Ecdysone signaling links germline differentiation to the overall body status

Kept on rich food, flies produce 60 times more eggs then on poor food (Drummond-Barbosa and Spradling, 2001). Thus, oogenesis is a highly energy demanding process that strongly depends on the flies nutritional situation.

3.3.1 Ecdysone signaling mediates effects of stress and starvation

Insulin signaling and other pathways were shown to mediate the response to food availability; GSCs and FSCs respond to the nutritional status: they adjust their proliferative rate and the progression of germ cells is slowed down upon food deprivation (reviewed in Jasper and Jones, 2010). However, the dramatically decreased egg production rate seems to also result from an increased degener-ation of egg chambers at stage 8–9 (Drummond-Barbosa and Spradling, 2001).

Ecdysone signaling is required to progress past this master checkpoint and we now show, that perturbation of ecdysone signaling also affects germline develop-ment in the germarium: if ecdysone signaling in the soma is altered, the interac-tion between germline and ECs is affected and the germline development is slowed down. Ecdysone is produced in older follicles that passed the stage 8–9 checkpoint (see Section 1.3.3, page 19): ecdysone control of germline development, therefore, presents a positive feedback mechanism, with which germline development in the germarium is synchronized with the presence or absence of older follicles.

Ecdysone function in the adult is complex and poorly defined Understand-ing the role of ecdysone signalUnderstand-ing in oogenesis is complicated by several findUnderstand-ings:

first, the levels of ecdysone in the adult were measured by multiple research groups (Ishimoto et al., 2009; Schwartz et al.; Schwedes and Carney, 2012; Terashima and Bownes, 2006; Tu et al., 2002). However, the results obtained are contradictory and it is, for example, unclear whether ecdysone titers increase or decrease upon starvation. Second, even though ecdysone signaling is clearly indispensable for oogenesis (Buszczak et al., 1999; Carney and Bender, 2000), high ecdysone titers also negatively affect oogenesis. Terashima et al., 2005 reported that ecdysone injection – similar to starvation – induces apoptosis of nurse cells at stage 8 and 9 (Terashima and Bownes, 2006). Presumably, ecdysone function depends on whether its levels are below or above a certain threshold. Supporting this hypoth-esis, accumulating evidence suggests that – depending on ecdysone presence – the EcR can carry out both, transcription activating and repressing functions, which is further discussed in Section 3.3.2, page 122. Third, while ecdysone is a major regulator at the larval–pupal transition, its role in the adult fly is remarkably different: high ecdysone signaling activity was shown to promote stress resistance and ecdysone levels are changed if flies are exposed to unfavorable conditions like heat, food or sleep deprivation (Ishimoto and Kitamoto, 2010; Rauschenbach

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et al., 2000; Simon et al., 2003; Terashima and Bownes, 2005; Terashima et al., 2005; Tricoire et al., 2009).

Ecdysone is a ”stress hormone” in the adult fly In adults, ecdysone signaling controls various aspects of behavior, reproduction, body size, longevity and mem-ory (see Section 1.3.3, page 19). Considering that the ecdysone titers, measured by several research groups in differently treated flies are remarkably different, it is reasonable to conclude that the ecdysone levels are highly dynamic and respon-sive to external conditions. In addition, we could show that the levels oflet-7, the fine-tuner of ecdysone signaling, are increased upon food or temperature stress.

In addition to the ecdysone dependent checkpoint at stage 8-9, we showed that ecdysone signaling also controls progression through the early stages of oogenesis (Buszczak et al., 1999; Carney and Bender, 2000; Terashima and Bownes, 2006;

Terashima et al., 2005). Ecdysone functions independently from the GSC main-tenance regulation by insulin signaling, suggesting that the hormones insulin and ecdysone control the speed of oogenesis in parallel (see Figure 3.1, page 117).

We, therefore, suggest that ecdysone signaling is a master regulator or ”stress hormone” that links vitellogenesis and the overall body status.

3.3.2 The tissue- and time-specific response to ecdysone signaling is controlled by a complex network of interacting partners

Given the diverse functions of ecdysone signaling in adults and larvae, it is under-standable, that the time- and tissue-specific actions of ecdysone are mediated by a variety of different interacting partners. EcR/Usp cofactors include chromatin remodelers, histone modifier and transcriptional cofactors (see Section 1.3.2, page 18).

