type clones.

(B) MTs with PMML clones that overexpress upd (PMML-UAS-upd). In the PMML-UAS-upd flies, the MT size was enlarged dramatically (compare [B] with [A]), and the number of GFP-marked clones increased greatly ([C]: anti-GFP, green; DAPI, blue).

(D) MTs with PMML clones that overexpress socs36E (PMML-UAS-socs36E). In the PMML-UAS-socs36E flies, only a few of the GFP-marked large nuclear cells were detected in the MTs (anti-Arm, red; anti-GFP, green; DAPI, blue).

(E) In the PMML-UAS-upd flies, the number of Stat92E-positive (red) small nuclear cells is dramatically increased (anti-Stat92E, red; anti-GFP, green; DAPI, blue). However, most Stat92E-positive cells are GFP-negative, indicating that the secreted-Upd from the GFP-positive clones stimulate cells proliferation in the neighbor cells.

(F–J) 2 days ACI in the PMML-UAS-upd flies, most GFPs mark cluster cells. Two examples of clones are shown in (G-J) (outlined by dashed lines). Each cluster has one RNSC (arrowhead), one RB (arrow), and one RC. Dashed arrows in (A–D) point from the ureter to upper tubules. Scale bars: in (A) and (B) represent 20 μm; in (C–E), 10 μm; in (F–J), 5 μm.

We further tested the effect of reducing the JAK-STAT signaling by generating GFP-marked clones that also overexpress socs36E using the PMML technique (PMML-UAS-socs36E; Figure 5D). In the PMML-UAS-socs36E flies, only GFP-marked large nuclear cells were detected in the MTs (Figure 5D), suggesting that reducing the JAK-STAT signaling caused the RNSC loss.

The JAK-STAT Signaling Autonomously Regulates the RNSCs' Self-renewal

To determine whether the JAK-STAT is directly required in the RNSCs, we generated Stat92E null clones, using the mosaic analysis with a repressible cell marker (MARCM) system (Lee and Luo, 1999). Marked clones homozygous for wild-type control (Figure 6A), Stat92E06346 (Figure 6B), Stat92Ej6C8 (Figure 6C), and Stat92Ej6C8 UAS-Stat92E (Figure 6D) were generated in the MTs and identified by GFP expression. In the control flies, the GFP marked primarily cell clusters with both Arm-positive small nuclear cells and large nuclear cells in the region of the lower tubules and ureters (Figure 6A); in the Stat92E06346 and Stat92Ej6C8 mutation flies, the GFP marks (∼100%, n = 45) a few large nuclear cells in the lower tubules (Figure 6B and C) and differentiated type I and type II cells in the upper tubules (data not shown), indicating that without the JAK-STAT signaling the RNSCs differentiated and moved out of their normal location in the region of lower tubules and ureters prematurely. Further, simultaneously expressing UAS-Stat92E in the Stat92Ej6C8 mutant clones rescued the Stat92Ej6C8 mutant phenotypes to those of the wild-type controls, the GFP marked cell clusters of both small and large nuclear cells in the UAS-Stat92E + Stat92Ej6C8 flies (Figure 6D).

Figure 6.

Figure 6

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The JAK-STAT Signaling Autonomously Regulates RNSC Self-renewal.

MTs with GFP-marked wild-type (A), Stat92E06346 (B), Stat92Ej6C8 (C), and Stat92Ej6C8 UAS-Stat92E (D) MARCM clones. Notice that the Arm-positive small nuclear RNSCs (white arrows) were lost in MTs with Stat92E mutant MARCM clones (B and C) and recovered in MTs with UAS-Stat92E rescue (D). Red arrows in A–D point to the large RCs. MTs with GFP-marked clones homozygous for wild type control (E), Stat92E06346 (F), Stat92Ej6C8 (G), and Stat92Ej6C8 UAS-Stat92E (H) were stained with anti-GFP and Apoptag kit to detect dead cells. Dashed arrows in (A–H) point from ureter to upper tubules. Scale bars in (A-H) represent 10 μm.

