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Rap Signaling Regulates Stem Cell Anchoring in Drosophila Testis
tem cells can either self-renew or differentiate into short-lived cell types. Cancer cells also possess the potential for self-renewal; tumors may originate from a few transformed cancer stem cells (Reya T et al. Nature 414: 105–11, 2001). Understanding the molecular mechanisms that control stem cell self-renewal versus differentiation is crucial to the use of stem cells in regenerative medicine and the development of effective anticancer therapies. Accumulated evidence suggests that stem cells are controlled by particular microenvironments known as niches (Spradling A et al. Nature 414: 98–104, 2001; Fuchs E et al. Cell 116: 769–78, 2004). A niche is a subset of neighboring stromal cells and extracellular substrates. The stromal cells often secrete growth factors to regulate stem cell behavior. The Drosophila testis provides an excellent in vivo system to study stem cells and niches at the cellular and molecular levels (Fuller MT. Semin Cell Dev Biol 9: 433–44, 1998; Yamashita YM et al. J Cell Sci 118: 665–72, 2005). At the tip of the Drosophila testis (the apex) is a germinal proliferation center, which contains the germline and somatic stem cells that maintain spermatogenesis. Each adult male fly testis has five to nine germline stem cells (GSCs), each encysted by two somatic stem cells (SSCs, also called cyst progenitor cells). Both GSCs and SSCs attach to a group of 12 nondividing somatic cells called the hub (Hardy RW et al. J Ultrastruct Res 69: 180–90, 1979; Gonczy P and DiNardo S. Development 122: 2437–47, 1996). The hub defines the stem cell niche by expressing the growth factor Unpaired (Upd), which activates the JAK/STAT pathway in GSCs to regulate the stem cell self-renewal process (Kiger AA et al. Science 294: 2542–5, 2001; Tulina N and Matunis E. Science 294: 2546–9, 2001). Meanwhile, a member of the transforming growth factor-β (TGF-β) family, glass bottom boat (Gbb), is also expressed in the hub and plays a part in regulating GSC self-renewal by activating its corresponding signal transduction pathway in the GSCs (Kawase E et al. Development 131: 1365–75, 2004). Because Upd and Gbb are expressed in the hub, they have very limited ability to diffuse; therefore, the GSCs must first be anchored to the hub to receive the signals and maintain their stem cell identity. The cell adhesion molecules E-cadherin and β-catenin (named Armadillo [Arm] in Drosophila) are concentrated at the hub-GSC interface and may anchor the stem cells to the niche (Yamashita YM et al. Science 301: 1547–50, 2003). However, how the adherens junctions are specifically formed at the hub-GSC interface is not clear. We recently identified in a genetic screen a Drosophila small GTPase Rap guanine nucleotide exchange factor (Gef26) as a major regulator of the anchoring of stem cells to their niche. The Gef 26 protein has a PDZ domain, a Ras-binding domain, a cAMP/cGMP-binding domain, and a Rap-binding domain. Mutations of Gef26 cause loss of both GSCs and SSCs in the fly testis. We demonstrated that the Rap-Gef (Gef26)/Rap signaling controls stem cell anchoring to the niche through regulation of E-cadherin–mediated cell adhesion. The Gef26 mutation specifically impairs adherens junctions at the hub–stem cell interface, which results in the stem cells “drifting away” from the niche and losing stem cell identity (Figure 1). Thus, the Rap signaling/E-cadherin pathway may represent one mechanism that regulates polarized niche formation and stem cell anchoring (Wang H et al. Dev Cell 10: 117–26, 2006). Figure 1. Germline stem cells (GSCs) “drift away” from the niche in Gef26 mutant testes because of impaired adherens junctions. The Wild-type (A), Gef264 (B), and Gef266 (C) testes of adult flies were immunostained with anti–E-cadherin (green), anti-Vasa (red), and DAPI (blue). In (A), all GSCs are anchored to the hub (star) through E-cadherin–positive adherens junctions. In (B), all Vasa-positive germ cells drifted away from the hub (star). In (C), only one GSC is still attached to the hub (star) through E-cadherin–positive adherens junctions (arrow). Rap1 was first identified as a gene that can reverse the transformed phenotype of fibroblasts by one of the mutated Ras genes, K-ras (Kitayama H et al. Cell 56: 77–84, 1989). Rap1 belongs to the Ras family of small GTP-binding proteins. Its apparent tumor suppressor properties were initially proposed to antagonize the activity of Ras by competing for a common target (or regulatory protein). However, recent studies have suggested that Rap1 may actually regulate adherens junctions. In a recent study, a Rap1 GTPase activator, Dock4, was identified as a tumor suppressor (Yajnik V et al. Cell 112: 673–84, 2003). Dock4 specifically activates Rap1 and regulates the formation of adherens junctions. Our recent results show that the Rap-Gef (Gef26)/Rap signaling controls the anchoring of stem cells to their niche through regulation of E-cadherin–mediated cell adhesion in the Drosophila testis. The mammalian homolog of Gef26 may regulate cancer stem cell anchoring and function as a tumor suppressor. We are in the process of knocking out the homologous Gef26 gene in mice (RapGef2) and searching for an association between RAPGEF2 and human diseases. Thus, the powerful genetic manipulations available in Drosophila in combination with the mouse knockout studies may make this an ideal system to study cancer stem cells and cancers.
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