ECs or – as they are sometimes called – inner germarial sheath cells line the germarium in regions 1 and 2a. They form long cytoplasmic extensions that envelope the developing CBs. It had been suggested, that ECs are maintained by a population of 4–6 escort stem cells and that their progeny moves along with the cysts through the germarium until they are lost by apoptosis (Decotto and Spradling, 2005). In contrast to this, it was recently shown that ECs show little movement and are stationary. Even though they are capable of dividing, they are mitotically quiescent most of the time, unless the ratio of ECs to germline cells increases. ECs do undergo some slow turnover, but the lost cells are replaced by dividing neighboring ECs and not by escort stem cells (Kirilly et al., 2011; Morris and Spradling, 2011).
Differentiating germline cells signal to the ECs ECs are of great importance for the differentiating germline cells: they form long cytoplasmic extensions that are believed to physically protect the differentiating CBs from the niche signaling.
In addition, ECs and germline cells actively communicate, and perturbing these interactions leads to malformations and differentiation defects in both ECs and germline: Stem cell tumor (Stet), a Rhomboid homolog is an intramembrane pro-tease that is required for the maturation of the epidermal growth factor receptor (EGFR) ligands Spitz, Gurken or Keren in the germline. Upon ligand secretion, the EGFR pathway is activated in the surrounding somatic ECs which leads to the activation of downstream signaling cascades including the mitogen-activated protein kinase (MAP kinase), the phosphatidylinositol 3-kinase and phospholi-pase C-γ pathways (Schulz et al., 2002; Yarden and Shilo, 2007). If stet function is removed from the germline, germaria with ectopic Dpp activity and a higher number of SSCs were observed, a phenotype that was accompanied by a disrupted formation of cytoplasmic extensions (Liu et al., 2010; Schulz et al., 2002). Inter-estingly, the EGFR signaling pathway in the somatic ECs is required to limit the expression of the glypican Dally, a Dpp stabilizing protein. Altogether, this pro-poses a model in which the activation of the EGFR pathway in the somatic ECs by ligands coming from the germline, is required to restrict the Dpp diffusion and thereby to enable CB differentiation. It was also shown that the differentiation status of the germline cells in the germarium is important for the maintenance of ECs extensions and ECs themselves. If GSCs are lost for example due to artifi-cial bam overexpression, germaria also loose all ECs, indicating that the presence of GSCs is required for EC maintenance (Margolis and Spradling, 1995; Xie and Spradling, 2000). But then, germaria full of undifferentiated germline cells due to bam loss of function or overexpression of dpp also do not show cytoplasmic ECs protrusions (Kirilly et al., 2011). And last, ECs at different positions in the ger-marium show different morphologies, depending on the germline cells that they are associated with (Kirilly et al., 2011). These examples illustrate, that ECs
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cannot properly form protrusions and even cannot be maintained in the absence of correctly differentiating germline cells.
ECs contribute to the GSC niche ECs are crucial for both the maintenance of GSCs and the differentiation of GSC progeny. They also express the TGF-β ligand Dpp which is mainly produced by CpCs and which is essential for GSC maintenance (Casanueva and Ferguson, 2004; Xie and Spradling, 2000; Zhu and Xie, 2003) and eliminating ECs from the germarium leads to GSC loss (Chen et al., 2011). Recently it was furthermore suggested, that ECs and CpCs cooperate via Hh signaling to regulate the levels of TGF-β signal that is transmitted to the GSCs. The CpCs are decorated with long filopodia or cytonemes via which the signal is transmitted to the ECs where it activates the transcription of gbb and dpp (Rojas-Rios et al., 2012). In summary, even though it seems to be clear, that CpCs are the main source for Dpp and Gbb, ECs also are an important part of the GSC niche in the D. melanogaster germarium.
Cytoplasmic EC protrusions are crucial for cyst differentiation Remarkably, besides its function for GSC maintenance, ECs are also required for cyst differ-entiation: as was mentioned before, it is thought that ECs send signals to the developing cysts and that EC protrusions also physically shield the germline from the niche signaling. Disturbing the formation of ECs extensions via downreg-ulating the actin-regulator cappuccino leads to an increase of SSCs, which is a hallmark of delayed or blocked germline differentiation (Kirilly et al., 2011). Sim-ilarly, knocking down the GTPase Rho specifically in ECs disturbs the formation of ECs extensions, which cell non-autonomously affects the germline differentia-tion (Kirilly et al., 2011). The JAK/STAT signaling pathway is active in CpCs and ECs and perturbed JAK/STAT signaling leads to a disturbed EC morphology that results in a higher number of germline cells (Decotto and Spradling, 2005;
Wang et al., 2008a). These results strongly suggest, that the proper formation of EC protrusions is required to create a microenvironment in which the germline cells receive differentiation promoting, but not TGF-β signaling from the CpCs and can differentiate. ECs therefore seem to have a dual role: they are required for both the maintenance of adult GSCs and for the differentiation of the GSC daughters. Also, it is becoming clear that germline progeny differentiation is not the default choice for germline cells that are not maintained as stem cells as the consequence of lacking signaling from the GSC niche. Instead, germline differ-entiation requires both appropriate physical interaction and communication with the surrounding somatic ECs. The nature of these interactions and the relevant signaling pathways remain poorly understood.
Systemic steroid hormone signaling is required for EC morphology In addi-tion to the pathways already known to be required for EC morphology, we were recently able to show that ecdysone, the main steroid hormone inD. melanogaster is required for proper EC morphology. If ecdysone signaling is perturbed, ECs
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loose their squamous shape and cytoplasmic protrusions and instead resemble a columnar-like epithelium. This affects the germline differentiation in a cell non-autonomous way, resulting in an increased number of cells at the GSC to CB transition, that are delayed in differentiation (K¨onig et al., 2011). This study is the first evidence for hormone signaling acting on the germline cells via ECs.
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Figure 1.3: GSC maintenance and germline differentiation are regu-lated by various signaling pathways The niche for the GSCs (purple) is formed by CpCs (gray), TFs (dark red), and ECs (yellow). The differentiating GSC daughter is the CB (turquoise) that is enveloped by cytoplasmic protru-sions, sent from the ECs. Transcription of the master differentiation gene bam is inhibited by TGF-βsignaling: Dpp and Gbb, sent from the niche lead to the phosphorylation of Mad in the GSCs. pMad partners with Med and translo-cates to the nucleus where it inhibitsbam expression. CBs do not receive TGF-β signaling from the niche andbam therefore becomes expressed, leading to differ-entiation. Bam forms a complex with its partner Bgcn and represses Nos, which – together with its partner Pum – is necessary for the maintenance of GSCs by repressing differentiation promoting mRNAs like Brat. Several other mecha-nisms contribute to the sharp gradient of TGF-β reception. The production of Dpp in the CpCs is regulated byLsd1 and the JAK/STAT signaling pathway, activated by the ligand Upd that is secreted from the TFs. Dpp diffusion is limited by type IY collagens whereas Dpp is stabilized by the glypican Dally that is itself downregulated in ECs by EGFR signaling. Several CB intrin-sic pathways limit the responsiveness to TGF-β signaling and thereby assure that only one cell diameter away from the GSCs, differentiation can start. The translational repressor complex consists of Pum and Brat and downregulates the levels of Mad. The serine/threonine Fu together with the E3 ligase Smurf leads to the ubiquitination and subsequent degradation of the TGF-βreceptor.
The homophilic cell adhesion proteins DE-Cad and Arm are found at high lev-els between CpCs and GSCs, where they attach GSCs to the niche cells and at lower levels between ECs and CBs and cysts. See the main text for details.
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