Ecdysone signaling is non cell-autonomously acting on the germline

We showed that ecdysone signaling is predominantly active in the somatic cells of the germarium, in CpCs and ECs (K¨onig et al., 2011). Interestingly, germline cells show a dramatic differentiation delay if ecdysone signaling is disturbed. Con-sequently, the ECs must convert the ecdysone signaling into another signal to communicate with the germline cells. However, the range of TGF-βsignaling, the main pathway for GSC maintenance in D. melanogaster ovaries, is not enlarged due to perturbed ecdysone signaling and, thus, it seems that ECs and GSCs com-municate via alternative pathways. Not much is known about the communication between ECs and differentiating germline cells (see Section 1.2.4, page 12) and therefore, how the systemic hormonal ecdysone signal is transmitted to a signal that cell non-autonomously acts on the germline, is a key question.

3.1.1 EC function is compromised upon loss of ecdysone signaling

Interestingly, shape and function of the ECs are dramatically impaired in ecdysone signaling mutants: the squamous ECs line the germarium and form long cyto-plasmic protrusions that envelop the developing germline cells. However, if the ecdysone signaling is perturbed, ECs form layers that resemble columnar epithe-lium. In addition, the thin cytoplasmic protrusions are no longer present and the

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levels of cell adhesion proteins are elevated. Earlier experiments already demon-strated how critical EC protrusions are: if the formation of protrusions is inhibited specifically in the ECs – for example via interfering with the cytoskeleton – the germline differentiation is affected and a larger number of SSCs was detected (De-cotto and Spradling, 2005; Kirilly et al., 2011; Wang et al., 2008a). A lack of EC protrusions therefore can cause a differentiation delay in the germline and we propose that the EC malformation and specifically the absence of protrusions contributes to the differentiation delay observed in the germline. How exactly EC protrusions enable germline differentiation is poorly understood; it is thought that they physically shield the differentiating germline cells against diffusible signals from the anterior CpCs, forming another barrier to locally restrict TGF-β signal-ing. Nevertheless, signaling processes most probably play a role in EC-germline communication, and so far, only few were identified.

Ecdysone signaling was suggested to act on the GSCs directly A recent report by Ables and Drummond-Barbosa, 2010 showed that ecdysone signaling regulates GSC self renewal and proliferation by functional interaction with the chromatin remodeling factors ISWI and NURF301. However, in contrast to our findings, Ables and Drummond-Barbosa, 2010 suggest that the ecdysone signaling is directly received by GSCs. While there are no conflicts regarding the results of experiments that overlap between our studies, the conclusions are different.

Interestingly, Gancz et al., 2011 reported a non cell-autonomous effect of ecdysone signaling on primordial germ cells – the germ line stem cell precursors. Upon perturbing the ecdysone signaling in the soma, primordial germ cells are delayed in differentiation and similar to what we observed in adult GSCs, do not display Bam or pMad.

3.1.2 ECs form a differentiation niche for the germline progeny

The diverse signals and factors that are required for the maintenance of GSCs in their somatic niche were examined by many studies in the past decades (reviewed in Spradling et al., 2011). It was shown that the two daughters of a stem cell do not differ inherently, but instead their fate is determined by their environment;

in order to be maintained as a stem cell, the germline cell has to be physically attached to the niche and receive the signaling from the CpCs. However, due to the limited size of the niche, stem cells compete for the available space and usually only one stem cell daughter can be maintained in the niche. The other one was thought to undergo differentiation simply because of the lack of niche contact and signaling. Only recently it was suggested that differentiation is not the default fate of cells that cannot be maintained as stem cells (Kirilly et al., 2011). Instead, it seems that the germline cells need specific microenvironments – consisting of signaling and cell contacts from the ECs – that enable differentiation to proceed.

The differentiation delayed germline cells in ecdysone signaling mutants lack the differentiation factor Bam TGF-β signaling is clearly the most important

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signaling pathway controlling the stem cell population in the female GSC niche (Song et al., 2004; Xie and Spradling, 1998). Activation of the TGF-β pathway by the ligands Gbb and Dpp, that are secreted by CpCs and ECs, is crucial for GSC maintenance. But is the loss of TGF-β signaling in CBs sufficient to in-duce the differentiation program or is another ”pro-differentiation” signal from the surrounding soma required? In wildtype germaria, expression of the differen-tiation factorbam is repressed by pMad and, hence, Bam becomes excluded from the GSCs and misexpression of bam is indeed sufficient to induce differentiation throughout the germline (McKearin and Ohlstein, 1995; McKearin and Spradling;

Ohlstein and McKearin, 1997). Ecdysone-deficient germaria display a population of differentiation delayed pre-CBs that – not surprisingly – do not express bam.