RNSC loss might be caused by cell death. We examined cell death in MTs with marked clones homozygous for in wild type control (Figure 6E), Stat92E06346 (Figure 6F), Stat92Ej6C8 (Figure 6G), and Stat92Ej6C8 UAS-Stat92E (Figure 6H) using an Apoptag kit. Some dying cells were detected in the whole MTs of the four genotypes, but the dying cells did not overlap with GFP and the numbers of dying cells was not significantly different among MTs of the four genotypes. This observation suggests that the JAK-STAT signaling functions in the RNSCs and regulates the stem cell self-renewal. Loss of the JAK-STAT signaling results in the RNSC differentiation rather than cell death.

The Relative Strength of the JAK-STAT Signaling May Regulate either Self-renewal or Differentiation in RNSCs

To gain insight into how the JAK-STAT signaling regulates RNSC self-renewal or differentiation, we further examined the activity of the JAK-STAT signaling in the MTs using a GFP reporter (Stat92E reporter-GFP) that detects the activation of the Drosophila JAK-STAT pathway in vivo (Bach et al., 2007). Stat92E reporter-GFP was expressed in the Arm-positive small nuclear cells from ureter to the lower part of the main segments (Figure 7A). From the ureter upward, the GFP signal is gradually reduced and completely diminished in the lower part of main segment. We may call the Stat92E reporter-GFP-positive segment (from the ureter to the lower part of main segment) the JAK-STAT signaling domain. Even in the JAK-STAT signaling domain, some small nuclear cells expressed strong GFP (white arrows in Figures 7A and 7B), while the other small nuclear cells expressed weak GFP (red arrows in Figures 7A and 7B). It is possible that the strong GFP cells are RNSCs and the weak GFP cells are RBs. The strong JAK-STAT signaling regulates RNSC self-renewal, and the weak JAK-STAT signaling prepares RB for differentiation.

Figure 7.

Figure 7

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The Relative Strength of the JAK-STAT Signaling may Regulate RNSCs Either Self-renewal or Differentiation.

(A and B) Stat92E reporter-GFP is expressed only in small nuclear cells in the region of the lower tubules and ureters (anti-Arm, red; anti-GFP, green; DAPI, blue). From the ureter upward, the GFP signal is gradually reduced and completely diminished in the lower part of main segment. We may call the Stat92E reporter-GFP-positive segment (from the ureter to lower part of main segment) the JAK-STAT signaling domain. Even in the JAK-STAT signaling domain, some small nuclear cells express strong GFP (white arrows), while the other small nuclear cells express weak GFP (red arrows). The strong GFP cells may identify RNSCs, and the weak GFP cells may identify RBs.

(C) Proposed model of the RNSC lineage (discussed in text). Dashed arrows in (A) and (B) point from the ureter to upper tubules. Scale bars in (A and B) represent 10 μm.

Discussion

The kidney has an inherent ability to recover and regenerate lost tissue following acute damage. It has been proposed that stem cells and progenitor cells are responsible for the repair and regeneration of the injured renal tissue. However, researchers differ as to the source of regenerating renal cells. The regenerating renal cells may come from one of the three possible sources, based on previous studies (Haller et al., 2005; Ricardo and Deane, 2005). First, the circulating blood contains bone marrow-derived stem cells able to differentiate into non-haematopoietic cells, such as cells of the kidney. Second, the differentiated glomerular and tubular cells may also be able to dedifferentiate into stem-like cells to repair the damaged tissues. Third, large numbers of slowly cycling cells have recently been identified in the mouse renal papilla region; these cells may be adult kidney stem cells and may participate in renal regeneration after ischemic injury (Oliver et al., 2004). Further, the ureter and the renal collecting ducts were formed from the epithelium originating from the ureteric bud, and the nephrons and glomerulli were formed from the metanephric mesoderm-derived portion during kidney development. Two distinguished stem cell types have been proposed as responsible for repairing the renal collecting tubules and the nephrons (Oliver et al., 2004). In this study, we identified a type of pluripotent stem cells (RNSCs) in the Drosophila renal organ. The stem cells are able to generate all cell types of the adult fly MTs (Figure 7C). In the region of lower tubules and ureters, autocrine JAK-STAT signaling regulates the stem cell self-renewal. Weak JAK-STAT signaling may convert an RNSC into an RB, which will differentiate into an RC in the region of lower tubules and ureters, and a type I or type II cell in the upper tubules. These data indicate that only one type of stem cell may be responsible for repair and regeneration of the whole damaged tissues in mammalian kidney.

