Original research
Defining gene networks controlling the maintenance and function of the differentiation niche by an in vivo systematic RNAi screen

https://doi.org/10.1016/j.jgg.2018.10.008Get rights and content

Abstract

In the Drosophila ovary, escort cells (ECs) extrinsically control germline stem cell (GSC) maintenance and progeny differentiation. However, the underlying mechanisms remain poorly understood. In this study, we identified 173 EC genes for their roles in controlling GSC maintenance and progeny differentiation by using an in vivo systematic RNAi approach. Of the identified genes, 10 and 163 are required in ECs to promote GSC maintenance and progeny differentiation, respectively. The genes required for progeny differentiation fall into different functional categories, including transcription, mRNA splicing, protein degradation, signal transduction and cytoskeleton regulation. In addition, the GSC progeny differentiation defects caused by defective ECs are often associated with BMP signaling elevation, indicating that preventing BMP signaling is a general functional feature of the differentiation niche. Lastly, exon junction complex (EJC) components, which are essential for mRNA splicing, are required in ECs to promote GSC progeny differentiation by maintaining ECs and preventing BMP signaling. Therefore, this study has identified the major regulators of the differentiation niche, which provides important insights into how stem cell progeny differentiation is extrinsically controlled.

Introduction

Stem cells continuously produce functional cells to replenish lost cells via differentiation. Studies in various systems, including Drosophila, have demonstrated that the niche and intrinsic factors work concertedly to control stem cell self-renewal (Li and Xie, 2005; Fuller and Spradling, 2007; Morrison and Spradling, 2008; Xie, 2013). We have recently proposed that a distinct niche is formed by escort cells (ECs; also known as inner sheath cells) in the Drosophila ovary to control germline stem cell (GSC) progeny differentiation, and we have termed it “the differentiation niche” (Kirilly et al., 2011). A recent study has also supported the existence of the somatic environment for controlling spermatogonial stem cell differentiation in mice (DeFalco et al., 2015). However, how the differentiation niche is maintained and regulated remains poorly understood. In this study, we used an in vivo systematic RNAi screen to identify the critical factors in ECs that control GSC maintenance and progeny differentiation.

In the adult Drosophila ovary, two or three GSCs are located in the germarium at the tip of each ovary, and are easily identified by their location, size and the presence of a germ cell-specific intracellular organelle known as the spectrosome (Lin and Spradling, 1993; Spradling, 1993). At each germarial tip, 5–7 cap cells (CPCs) and 4–6 GSC-contacting ECs (previously known as escort stem cells) form the niche for controlling GSC self-renewal (Xie and Spradling, 2000, 2001; Kirilly et al., 2011; Wang et al., 2011; Sun et al., 2015; Yang et al., 2017). The CPC-derived BMP ligand Dpp directly activates BMP signaling in GSCs for promoting self-renewal by repressing the expression of the master differentiation factor bam (Xie and Spradling, 1998; Chen and McKearin, 2003a; Song et al., 2004), and CPC-expressed E-cadherin helps stabilize GSCs in the niche (Song et al., 2002). Different classes of intrinsic factors promote GSC self-renewal by strengthening BMP signaling and E-cadherin-mediated cell adhesion and thus repress differentiation (Xie, 2013). Therefore, niche signaling and intrinsic factors work concertedly to promote GSC self-renewal by inhibiting differentiation pathways. However, it remains unclear how the anterior ECs control GSC maintenance at the molecular level.

GSC progeny differentiation is also controlled intrinsically and extrinsically (Xie, 2013). GSCs continuously produce differentiated cystoblasts (CBs), which further undergo four synchronous divisions with incomplete cytokinesis to form 16-cell cysts with 2-cell, 4-cell and 8-cell mitotic cysts (MCs) as intermediates. ECs on the surface of the germarium extend long cellular processes to wrap up CBs, MCs and 16-cell cysts, promoting their development (Kirilly et al., 2011; Morris and Spradling, 2011). Due to the loss of Dpp signaling-mediated repression, bam is upregulated in CBs and MCs to drive cyst division and germ cell differentiation (Chen and McKearin, 2003a; Song et al., 2004). Bam is necessary and sufficient for promoting CB differentiation (McKearin and Spradling, 1990; Ohlstein and McKearin, 1997; Ohlstein et al., 2000). In addition, Bam promotes GSC progeny differentiation by working with different factors, including Bgcn, Sxl, Mei-P26, COP9 and the CCR4-Not complex (Li et al., 2009, 2013; Shen et al., 2009; Pan et al., 2014; Fu et al., 2015). Recently, we have shown that ECs themselves and cellular process-mediated interactions are critical for GSC progeny differentiation (Kirilly et al., 2011; Wang et al., 2011, 2015; Lu et al., 2015). Therefore, GSC progeny differentiation is controlled intrinsically and extrinsically.

