Sex-lethal enables germline stem cell differentiation by down-regulating Nanos protein levels during Drosophila oogenesis

Abstract

Drosophila ovarian germ cells require Sex-lethal (Sxl) to exit from the stem cell state and to enter the differentiation pathway. Sxl encodes a female-specific RNA binding protein and in somatic cells serves as the developmental switch gene for somatic sex determination and X-chromosome dosage compensation. None of the known Sxl target genes are required for germline differentiation, leaving open the question of how Sxl promotes the transition from stem cell to committed daughter cell. We address the mechanism by which Sxl regulates this transition through the identification of nanos as one of its target genes. Previous studies have shown that Nanos protein is necessary for GSC self-renewal and is rapidly down-regulated in the daughter cells fated to differentiate in the adult ovary. We find that this dynamic expression pattern is limited to female germ cells and is under Sxl control. In the absence of Sxl, or in male germ cells, Nanos protein is continuously expressed. Furthermore, this female-specific expression pattern is dependent on the presence of canonical Sxl binding sites located in the nanos 3' untranslated region. These results, combined with the observation that nanos RNA associates with the Sxl protein in ovarian extracts and loss and gain of function studies, suggest that Sxl enables the switch from germline stem cell to committed daughter cell by posttranscriptional down-regulation of nanos expression. These findings connect sexual identity to the stem cell self-renewal/differentiation decision and highlight the importance of posttranscriptional gene regulatory networks in controlling stem cell behavior.

Fig. 1.

Nanos and Bam protein expression in ovaries, testis, and snf¹⁴⁸ ovarian tumors. (A–C) Confocal images of gonads from WT animals and tumorous ovaries from snf¹⁴⁸/snf¹⁴⁸ females carrying fully functional copies of a Bam-GFP fusion protein and a Nanos-Myc fusion protein costained for GFP and MYC. (A) In the ovary, Bam and Nanos protein are expressed in nonoverlapping domains in early germ cells. The GSCs are located adjacent to the somatic cap cells, marked with an asterisk. Domains are numbered as in D. (B) In the testis, Nanos and Bam are coexpressed in early germ cells, except for the presumptive GSCs located adjacent to the hub, marked with an asterisk. (C) In the tip of the snf¹⁴⁸/snf¹⁴⁸ ovarian tumor, Nanos and Bam are coexpressed, except for the presumptive GSCs located adjacent to the somatic cap cells marked with an asterisk. Images in A–C are the same magnification.

Fig. 2.

The nanos gene is a Sxl target gene. (A) Schematic representation of the 5.8-kb genomic myc-tagged nanos constructs. Gray boxes indicate the 5′ and 3′ UTRs, white boxes represent the ORFs, and green box indicates the 6× MYC-tag inserted in frame. The location of the wild-type and mutated Sxl binding sites are shown above the diagram. (B) Sxl associates with nanos RNA. RNA IP assays were carried out in whole-cell extracts from ovaries or testis. The presence of nanos RNA in the IP pellet was detected by RT-PCR. (C) Mutagenesis of the S5, S1 and S3 binding sites impairs Sxl binding to nanos RNA. RNA IP assays were carried out in whole-cell extracts from ovaries carrying either the WT nanos-myc^WT construct, the mutant nanos-myc^ALL construct, or no transgene. The presence of nanos-myc RNA in the IP pellet was detected by RT-PCR. (C) Confocal images of ovaries carrying either a WT or mutant myc-tagged nanos construct stained for Myc. The staining pattern of the nanos-myc^WT parent construct resembles WT, whereas the mutant nanos-myc^ALL construct in which all of the S5, SI, and S3 binding sites are mutated is not down-regulated in the early differentiating germline cells. The location of the somatic cap cells is marked with an asterisk, and the regions are marked as in Fig. 1. Scale bars = 25 μm.

Fig. 3.

The nanos 3′ UTR Sxl binding site is essential for Nanos protein down-regulation. (A–E) Confocal images of ovaries from animals carrying a fully functional copy of a Bam-GFP fusion protein and one of the control or experimental Nanos-Myc fusion protein constructs costained for GFP and MYC. Arrows mark the location of the Bam-expressing cells. Scale bars = 25 μm.

Fig. 4.

Impact of ectopic Nanos expression on germ cell differentiation. (A) Phenotypic impact of ectopic Nanos expression driven by two copies of the nanos-myc^ALL transgene is more pronounced in a bam^Δ86/+ background. Confocal images of ovaries from 3-d-old animals stained for Vasa (red) to mark germ cells and 1B1 (green) to label the germ cell–specific spectrosomes and fusomes. 1B1 also labels somatic cell membranes. Scale bars = 25 μm. (B) Average number of spectrosomes/fusomes per germarium in different genetic backgrounds scored 3 and 20 d after eclosion. Spectrosomes/fusomes were classified as Round (R, spherical and not protruding into other cells), Short (S, extends to one adjacent cell), or Long (L, branches into two or more adjacent cells). Error bars represent SDs (n = 20 for each genotype/age group). Asterisks indicate significant differences by Student t test (***P < 0.001). (C) Relative distribution of the different types of spectrosome/fusomes structures per germarium. Asterisks indicate significant differences by the χ² test (*P < 0.05, ***P < 0.001).

Fig. 5.

snf¹⁴⁸, nanos¹⁸ Double mutant analysis. Comparison of germaria from (A–C) nanos¹⁸/Df(3R) Exel6183, (D–F) snf¹⁴⁸/snf¹⁴⁸, and (G–I) double-mutant nanos¹⁸/Df(3R)Exel6183; snf¹⁴⁸/snf¹⁴⁸ females stained 0–1 d or 12–14 d after eclosion for Vasa, Hu Li Tai Shao (Hts) (C, F, and I), and DNA (A, B, D, E, G, H). Arrow in C, F, and I shows the region enlarged in C', F', and I'), illustrating the difference between the thin branching fusome structure seen in nanos¹⁸/Df(3R)Exel6183 and the abnormal long branching fusome structures visible in snf¹⁴⁸/snf¹⁴⁸ and double-mutant nanos¹⁸/Df(3R)Exel6183; snf¹⁴⁸/snf¹⁴⁸ females. Scale bars: 50 μm (A, B, D, E, G, and H); 25 μm (C, F, and I). Asterisks indicate germaria with few or no germ cells.