Drosophila RNSCs Are Not Regulated by a Fixed Niche

The Drosophila RNSCs represent a unique model to study the molecular mechanisms that regulate stem cell or cancer stem cell behavior. In most of the stem cell systems that has been well characterized to date, stem cells always reside in a specialized microenvironment, called a niche (Fuchs et al., 2004; Spradling et al., 2001). A niche is a subset of neighboring stromal cells and has a fixed anatomical location. The stromal cells often secrete growth factors to regulate stem cell behavior. The stem cell niche plays an essential role in maintaining stem cells, and stem cells will lose stem cell status once they are detached from the niche (Wang et al., 2006). The niche often provides the balanced (proliferation-inhibiting and proliferation-stimulating) signals that keep the stem cells dividing slowly. The inhibitory signals keep the stem cell quiescent most of the time while the stimulating signals promote stem cell division, to replenish lost differentiated cells. Maintaining the balance between proliferation-inhibiting and proliferation-stimulating signals is the key to maintaining tissue homeostasis (Li and Neaves, 2006).

Drosophila RNSCs are controlled differently. We demonstrated that the JAK-STAT signaling regulates the stem cell self-renewal. We further found that both the ligand Upd and the receptor Dome are expressed in the RNSCs and the autocrine JAK-STAT signaling regulates the stem cell self-renewal; thus, the self-sufficient stem cells control their self-renewal or differentiation and do not need to constrained to a fixed niche. However, the RNSCs are still confined to the region of lower tubules and ureters even in the Upd overexpressed flies, suggesting that some other factors besides the JAK-STAT signaling may restrict the RNSCs to the region of the lower tubules and ureters.

JAK-STAT Signaling and Cancer Stem Cells

Recent studies also suggest that tumors may arise from small populations of so-called cancer stem cells (CSCs; Li and Neaves, 2006; Pardal et al., 2003; Wang and Dick, 2005). The CSCs probably have arisen from mutations that dysregulate normal stem cell self-renewal. For example, mutations that block the proliferation-inhibiting signals or promote the proliferation-stimulating signals can convert the normal stem cells into CSCs. We demonstrated that amplifying the JAK-STAT signaling by overexpressing its ligand Upd stimulates the RNSCs to proliferate and also to differentiate into RC, which results in tumorous overgrowth in the MT. Therefore, the Drosophila RNSC system may also be a valuable in vivo system in which to study CSC regulation.

Comparison of the Drosophila Renal and Nephric Stem Cells (RNSCs) with Intestinal Stem Cells (ISCs)

The RNSCs are located in the region of the lower tubules and ureter of the MTs, while ISCs are located at the posterior midgut. The MTs' ureters connect to the posterior midgut. The two types of stem cells are at close anatomical locations in the adult fly digestion system and also share some properties. For example, both of them are small nuclear cells, Arm-positive, and express esg. However, RNSCs and ISCs produce distinctly different progenies. ISCs produce progenies that include either Su(H)GBE-lacZ- or Pros-positive cells, which are not among the progenies of RNSCs because Su(H)GBE-lacZ and Pros are not expressed in the MTs. RNSCs produce progenies that include Cut- or TSH-positive cells, which are not among the progenies of ISCs because Cut and TSH are not expressed in the posterior midgut. One possibility for this difference is that, although RNSCs and ISCs originate from the same stem cell pool, their particular environments restrict their differentiation patterns. Future experiments, such as transferring RNSCs to the posterior midgut and vice versa, should be able to test this model.

JAK-STAT Signaling May Be a General Stem Cell Signaling and esg May Be a General Stem Cell Marker

The JAK-STAT signaling regulates self-renewal of the male germline, the male somatic, female escort stem cells in fly (Decotto and Spradling, 2005; Kiger et al., 2001; Nystul and Spradling, 2006; Tulina and Matunis, 2001). The signaling also regulates self-renewal and maintenance of mammalian embryonic stem cells (Matsuda et al., 1999; Ying et al., 2003). In this study, we reported that the JAK-STAT signaling regulates self-renewal of RNSCs. The JAK-STAT signaling may be a general stem cell signaling and also regulate stem cell self-renewal in other, un-characterized stem cell systems.