ECs promote GSC progeny differentiation as a niche through multiple mechanisms. The first mechanism is to prevent BMP signaling in GSC progeny (Xie, 2013). EGFR signaling has been shown to operate in ECs to prevent BMP signaling by repressing the expression of dally, which encodes a proteoglycan for facilitating Dpp diffusion and signaling (Liu et al., 2010), and Wnt signaling is also required in ECs to prevent BMP signaling by maintaining the expression of Tkv (a BMP type I receptor) and EC survival or by repressing dpp expression (Wang et al., 2015; Luo et al., 2015; Mottier-Pavie et al., 2016). In addition, Rho is required in ECs to prevent BMP signaling by repressing the expression of dally and dpp; Eggless, Piwi, Hh signaling and the COP9 complex are required in ECs to prevent BMP signaling by repressing dpp expression (Kirilly et al., 2011; Wang et al., 2011, 2015; Jin et al., 2013; Ma et al., 2014; Liu et al., 2015; Lu et al., 2015). Furthermore, Lsd1 is required in ECs to prevent BMP signaling by controlling the expression of engrailed, which is known to be important for maintaining Hh signaling (Eliazer et al., 2011, 2014). Another mechanism demonstrated by our lab, in addition to BMP signaling repression, is the promotion of GSC progeny differentiation via direct signaling, but the signal remains to be identified (Wang et al., 2011; Ma et al., 2014; Lu et al., 2015). Likely, the direct signal could be mediated by EC long cellular process-mediated physical interactions, which are known to be important for GSC progeny differentiation (Kirilly et al., 2011; Wang et al., 2011). For instance, the Woc-Stat-Zfh1 axis and the ecdysone-let7 miRNA-Wnt signaling axis also operate in ECs to regulate the cellular process-mediated interactions, thereby promoting GSC progeny differentiation (Morris and Spradling, 2012; Maimon et al., 2014; Konig and Shcherbata, 2015). For direct material transfer, physical interactions between ECs and GSC progeny might facilitate the formation of gap junctions between them because gap junction protein Inx2 is required in ECs to promote GSC progeny differentiation (Mukai et al., 2011). Therefore, ECs promote GSC progeny differentiation extrinsically through modulating multiple mechanisms.

In this study, we systematically used an in vivo RNAi screen to identify 173 genes functioning in ECs to promote GSC maintenance and progeny differentiation by maintaining their survival and regulating their functions. The identified genes fall into different functional categories, likely forming interacting genetic networks to control the maintenance, the function or both of the differentiation niche. Our findings confirmed that the EC-mediated repression of BMP signaling is a general mechanism whereby the differentiation niche controls GSC progeny differentiation. In addition, we showed that the niche promotes GSC progeny differentiation by indirectly enhancing Bam function. The identification of these genes provides a solid foundation for us to gain important molecular insights into how the niche controls GSC progeny differentiation extrinsically. Altogether, we identified novel regulators and defined the networks that control GSC behavior. Specifically, we performed an effective genetic screen and successfully identified hundreds of genes in ECs that non-autonomously regulate GSCs. Following functional analysis of the screened genes, we found that RNAi of those housekeeping genes that are essential for EC survival often produced severe phenotypes. Interestingly, we found that many genes affect BMP signaling and thereby regulate GSC through a Bam-dependent manner. Finally, we focused on the exon junction complex (EJC) that is involved in the mRNA splicing process, and confirmed the non-autonomous role of the EJC in GSC maintenance. To our knowledge, all of these findings are reported for the first time in Drosophila, and will draw wide attentions in the large fly community. This study has therefore advanced our understanding of the maintenance and function of the differentiation niche in the Drosophila ovary, and the results will also help to understand how the differentiation of other stem cell types is controlled.

Section snippets

A genome-wide RNAi strategy for identifying the genes required in ECs for promoting GSC progeny differentiation

Earlier studies by others and us have shown that GSC progeny differentiation is controlled by ECs, but the underlying mechanisms remain poorly defined (Xie, 2013). Initially, we employed a strategy using the combined nos-gal4 and c587-gal4 to knock down gene functions simultaneously in germ cells and ECs for identification of the genes operating to control GSC progeny differentiation (Fig. 1A). nos-gal4 is known to express GAL4 in germ cells, whereas c587-gal4 is shown to express GAL4 in ECs

Discussion

Recent studies have demonstrated that ECs function as the niche to promote GSC progeny differentiation partly by preventing BMP signaling (Liu et al., 2010, 2015; Eliazer et al., 2011, 2014; Kirilly et al., 2011; Wang et al., 2011, 2015; Ma et al., 2014; Lu et al., 2015; Luo et al., 2015). Additionally, several signaling pathways, such as EGFR, Wnt, Hh and Jak-Stat, have been shown to act in ECs to promote GSC progeny differentiation (Schulz et al., 2002; Maimon et al., 2014; Liu et al., 2010,

Fly stocks

The Drosophila stocks are maintained at room temperature on standard cornmeal media unless specified. Information on the following stocks is available from http://flybase.org/: c587-gal4, PZ1444, bam-GFP, nos-gal4, tub-gal80ts and Dad-lacZ. The UAS-RNAi knockdown strains used in this study were generated by the Ni and Perrimon laboratories (Ni et al., 2008). To maximize the RNAi-mediated knockdown effect, newly eclosed flies were cultured at 29 °C for one week before the analysis of ovarian

Acknowledgments

We would like to thank Tsinghua Fly Center for reagents, and the Ni and Xie lab members for comments and discussions. This work was supported by the National Key Technology Research and Development Program of the Ministry of Science and Technology of the People's Republic of China (2016YFE0113700), and the National Natural Science Foundation of China (31571320, 31872818, and 31801079).

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