Ab is a potent transcription factor whose function depends of its concentra-tion Ab inhibits the strength of the ecdysone signaling by interacting with the cofactor Tai and interestingly, is targeted by the miRNA let-7 that is itself regu-lated by ecdysone signaling. Based on the analysis of penetrance and severity of ab mutant phenotypes, it was suggested that Ab controls dendrite branching and formation of other adult structures in a dosage dependent way (Hu et al., 1995; Li et al., 2004). Ab contains the BTB/POZ protein domain, that is highly conserved among metazoans and defines a protein-protein interaction interface (Zollman et al., 1994). Transcription factors containing BTB/POZ domains are involved in diverse cellular functions including regulation of transcription and cytoskeleton dynamics, ion channels and protein degradation (reviewed in Stogios et al., 2005).

Ab belongs to the ”tkk” subgroup that also contains Bab, Br, Pipsqueak, the GAGA factor and Batman. Whereas the sequence homology between members of the tkk subgroup and other BTB proteins is 24%, it is 49% on average within members of the tkk subgroup (Bonchuk et al., 2011). Recently it was shown, that

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the BTB domains of the tkk subgroup members Batman, Mod(mdg4), Pipsqueak, Tramtrack and GAF are able to form multimers (Bonchuk et al., 2011). Interest-ingly, Bonchuk et al., 2011 performed cross-linking experiments that suggest that the Ab BTB domain is also able to form major multimers. Given the similarity between the members of the ttk subgroup, the reported interaction between BTB domains of different proteins and the ability of Ab to form dimers, it is very likely that the Ab BTB domain is able to mediate the formation of Ab multimers. This provides a possible explanation for the observed dosage dependent action of Ab.

Importantly, Ab was shown to be a transdetermination factor, since overexpressing Ab in antennal imaginal discs leads to the transformation of arista into putative legs (Grieder et al., 2007). In addition, Ab is a global transcriptional regulator in the epithelium (Turkel et al., 2013). Altogether, Ab is a potent transcription factor that acts in a dosage dependent way to modulate the ecdysone signaling response, explaining the necessity for its precise regulation by the miRNA let-7.

Apart from the various interacting factors, the EcR/Usp itself contributes to the complexity of ecdysone signaling in D. melanogaster: the unliganded EcR/Usp complex is not only transcriptionally inactive, but even repressive.

The unliganded EcR complex represses transcription Previously it was shown that in the absence of ecdysone, the basal expression of a test vector was lower in vectors containing ecdysone responsive elements compared to empty ones. In addition, loss of Usp or EcR function was shown to lead to precocious differen-tiation of sensory neurons and expression of Br-Z1 (an isoform of Br) even at developmental stages with low ecdysone titers. Based on these results, it was suggested that the EcR/Usp complex can repress the expression of target genes if unliganded (Cherbas et al., 1991; Dobens et al., 1991; Schubiger and Truman, 2000; Schubiger et al., 2005), a phenomenon that has also been described for the retonoid X and retinoic acid receptors (reviewed in Glass and Rosenfeld, 2000).

We observed that overexpression of theEcRand lowering the levels of ecdysone in the adult led to similar phenotypes: an increased number of SSCs and a reduced cysts/single spectrosome ratio. We, therefore, speculated that the similarity of the phenotypes may arise because in both cases the activating function of the EcR was inhibited: either because of an artificially high level of EcR that was shown in vitro to form homodimers (Elke et al., 1997) or because of low ecdysone levels.

In order to prove this hypothesis, we fed ecdysone toEcR overexpressing flies: as expected, this led to a partial rescue of the observed phenotypes. Our results, therefore, support the hypothesis that unliganded receptor complexes can have a repressive function. Indeed Gancz et al., 2011 also reported that the repressive function of EcR/Usp in early third instar larvae – when ecdysone levels are low – is required for the correct formation of the gonad. Later in mid and late third instar larvae, active ecdysone signaling is required for niche formation and trig-gers primordial germ cell differentiation (Gancz et al., 2011). Recently Johnston et al., 2011 showed that in salivary glands the unliganded EcR does not repress the transcription of target genes, but is localized to the cytoplasm. Rising ecdysone titers lead to the translocation of the EcR/Usp complex to the nucleus, activating

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the transcription of target genes, for example E75A, a nuclear hormone receptor itself. As ecdysone titers increase at the larval–pupal transition, E75A replaces the EcR/Usp complex and represses the expression of target genes, including the EcR itself. The results provided by Johnston et al., 2011 present an additional mechanism by which tissue- and time-specific actions of the ecdysone regulated networks could be provided.