esg has been used as a marker of both male germline stem cells (Kiger et al., 2001; Wang et al., 2006) and ISCs (Micchelli and Perrimon, 2005). In this study, we demonstrated that the esg-Gal4. UAS-GFP transgene is specifically expressed in RNSCs. The function of the esg gene is to maintain cells as diploid in Drosophila imaginal cells (Fuse et al., 1994). Stem cells may have to be diploid, and esg may be a general stem cell factor. Identifying a stem cell signaling pathway (such as the JAK-STAT signal transduction pathway) and a stem cell factor (such as esg) will provide useful tools for identifying stem cells in other systems and for understanding stem cell regulation in general.

Experimental Procedures

Drosophila Stocks

Oregon R was used as wild type. Other fly stocks used in this study, described either in FlyBase or as otherwise specified, were as follows: hs-FLP UAS-srcEGFP, FRT52B(y) (yellow-FRT-Gal4), UAS-upd FRT52B(y), FRT52B(w)(white-Actin5C-FRT) UAS-EGFP, and upd-Gal4 were provided by T. Xie (Kirilly et al., 2005); esg-Gal4 was provided by S. Hyashi; dome-Gal4 was provided by S. Noselli; UAS-socs36E-45 was a gift from B. Mathey-Prevot; Stat92E reporter-GFP was provided by G. Baeg (Bach et al., 2007); UAS-upd, FRT82B-Stat92Ej6C8, and FRT82B-Stat92E06346 were described previously (Hou et al., 1996; Chen et al., 2002); UAS-Stat92E was generated in Hou's laboratory; Kr-Gal4 UAS-GFP, tsh-lacZ (tsh04519), AyGal4 UAS-GFP, SM6, hs-Flp, FRT82B-tub-Gal80, and FRT82B were obtained from the Bloomington stock center.

Flies were raised on standard Drosophila media at 25°C unless otherwise indicated. Chromosomes and mutations that are not described in the text can be found in Flybase (http://flybase.bio.indiana.edu).

Generating GFP-positive Marked Clones with MARCM and PMML

To use the MARCM system to generate GFP-marked wild-type or Stat92E mutant clones, AyGal4 UAS-GFP/+; FRT82B +/Ly, AyGal4 UAS-GFP/+; FRT82B Stat92E06346/Ly, AyGal4 UAS-GFP/+; FRT82B Stat92Ej6C8/Ly, AyGal4 UAS-GFP/+; FRT82B Stat92Ej6C8 UAS-Stat92E/Ly males were mated with virgin females of genotype SM6, hs-Flp/+; FRT82B-tub-Gal80/TM3, Sb, respectively. To use the PMML system to generate GFP-marked clones that also overexpress their respective genes, hs-Flp UAS-srcEGFP; FRT52B(w) UAS-EGFP/Cyo virgin females were mated with males of genotypes FRT52B(y)/Cyo, UAS-upd FRT52B(y)/Cyo or UAS-socs36E FRT52B(y)/Cyo, respectively. One- or two-day-old adult non-TM3, Sb and non-Ly or non-Cyo females were heat-shocked one (for PMML) or six (for MARCM) times (37°C, 60 min) for three consecutive days, with an interval of 8–12 hours between heat shocks. The flies were transferred to fresh food daily after heat shocks, and their MTs were processed for staining at indicated times.

BrdU Labeling

Female flies were starved at 25°C for 16 hours and then fed 100 mM BrdU (Sigma) in a paste of yeast granule and sucrose in water for 4 days. MT was dissected on the 5th day, fixed with 4% formaldehyde, and stained with anti-BrdU antibodies.

Immunofluorescence Staining and Microscopy

MTs were dissected and stained as described previously (Singh et al., 2006). Confocal images were obtained by using a Zeiss LSM510 system, and processed using Adobe Photoshop CS2.

