Maintaining sexual identity is required for germline differentiation

Male and female GSCs are in principle controlled by similar mechanisms; however, there are sex specific differences with respect to how, when and to which extent certain signaling pathways are used (for review see Fuller and Spradling, 2007;

Spradling et al., 2011). The D. melanogaster testis is a paired tubular organ;

the GSCs reside at the apical tip and are attached to somatic hub cells. The cyst stem cells give rise to somatic cells that – similar to the ECs in the female ovaries – encapsulate the differentiating germline cells. The TGF-β ligands Dpp and Gbb are produced in the somatic hub and cyst cells and – like in the ovary – are required for GSC maintenance. Different from the situation in females, ectopic activation of the TGF-βpathway is not sufficient to induce self-renewal of germline cells (reviewed in Matunis et al., 2012). Bam is a differentiation factor in males as well, but acting at a different step than in the females: it is expressed in differentiating spermatocytes and is required to cease mitotic amplification divisions (reviewed in Spradling et al., 2011).

1.7.1 Sex is determined by a series of alternative splicing events in D. melanogaster

Different species use a variety of different mechanisms to establish and maintain the somatic sex. The X chromosome to autosome ratio determine the sexual iden-tity: two X chromosomes (XX:AA) determines female, one X chromosome (X:AA) yields male identity. The Y chromosome has no influence on the sexual identity, but is required for spermatogenesis. Regulatory proteins lead to the activation of the gene encoding the RNA binding protein Sex lethal (Sxl) in XX:AA animals only. After the Sxl activity was set up early in embryogenesis, a positive feed-back splicing mechanism is used, in which Sxl controls its own splicing. X:AA cells lack Sxl protein and allSxl transcripts, therefore, contain an exon containing a translation termination codon. In XX:AA cells, however, Sxl protein affects splicing and thus, the male specific translation terminating codon is skipped and functional Sxl mRNA is generated. Sxl is both necessary and sufficient to pursue its own splicing loop and is on top of the cascade determining the female trait, directly or indirectly controlling all female specific programs (reviewed in Salz, 2011; Salz and Erickson, 2010). Sxl controls the splicing oftransformer (tra). Tra – a female specific RNA binding protein as well – in turn, controls splicing of the transcription factors Doublesex (Dsx) and Fruitless. Dsx is the main factor con-trolling the sexual dimorphism of the somatic gonads. It is expressed in a subset of somatic cells and controls the sexually dimorphic development of the others via cell non-autonomous processes (reviewed in Murray et al. (2010)).

The germline sexual identity is determined in collaboration with the soma Interestingly, the sexual identity of germline cells is determined differently from

32

1 Introduction

the somatic cells in many species. In some, the somatic cells solely control the germline sexual fate. In others, such as fruit flies, mice or humans the sexual identity of the germ cells is important for the germ cells sex. Tra and Dsx, the main determinants of somatic sexual identity are not required in the germline for specifying the sexual identity. The role of Sxl in the germline is discussed controversial; while it was thought to act and be activated differently then in the soma (reviewed in Casper and Van Doren, 2009), it was recently shown that ectopic expression ofSxl is sufficient to induce female development in the pole cells (Hashiyama et al., 2011). Hence, Sxl is a key player in establishing sexual identity of both soma and germline, nevertheless a comprehensive analysis of downstream effectors is missing (reviewed in Murray et al., 2010; Salz and Erickson, 2010). The germline sexual identity is first detected after formation of the embryonic gonad and is mainly controlled by the soma at this point (Casper and Van Doren, 2009;

Wei et al., 1994). As development goes on, the soma is not sufficient for keeping the germline sex; XX:AA or X:AA germ cells in the soma of the opposite sex do not follow the somatic sex. Transplant studies demonstrated, that XX:AA germ cells cannot form oocytes in the male soma and XY:AA germ cells do not give rise to spermatocytes when transferred into the female soma, but rather display a confused sexual identity (reviewed in Murray et al., 2010). It is therefore critical to understand, which pathways are used by the soma to control the germline sex. The JAK/STAT signaling pathway that is known to be one of the main mechanisms that control GSC maintenance in the testis also has a masculinizing effect on the germ cells (Wawersik et al., 2005). The mechanisms that are used by the female soma to control the germline sex remain undiscovered.

1.7.2 Germline tumor arise as a consequence of confused sexual identity

Mutations inSxl lead to tumors in XX:AA germline cells that are blocked between GSC and CB stage. The tumorous cells not only express the differentiation factor Bam, but also the GSC specific marker Nos (Chau et al., 2009, 2012). Together with its partner Pum, Nos inhibits differentiation-promoting mRNAs, including brat and it is therefore critical to restrict Nos function to the GSC. It is known that Bam itself downregulates the levels of Nos (Li et al., 2009) and, in addition, Chau et al., 2009, 2012 showed that the translation of nos is directly repressed by Sxl. Sxl, thus, promotes differentiation of the GSC progeny; since Sxl is also present in the GSCs themselves, this raises the question how the Nos repression by Sxl is restricted to CBs. Based on epistasis experiments, Chau et al., 2009, 2012 suggest, that Bam itself is required for this germline cell specificity and that nos is ultimately repressed by Bam and Sxl functioning together. The misdirected expression of nos, however, is not sufficient to explain the described tumorous phenotypes (Chau et al., 2012; Li et al., 2009).

Mismatch of germline and somatic sexual identity leads to differentiation failure Interestingly, germline cells lackingSxl orbam express a set of commonly

33

1 Introduction

testis-specific markers, suggesting that their sexual identity is confused (Chau et al., 2009; Staab et al., 1996; Wei et al., 1994). Such ”ovarian tumors”, consisting of germline cells displaying both male and female characteristics are also observed when XX:AA germline cells are transplanted into a female soma (reviewed in Casper and Van Doren, 2006). Furthermore, mutations in ovarian tumor or ovo, which are thought to promote female identity, lead to germline overproliferation (King; Oliver et al., 1987). Altogether, there are many examples showing that the sexual identity of soma and germline have to match in order to allow for proper germline development. Considering that steroid hormones are key determinants for sexual development in mammals, these findings raise the question whether defects in maintaining the sexual identity contribute to the differentiation delay observed upon perturbed ecdysone signaling.

34

1 Introduction