3.3.3 Maintaining the sexual identity requires intact ecdysone signaling

Steroid hormones are major regulators of mammalian sex determination and ga-metogenesis (reviewed in Arnold, 2012; Sim et al., 2008). Interestingly, we and others could show that ecdysone signaling regulates different aspects of gonadoge-nesis and adult gonad function in femaleD. melanogaster (Buszczak et al., 1999;

Carney and Bender, 2000; Hackney et al., 2007; K¨onig et al., 2011; Morris and Spradling, 2012). Data for a differential expression of ecdysone signaling pathway genes between male and female gonads remain poor, but there is evidence for sex specific differences of ecdysone levels (reviewed in Schwedes and Carney, 2012).

Morris and Spradling, 2012 recently reported that reducing ecdysone signaling in the male testis for eight days does not lead to visible effects on GSCs, developing cysts or primary spermatocyte clusters. In contrast, Garen et al., 1977 reported that ecdysone is essential for male fertility. Our own analysis reveal that ecdysone signaling mutant testes exhibit several severe phenotypes including a germline differentiation delay and formation of somatic epithelia (Fagegaltier et al.). Im-portantly, we have observed similar phenotypes in males lacking the miRNA let-7 that is a downstream effector of ecdysone signaling.

A confused sexual identity can contribute to the observed germline differen-tiation delay The soma has an important function in controlling the germlines sexual identity, but the genetic sex of the germline also is important. Interest-ingly, germline differentiation defects and tumors can also be the consequence of non-matching sexual identities in germline and soma; this happens for example if XX:AA germline cells are transplanted into male soma. JAK/STAT signaling is used by the male soma to masculinize the associated germline cells (reviewed in Murray et al., 2010). Furthermore, mutants of the master differentiation factor bam display a confused sexual identity as well: ovaries express a set of normally testis specific markers (Chau et al., 2009; Staab et al., 1996; Wei et al., 1994).

We, thus, analyzed ovaries and testes with respect to the expression pattern of sex specific transcripts. Interestingly, especially let-7 deficient males strongly up-regulate transcripts of the opposite sex; however, the phenotypes observed upon ecdysone signaling loss in testes and ovaries seem to be equally strong (Fage-galtier et al.). The ecdysone signaling target let-7 is expressed at higher levels in males, but whether ecdysone titers itself differ between sexes is not clear (re-viewed in Schwedes et al., 2011). We, thus, speculate that downstream effectors of

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ecdysone signaling affect the maintenance of the sexual identity and that the im-portance of these downstream effectors differs between sexes. The ecdysone/let-7 cascade cooperates with the JAK/STAT pathway in neuronal cell fate determina-tion (Kucherenko and Shcherbata, 2013) and it is tempting to speculate and will be subject of further analysis that the ecdysone/let-7 cascade may affect the male sexual identity via JAK/STAT.

3.3.4 The ecdysone/let-7 /Ab signaling cascade modulates oogenesis in response to different conditions

The BTB protein Ab is not only the negative regulator of ecdysone signaling, its levels itself are negatively regulated by steroids and stress. ab mRNA levels are increased in ecdysone-depleted ovaries; in addition, the cellular localization of Ab is variable, depending on ecdysone availability and external conditions. Given the severity of phenotypes induced by alterations of Ab on the one hand and the changes in localization that we observed upon stress on the other hand, it is understandable that changes in level and localization of this potent epithelial reg-ulator have to be robustly buffered, which is provided by the miRNA let-7. let-7 is induced by steroids in the D. melanogaster germarium andlet-7 itself promotes ecdysone signaling by reducing the levels of the ecdysone signaling inhibitor Ab.

We, thus, showed that the ecdysone signaling/let-7/Ab cascade regulates oogene-sis in response to stress. While the purpose of this regulation is to flexibly adjust gene expression in response to external stimuli, ”overshooting” reactions have to be prevented. miRNAs can ensure biological robustness and provide a buffer against stochastic fluctuations or ”overshooting” gene expression in a system (reviewed in Siciliano et al., 2013); which – considering the strong concentration dependent effects of Ab – is extremely important.

3.4 The D. melanogaster germarium provides a