The following antisera were used: rabbit polyclonal anti-STAT92E antibody (1:500; Chen et al., 2002); rabbit polyclonal anti-TSH antibody (1:500; gift from S. Cohen); rabbit polyclonal anti-β-Gal antibody (1:1000; Cappel); rabbit polyclonal anti-phophohistone H3 (1:1000; Upstate Biotechnology); mouse monoclonal anti-Armadillo N7A1 [1:4; Developmental Studies Hybridoma Bank (DSHB)]; mouse monoclonal anti-Cut antibody (1:10; DSHB); mouse monoclonal anti-BrdU antibody (1:100; Becton Dickson); rabbit polyclonal anti-GFP antibody (1:200; Molecular Probes); mouse monoclonal anti-GFP antibody (1:100; Molecular Probes). Secondary Abs were goat anti-mouse and goat anti-rabbit IgG conjugated to Alexa 488 or Alexa 568 (1:400; Molecular Probes). DAPI (Sigma) was used to stain DNAs. Actins were stained with phalloidin stain solution (Phalltoxin; Molecular Probes).

Detection of Apoptosis

We used an Apoptag Red In Situ Detection Kit (Chemicon) to detect cell death in the MTs. The MTs were dissected and fixed in 4% formaldehyde in PBX as described above. Fixed MTs were washed in PBX and cell death detected according to the manufacturer's instruction.

Acknowledgments

We thank T. Xie, S. Hyashi, S. Noselli, B. Mathey-Prevot, G. Baeg, and Bloomington stock center for fly stocks; S. Cohen for antibody; and S. Lockett for help on the confocal microscope. M. Grau for help on preparation of the manuscript. This research was supported by the Intramural Research Program of NIH, National Cancer Institute.