The pMad/Med complex that forms upon TGF-β activation is to date the only known repressor of bam; it was, therefore, rather unexpected that the pMad ex-pression radius is unchanged in ecdysone deficient germaria. Isbam expression not allowed because of another yet unidentified factor? Or is an unknown activator missing that would promote bam expression? Apart from intrinsic and extrin-sic signaling pathways that control stem cell fate, chromatin modifying factors have been shown to be indispensable for stem cell maintenance and differentia-tion (reviewed in Buszczak and Spradling, 2006). Chromatin modificadifferentia-tions are, therefore, great candidates for a mechanistic link between ecdysone signaling and Bam-driven differentiation.

Histone H2B ubiquitination precedes germline differentiation Which epige-netic features define the stem cell state and how they change upon differentiation are key questions that need to be resolved in order to fully understand and to po-tentially manipulate stem cell maintenance and differentiation. Stem cells exhibit a certain set of chromatin modifications, governing their unique gene expression patterns (reviewed in Buszczak and Spradling, 2006). At the same time, the epigenetic state has to be flexible enough to allow for differentiation (reviewed in Buszczak and Spradling, 2006). A number of chromatin remodelers, like ISWI and NURF301 were shown to be required for GSC maintenance (Buszczak et al., 2009;

Xi and Xie, 2005 and Section 1.6, page 30). The role of chromatin modifications in germline differentiation is, however, far less understood. Many developmental genes exhibit both ”active” and ”repressive” chromatin modifications at the same time. Interestingly, the monoubiquitination of histone H2B was shown to parallel resolving this bivalency during differentiation in human mesenchymal stem cells and the patterns of H2Bub1 andbamcorrelate (Karpiuk et al., 2012). We therefore suggest that the monoubiquitination of H2B precedes differentiation and bam ex-pression. Furthermore, ecdysone deficient flies show non-differentiating germline cells that lack H2Bub1, indicating that this epigenetic mark is regulated in re-sponse to steroid hormone signaling. The exact mechanism by which H2Bub1 and possibly other chromatin remodelers regulate bam expression, and thus germline differentiation, is a key question that will be subject of further studies.

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Adhesion complexes are affected by hormonal ecdysone signaling DE-Cad and Arm are normally found at high levels in the junctions between CpCs and between CpCs and GSCs where they are necessary to maintain GSCs (Song et al., 2002). More towards the posterior in CBs and ECs, Arm and DE-Cad levels are strongly reduced in wildtype germaria. Adhesive junctions are critical for germline-soma interaction: subtle differences in the adhesive strength determine which GSCs are maintained and which ones are outcompeted (Jin et al., 2008).

The local levels of adhesion proteins are controlled by other signals like insulin or JAK/STAT signaling and, thus, are adjusted to the overall body status (Hsu and Drummond-Barbosa, 2009; Leatherman and Dinardo, 2010). The intensity of the adhesive connections, therefore, provide a mechanism by which soma and germline communicate and govern each other and serves as an interface for other signaling pathways.

Increased levels of cell adhesion proteins affect EC protrusions We now de-scribe, that the ecdysone signaling also affects the levels or turnover of the cell adhesion proteins Arm and DE-Cad, that were found in abnormally high levels between ECs and the germline. In order to escort the constantly moving germline cells, ECs and germline cells have to attach and detach frequently, and especially the formation and movement of cytoplasmic EC protrusions have to be highly dynamic. During these rearrangements, the adhesive complexes between ECs and the germline have to assemble and disassemble repeatedly and we suggest, that ECs protrusions cannot form properly if this process is defective. The ecdysone signaling pathway regulates the expression of various genes; in addition to di-rectly acting on the levels of DE-Cad and Arm it may also interfere with their turnover. Bai et al., 2000 observed elevated levels of DE-Cad in border cells in which ecdysone signaling was reduced. Artificial overexpression of DE-Cad how-ever did not phenocopy the ecdysone signaling mutant phenotype, nor did it lead to visible accumulation of DE-Cad at the cell membrane, suggesting that the cell can compensate for high DE-Cad and degrade it (Bai et al., 2000). Thus, ecdysone signaling acts on the formation of adhesive complexes, which dampens EC abil-ity to form cytoplasmic protrusions and ultimately affects germline differentiation (see Figure 3.1, page 117).

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Figure 3.1: Ecdysone signaling acts on germline differentiation in a cell non-autonomous way

(A)GSC division and germline differentiation are controlled by sys-temic hormonal signaling: insulin signaling regulates GSC main-tenance and division and integrates the overall nutritional status.

Ecdysone signaling controls a stress and starvation dependent check-point at which egg chambers undergo apoptosis if applicable.

(B) Ecdysone signaling acts on the levels or turn-over of the cell adhesion molecules DE-Cad and Arm and thereby also influences EC shape and function. Altered Arm levels in the germline alter the cells responsiveness to canonical Wg signaling. Thus, via the ECs, ecdysone signaling cell non-autonomously regulates the differ-entiation of the germline cells. The miRNAlet-7 attenuates the cell specific response to ecdysone.

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3.2 Wg signaling – a connection between ECs and