The capacity to delay cell cycle progression at

The capacity to delay cell-cycle progression at the G1/S transition is central to tumor suppression by RB proteins, predominantly via interaction with, and inhibition of, the E2F family of S-phase transcriptional activators. In Drosophila, the role of Rbf proteins in cell-cycle regulation is considerably less complex than for mammals, with just two E2F subunits (compared with at least eight in mammals) and one DP cofactor (compared with two in mammals) (Dynlacht et al., 1994; van den Heuvel and Dyson, 2008). Rbf and RB1 share capacity to bind to E2F transcriptional activators, similarly RBL1/p107, RBL2/p130, and Rbf2 bind E2F repressor complexes (Du and Pogoriler, 2006). Drosophila E2F1 activates transcription by forming heterodimers with the DP transcriptional cofactor. In the absence of developmental growth signals, hypophosphorylated Rbf represses E2F-mediated transcription by binding and blocking the transcriptional activation domain of E2F/DP (Giacinti and Giordano, 2006). In response to mitogenic signals, G1-S Cyclin/cyclin-dependent kinase (CDKs) (e.g., CycD and CycE) can hyperphosphorylate Rbf, releasing the E2F1-DP complex to promote S-phase gene transcription (reviewed in Giacinti and Giordano, 2006). Flies have just one CDK inhibitor, Dacapo (Dap), which selectively inhibits CycE/Cdk2, but not CycD/Cdk4 (de Nooij et al., 1996). The Drosophila testis provides a system for analysis of gene function in two distinct cell populations derived from adjacent stem cell types (the germline and somatic lineage) within their endogenous niche. The testis produces sperm throughout the lifetime of the adult male fly. From the L1 stage, the stem cell niche is composed of a cluster of somatic transketolase (the hub) that supports two stem cell populations: the germline stem cells (GSCs) and the somatic stem cells, also known as cyst stem cells (CySCs) (Gönczy and DiNardo, 1996; Hardy et al., 1979). Each GSC is enclosed by two CySCs, and both populations undergo asymmetric divisions to (1) maintain the stem cell pool and (2) differentiate into gonialblast daughter or somatic cyst cells, respectively (Fuller and Spradling, 2007; Hardy et al., 1979; Yamashita et al., 2003) (Figures 1A and 1B). The gonialblast exits the niche enclosed by a pair of cyst cells and, after four rounds of transit-amplifying (TA) mitotic divisions with incomplete cytokinesis, generates a 16-cell spermatogonial cyst (Hardy et al., 1979). Upon further growth and differentiation, spermatogonial cysts develop into spermatocytes, which undergo meiosis to produce sperm (Fuller and Spradling, 2007) (Figures 1A and 1B). Here, we demonstrate that although Rbf mutants display cell-cycle exit and differentiation defects in both the germline and somatic lineages, Rbf function was only required in the somatic lineage for testes development. Thus, Rbf function in the somatic cell lineage is required non-autonomously for regulating cell-cycle exit and differentiation in the germline.
Results
Discussion Analysis of EMS-induced male-sterile mutant collections suggest most alleles elicit meiosis or spermiogenesis phenotypes, but relatively few hits disrupt the stem cell niche (Wakimoto et al., 2004). This is not surprising, as many stem cell determinants and signaling pathways essential to niche function also have critical functions in earlier development, hence loss-of-function alleles are associated with embryonic or larval lethality and will be absent from fertility screens. In contrast, the unique features of meiotic cells (only spermatocytes and oocytes undergo meiosis) or post-meiotic spermatids often derive from factors specific to these processes. Thus, although several genetic screens for male-sterile alleles have identified genes that function in the stem cell niche to regulate maintenance, proliferation, and differentiation of GSCs and/or somatic stem cells, these are often hypomorphic alleles (Castrillon et al., 1993; Kiger et al., 2001).