Footnotes

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References

  1. Arbouzova NI, Zeidler MP. JAK/STAT signaling in Drosophila:insights into conserved regulatory and cellular functions. Development. 2006;133:1605–1616. doi: 10.1242/dev.02411. [DOI] [PubMed] [Google Scholar]
  2. Bach EA, Ekas LA, Ayala-Camargo A, Flaherty MS, Lee H, Perrimon N, Baeg G. GFP reporters detect the activation of the Drosophila JAK/STAT pathway in vivo. Gene Exp Patterns. 2007;7:323–331. doi: 10.1016/j.modgep.2006.08.003. [DOI] [PubMed] [Google Scholar]
  3. Binari R, Perrimon N. Stripe-specific regulation of pair-rule genes by hopscotch, a putative Jak family tyrosine kinase in Drosophila. Genes Dev. 1994;8:300–312. doi: 10.1101/gad.8.3.300. [DOI] [PubMed] [Google Scholar]
  4. Brown S, Hu N, Hombria JC. Identification of the first invertebrate interleukin JAK/STAT receptor, the Drosophila gene domeless. Curr Biol. 2001;11:1700–1705. doi: 10.1016/s0960-9822(01)00524-3. [DOI] [PubMed] [Google Scholar]
  5. Callus BA, Mathey-Prevot B. SOCS36E, a novel Drosophila SOCS protein, suppresses JAK/STAT and EGF-R signaling in the imaginal wing disc. Oncogene. 2002;21:4812–4821. doi: 10.1038/sj.onc.1205618. [DOI] [PubMed] [Google Scholar]
  6. Chen HW, Chen X, Oh S, Marinissen MJ, Gutkind JS, Hou XS. mom identifies a receptor for the Drosophila JAK/STAT signal transduction pathway and encodes a protein distantly related to the mammalian cytokine receptor family. Genes Dev. 2002;16:388–398. doi: 10.1101/gad.955202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Decotto E, Spradling AC. The Drosophila ovarian and testis stem cell niches: similar somatic stem cells and signals. Dev Cell. 2005;9:501–510. doi: 10.1016/j.devcel.2005.08.012. [DOI] [PubMed] [Google Scholar]
  8. Denholm B, Sudarsan V, Pasalodos-Sanchez S, Artero R, Lawrence P, Maddrell S, Baylies M, Skaer H. Dual origin of the renal tubules in Drosophila mesodermal cells integrate and polarise to establish secretory function. Curr Biol. 2003;13:1052–1057. doi: 10.1016/s0960-9822(03)00375-0. [DOI] [PubMed] [Google Scholar]
  9. Edgar BA, Orr-Weaver TL. Endoreplication cell cycles: More for less. Cell. 2001;105:297–306. doi: 10.1016/s0092-8674(01)00334-8. [DOI] [PubMed] [Google Scholar]
  10. Elger M, Hentschel H, Litteral J, Wellner M, Kirsch T, Luft FC, Haller H. Nephrogenesis is induced by partial nephrectomy in the elasmobranch Leucoraja erinacea. J Am Soc Nephrol. 2003;14:1506–1518. doi: 10.1097/01.asn.0000067645.49562.09. [DOI] [PubMed] [Google Scholar]
  11. Fuchs E, Tumbar T, Guasch G. Socializing with the neighbors: stem cells and their niche. Cell. 2004;116:769–778. doi: 10.1016/s0092-8674(04)00255-7. [DOI] [PubMed] [Google Scholar]
  12. Fuse N, Hirose S, Hayashi S. Diploidy of Drosophila imaginal cells is maintained by a transcriptional repressor encoded by escargot. Genes Dev. 1994;8:2270–2281. doi: 10.1101/gad.8.19.2270. [DOI] [PubMed] [Google Scholar]
  13. Haller H, Groot KD, Bahlmann F, Elger M, Fliser D. Stem cells and progenitor cells in renal disease. Kidney Int. 2005;68:1932–1936. doi: 10.1111/j.1523-1755.2005.00622.x. [DOI] [PubMed] [Google Scholar]
  14. Harrison DA, McCoon PE, Binari R, Gilman M, Perrimon N. Drosophila unpaired encodes a secreted protein that activates the JAK signaling pathway. Genes Dev. 1998;12:3252–3263. doi: 10.1101/gad.12.20.3252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Harrison DA, Perrimon N. A simple and efficient generation of marked clones in Drosophila. Curr Biol. 1993;3:424–433. doi: 10.1016/0960-9822(93)90349-s. [DOI] [PubMed] [Google Scholar]
  16. Hou XS, Melnick MB, Perrimon N. marelle acts downstream of the Drosophila hop/JAK kinase and encodes a protein similar to the mammalian STATs. Cell. 1996;84:411–419. doi: 10.1016/s0092-8674(00)81286-6. [DOI] [PubMed] [Google Scholar]
  17. Jung A, Denholm B, Skaer H, Affolter M. Renal tubule development in Drosophila: a closer look at the cellular level. J Am Soc Nephrol. 2005;16:322–328. doi: 10.1681/ASN.2004090729. [DOI] [PubMed] [Google Scholar]
  18. Kiger AA, Jones DL, Schulz C, Rogers MB, Fuller MT. Stem cell self-renewal specified by JAK-STAT activation in response to a support cell cue. Science. 2001;294:2542–2545. doi: 10.1126/science.1066707. [DOI] [PubMed] [Google Scholar]
  19. Kirilly D, Spana EP, Perrimon N, Padgett RW, Xie T. BMP signaling is required for controlling somatic stem cell self-renewal in the Drosophila ovary. Dev Cell. 2005;9:651–662. doi: 10.1016/j.devcel.2005.09.013. [DOI] [PubMed] [Google Scholar]
  20. Lee T, Luo L. Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron. 1999;22:451–461. doi: 10.1016/s0896-6273(00)80701-1. [DOI] [PubMed] [Google Scholar]
