tudor-domain containing protein 5-prime promotes male sexual identity in the Drosophila germline and is repressed in females by Sex lethal

For sexually reproducing organisms, production of male or female gametes depends on specifying the correct sexual identity in the germline. In D. melanogaster, Sex lethal (Sxl) is the key gene that controls sex determination in both the soma and the germline, but how it does so in the germline is unknown, other than that it is different than in the soma. We conducted an RNA expression profiling experiment to identify direct and indirect germline targets of Sxl specifically in the undifferentiated germline. We find that, in these cells, Sxl loss does not lead to a global masculinization observed at the whole-genome level. In contrast, Sxl appears to affect a discrete set of genes required in the male germline, such as Phf7. We also identify tudor domain containing protein 5-prime (tdrd5p) as a target for Sxl regulation that is important for male germline identity. tdrd5p is repressed by Sxl in female germ cells, but is highly expressed in male germ cells where it promotes proper male fertility and germline differentiation. Additionally, Tdrd5p localizes to cytoplasmic granules with some characteristics of RNA Processing (P-) Bodies, suggesting that it promotes male identity in the germline by regulating post-transcriptional gene expression. Author summary Like humans, all sexually reproducing organisms require gametes to reproduce. Gametes are made by specialized cells called germ cells, which must have the correct sexual identity information to properly make sperm or eggs. In fruit flies, germ cell sexual identity is controlled by the RNA-binding protein Sxl, which is expressed only in females. To better understand how Sxl promotes female identity, we conducted an RNA expression profiling experiment to identify genes whose expression changes in response to the loss of Sxl from germ cells. Here, we identify tudor domain containing protein 5-prime (tdrd5p), which is expressed 17-fold higher in ovaries lacking Sxl compared to control ovaries. Additionally, tdrd5p plays an important role in males as male flies that are mutant for this gene cannot make sperm properly and thus are less fertile. Moreover, we find that tdrd5p promotes male identity in the germline, as several experiments show that it can shift the germ cell developmental program from female to male. This study tells us that Sxl promotes female identity in germ cells by repressing genes, like tdrd5p, that promote male identity. Future studies into the function of tdrd5p will provide mechanistic insight into how this gene promotes male identity.


Introduction
protein 7 (Phf7, [20] as being regulated downstream of Sxl, as has previously been 126 described [21]. However, based on this analysis, it does not appear that regulation of 127 alternative splicing is a primary mechanism for Sxl regulation of gene expression in the 128 germline. 129 We also analyzed changes in overall gene expression levels in the presence of differentially expressed between bam Sxl-RNAi and control samples to determine 160 whether there was a "male" signature. We determined whether genes differentially 161 expressed in Sxl-RNAi compared to controls were also differentially expressed in bam 162 males compared to bam females. Of the 94 genes differentially expressed in Sxl RNAi, a 163 high fraction (44 genes) were also differentially expressed between bam male vs. female 164 samples (47%, which is considerably higher than the fraction of total genes in the 165 genome called as different between bam male and female, 13%). However, these genes 166 did not always change in the expected direction; only 61% of genes altered in both Sxl 167 RNAi and bam males changed in the same direction in both, while 39% changed in the 168 opposite direction (Fig 1E). Thus, the Sxl-RNAi sample does not appear globally 169 "masculinized" relative to controls and it may be that Sxl's role in repressing male 170 identity in the early germline is restricted to a few specific targets that are important for 171 the male germline. 172 One such candidate is a previously uncharacterized gene, CG15930, that was 173 strongly upregulated in Sxl-RNAi ovaries and is normally enriched in testes. CG15930 174 exhibits strong homology to mouse Tdrd5 [24], which is essential for male germ cell  The RNA-seq expression profiles show that tdrd5p has a dynamic expression 182 pattern characteristic of genes with sex-specific functions. tdrd5p is 18-fold enriched in 183 bam testes compared to bam ovaries, and is upregulated 17-fold in bam, Sxl-RNAi 184 ovaries relative to bam, control-RNAi ovaries (Fig 2A), and is clearly enriched in bam, 185 Sxl-RNAi ovaries by RT-PCR ( Fig 2B). This is in contrast to nanos, which is expressed at 186 similar levels in all of the genotypes in our RNA-seq experiment (Fig 2A), consistent 187 with its role in the germline of both sexes. Changes in tdrd5p expression were restricted 188 to total RNA levels, and no change in exon usage was detected. In situ hybridization to 189 wild-type gonads revealed that tdrd5p expression is highly enriched in the testis, particularly at the apical tip of the testis where the germline stem cells and proliferating 191 gonial cells reside (Fig 2C-D). The finding that tdrd5p is expressed at high levels in testes 192 relative to ovaries, and is repressed by Sxl in the ovary, suggests that it plays a role in 193 male germline development or function.

194
To determine the expression pattern of Tdrd5p protein, we generated a genomic As DCP-1 is localized to "Processing bodies" (P-211 bodies), Tdrd5p appears to be present in a subset of these structures.

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HA:Tdrd5p protein expression is upregulated in ovaries that are mutant for Sxl 213 function in the germline (Fig 3A-B). Note that the punctae seen in males are also present 214 in Sxl RNAi ovaries, though they are fewer in number (Fig 3D, arrows). Thus, the de-215 repression of tdrd5p in Sxl RNAi ovaries is also detected at the protein level.    To characterize the germ cell defects that may lead to decreased fecundity, we 243 evaluated several aspects of germ cell differentiation in young and aged mutant males.

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While testes of newly eclosed males appeared similar to wild-type, testes of older males 245 (15-20 days old) exhibited a dystrophic "skinny testis" phenotype with a dramatic 246 reduction in the germline (Fig 4A-C). This was observed in 7% of animals raised at 25 0 C 247 and 18% of animals raised at 29 0 C. Additionally, 15% of males exhibited a displaced hub 248 phenotype (Fig 4E arrow), where the hub is no longer located at the apical tip of the 249 testis as seen in wild-type (Fig 4D arrow). These combined phenotypes suggest a defect determination pathway-undergo a transformation so that the somatic gonad of XX tra 282 mutants develops as male instead of female. However, because the germline is XX, and 283 therefore incompatible with spermatogenesis, the germline of these testes is highly 284 undeveloped, causing these animals to be sterile (Fig 5B). A strong test of a gene's 285 ability to promote male identity in the germline is to determine whether it is sufficient     374 We show here that tdrd5p is both a target for Sxl regulation and is important for 375 male germline identity and spermatogenesis. tdrd5p expression is highly male-biased, 376 both at the RNA and protein levels (Fig 2). When female germ cells are sensitized by 377 partial loss of female sex determination genes, expression of tdrd5p exacerbates the 378 masculinized phenotype in these germ cells (Fig 5). Significantly, expression of tdrd5p is 379 sufficient to promote spermatogenesis in XX germ cells present in a male soma (XX tra-380 mutant testes, Fig 5). Thus, tdrd5p clearly has a role in promoting male germline 381 identity.

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Loss of tdrd5p also has a strong effect on male fecundity, even though it is not 383 absolutely required for spermatogenesis. The 50% reduction in fecundity is a strong