  21. Li L, Neaves WB. Normal stem cells and cancer stem cells: The niche matters. Cancer Res. 2006;66:4553–4557. doi: 10.1158/0008-5472.CAN-05-3986. [DOI] [PubMed] [Google Scholar]
  22. Matsuda T, Nakamura T, Nakao K, Arai T, Katsuki M, Heike T, Yokota T. STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells. EMBO J. 1999;18:4261–4269. doi: 10.1093/emboj/18.15.4261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Micchelli CA, Perrimon N. Evidence that stem cells reside in the adult Drosophila midgut epithelium. Nature. 2005;439:475–479. doi: 10.1038/nature04371. [DOI] [PubMed] [Google Scholar]
  24. Moore KL. The urogenital system. In: Moore KL, editor. The Developing Human: Clinically Oriented Embryology. Philadephia: W.B. Saunders Company; 1988. pp. 246–286. [Google Scholar]
  25. Nystul TG, Spradling AC. Breaking out of the mold: diversity within adult stem cells and their niches. Curr Opin Genet Dev. 2006;16:463–468. doi: 10.1016/j.gde.2006.08.003. [DOI] [PubMed] [Google Scholar]
  26. Ohlstein B, Spradling A. The adult Drosophila posterior midgut is maintained by pluripotent stem cells. Nature. 2005;439:470–474. doi: 10.1038/nature04333. [DOI] [PubMed] [Google Scholar]
  27. Oliver JA, Maarouf O, Cheema FH, Martens TP, Al-Awqati Q. The renal papilla is a niche for adult kidney stem cells. J Clin Invest. 2004;114:795–804. doi: 10.1172/JCI20921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pardal R, Clarke MF, Morrison SJ. Applying the principles of stem cell biology to cancer. Nat Rev Cancer. 2003;3:895–902. doi: 10.1038/nrc1232. [DOI] [PubMed] [Google Scholar]
  29. Pugatcheva OM, Mamon LA. Genetic control of development of Malpighian tubules in Drosophila melanogaster. Russian J Dev Biol. 2003;34:269–284. [Google Scholar]
  30. Ricardo SD, Deane JA. Adult stem cells in renal injury and repair. Nephrology. 2005;10:276–282. doi: 10.1111/j.1440-1797.2005.00373.x. [DOI] [PubMed] [Google Scholar]
  31. Saxen L. Organogenesis of the kidney. New York: Cambridge University Press; 1987. [Google Scholar]
  32. Singh SR, Zhen W, Zheng ZY, Wang H, Oh SW, Liu W, Zbar B, Schmidt LS, Hou SX. The Drosophila homologue of the human tumor suppressor gene BHD regulates male germline stem cell maintenance and functions downstream of Dpp. Oncogene. 2006;25:5933–5941. doi: 10.1038/sj.onc.1209593. [DOI] [PubMed] [Google Scholar]
  33. Skaer H. The alimentary canal. In: Bate M, Martinez A, editors. The Development of Drosophila melanogaster. New York: Cold Spring Harbor Lab; 1993. pp. 941–1012. [Google Scholar]
  34. Sorokin L, Klein G, Mugrauer G, Fecker L, Ekblom M, Ekblom P. Development of kidney epithelial cells. In: Fleming TP, editor. Epithelial Organization and Development. London: Chapman and Hall; 1995. pp. 163–190. [Google Scholar]
  35. Sözen MA, Armstrong JD, Yang M, Kaiser K, Dow JAT. Functional domains are specified to single-cell resolution in a Drosophila epithelium. Proc Natl Acad Sci USA. 1997;94:5207–5212. doi: 10.1073/pnas.94.10.5207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Spradling A, Drummond-Barbosa D, Kai T. Stem cells find their niche. Nature. 2001;414:98–104. doi: 10.1038/35102160. [DOI] [PubMed] [Google Scholar]
  37. Tulina N, Matunis E. Control of stem cell self-renewal in Drosophila spermatogenesis by JAK-STAT signaling. Science. 2001;294:2546–2549. doi: 10.1126/science.1066700. [DOI] [PubMed] [Google Scholar]
  38. Wang H, Singh SR, Zheng ZY, Oh SW, Chen X, Edwards K, Hou SX. A Rap-GEF/Rap GTPase signaling controls stem cell maintenance through regulating adherens junction positioning and cell adhesion in Drosophila testis. Dev Cell. 2006;10:117–126. doi: 10.1016/j.devcel.2005.11.004. [DOI] [PubMed] [Google Scholar]
  39. Wang JCY, Dick JE. Cancer stem cells: lessons from leukemia. Trends Cell Biol. 2005;15:494–501. doi: 10.1016/j.tcb.2005.07.004. [DOI] [PubMed] [Google Scholar]
  40. Wessing A, Eichelberg D. Malpighian tubules, rectal papillae and excretion. In: Ashburner A, Wright TRF, editors. The Genetics and Biology of Drosophila. 2c. London: Academic Press; 1978. pp. 1–42. [Google Scholar]
  41. Yan R, Small S, Desplan C, Dearolf CR, Darnell JJ. Identification of a Stat gene that functions in Drosophila development. Cell. 1996;84:421–430. doi: 10.1016/s0092-8674(00)81287-8. [DOI] [PubMed] [Google Scholar]
  42. Ying QL, Nichols J, Chambers I, Smith A. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell. 2003;115:281–292. doi: 10.1016/s0092-8674(03)00847-x. [DOI] [PubMed] [Google Scholar]

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