GENETICS OF SEX A Genome-Wide Survey of Sexually Dimorphic Expression of Drosophila miRNAs Identifies the Steroid Hormone-Induced miRNA let-7 as a Regulator of Sexual Identity

MiRNAs bear an increasing number of functions throughout development and in the aging adult. Here we address their role in establishing sexually dimorphic traits and sexual identity in male and female Drosophila. Our survey of miRNA populations in each sex identifies sets of miRNAs differentially expressed in male and female tissues across various stages of development. The pervasive sex-biased expression of miRNAs generally increases with the complexity and sexual dimorphism of tissues, gonads revealing the most striking biases. We find that the male-specific regulation of the X chromosome is relevant to miRNA expression on two levels. First, in the male gonad, testis-biased miRNAs tend to reside on the X chromosome. Second, in the soma, X-linked miRNAs do not systematically rely on dosage compensation. We set out to address the importance of a sex-biased expression of miRNAs in establishing sexually dimorphic traits. Our study of the conserved let-7-C miRNA cluster controlled by the sex-biased hormone ecdysone places let-7 as a primary modulator of the sex-determination hierarchy. Flies with modified let-7 levels present doublesex-related phenotypes and express sex-determination genes normally restricted to the opposite sex. In testes and ovaries, alterations of the ecdysone-induced let-7 result in aberrant gonadal somatic cell behavior and non-cell-autonomous defects in early germline differentiation. Gonadal defects as well as aberrant expression of sex-determination genes persist in aging adults under hormonal control. Together, our findings place ecdysone and let-7 as modulators of a somatic systemic signal that helps establish and sustain sexual identity in males and females and differentiation in gonads. This work establishes the foundation for a role of miRNAs in sexual dimorphism and demonstrates that similar to vertebrate hormonal control of cellular sexual identity exists in Drosophila. SEXUAL dimorphism is pervasive throughout the animal kingdom. From insects, fishes, reptiles, and birds to mammals, hormones and genes shape the morphological, behavioral, and reproductive potential of each sex throughout development and adult life. Drosophila is no exception, with males and females differing in many ways: anatomical differences include the number of abdominal segments and their pigmentation, the proboscis, labial parts, dimorphic reproductive organs, the formation of sex combs exclusively in males, and 25% larger size in females. Differences that affect male and female behavior exist also in the nervous system and the brain. Y chromosome aside, male and female cells possess a strictly identical genomic content. Most of the differences between the sexes arise and persist via the regulation of sets of genes in a sex-specific manner. The question of how hundreds if not thousands of genes are differentially expressed in males and females to produce Copyright © 2014 by the Genetics Society of America doi: 10.1534/genetics.114.169268 Manuscript received May 20, 2014; accepted for publication July 14, 2014; published Early Online July 31, 2014. Supporting information is available online at http://www.genetics.org/lookup/suppl/ doi:10.1534/genetics.114.169268/-/DC1. Available freely online through the author-supported open access option. miRNA libraries from male and female tissues have been submitted to the GEO database at NCBI as series GSE57029. Corresponding authors: Cold Spring Harbor Laboratory, McClintock Bldg., 1 Bungtown Road, Cold Spring Harbor, NY 11724. E-mail: [email protected]; Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, Göttingen 37077, Germany. E-mail: [email protected] Genetics, Vol. 198, 647–668 October 2014 647 sexually dimorphic individuals is extensively studied. Refined genomic and genetic studies have converged toward a model of differential expression that requires that both spatial and temporal programs be established throughout development (Arbeitman et al. 2002; Parisi et al. 2004; Lebo et al. 2009; Chatterjee et al. 2011). Probably the most important of these programs in flies is the sex-determination hierarchy (Baker et al. 1989; Christiansen et al. 2002; Camara et al. 2008; Clough and Oliver 2012). The primary determinant of Drosophila sex is the X chromosome to autosome (X:A) ratio (Bridges 1921), which determines the production of alternative splice variants of Sex lethal (Sxl) to generate an active SXL protein in females and a nonfunctional truncation in males (Cline 1978). Sxl activity is sufficient to direct the entire developmental programs of both somatic and germline sex determination (Christiansen et al. 2002; Robinett et al. 2010; Salz 2011; Whitworth et al. 2012). Sxl serves two essential functions: it restricts dosage compensation to males and controls the sex-determination hierarchy in each sex. Dosage compensation is the process by which males double the transcription of genes on their single X chromosome to match the levels found in diplo-X females. This process requires a ribonucleoprotein complex, the compensasome, composed of two noncoding RNAs (roX1 and roX2) and six proteins (male-specific lethals MSL-1, -2, -3, the helicase/ATPase MLE, histone acetyltransferase MOF, and histone kinase JIL1). In females, SXL represses the production of MSL-2 at both the transcriptional and translational level, therefore preventing dosage compensation. In males, lack of SXL function allows the male-specific expression of MSL-2 and its assembly into compensasomes to initiate dosage compensation (Bashaw and Baker 1997; see Duncan et al. 2006 for review). At the top of the sex-determination hierarchy, SXL controls which sex-specific isoform is being processed from the doublesex (dsx) transcripts (reviewed in Christiansen et al. 2002). If the X:A ratio is 1, Sxl produces a femalespecific splicing factor that causes female-specific splicing of the transformer (tra) transcript. TRA interacts with the transformer-2 (TRA2) splicing factor to produce a femalespecific splice variant of dsx (Belote et al. 1989; Sosnowski et al. 1989; Ryner and Baker 1991). The female-specific DSXF protein then activates female and inhibits male development. Because males lack SXL and subsequently TRA, a “default” male-specific splicing of dsx transcript generates the DSXM protein, which inhibits female and promotes male traits. Loss-of-function mutations in Sxl, tra, and tra2 transform XX individuals into males, but have no effect in XY males. In contrast, the dsx gene is important for the sexual differentiation of both sexes—in the absence of dsx, both XX and XY flies are anatomically and behaviorally intersex (Baker and Ridge 1980; Belote et al. 1985). Only a few transcriptional targets through which DSX ultimately functions are known (Luo et al. 2011). DSX regulates sex-specific pigmentation patterns with abdominal-B (Abd-B) and bric-a-brac1 (bab1), resulting in males’ darker abdomen (Williams et al. 2008). DSXM controls the development of male-specific bristles or sex combs on the forelegs with sex-comb reduced (Scr) (Tanaka et al. 2011). In each sex, DSX orchestrates the differentiation of larval genital discs into mature dimorphic reproductive organs, external genitalia, and analia (Hildreth 1965; Chatterjee et al. 2011). DSXF directly upregulates the expression of yolk proteins (Yp1, Yp2) (Burtis et al. 1991), and DSXM downregulates their transcription. The thorough dissection of dsx expression reveals that DSX presents two main characteristics (Lee et al. 2002; Hempel and Oliver 2007; Rideout et al. 2010; Robinett et al. 2010). First, the levels of DSX protein vary greatly throughout development within cells and tissues, implying a tight regulation of its steady states. Second, DSX is not present in all cells in a given tissue, so only some cells know their sex while others remain asexual. MicroRNAs (miRNAs) appear as critical regulators of development and are themselves highly regulated (Ambros and Chen 2007; Bartel 2009; Smibert and Lai 2010; Dai et al. 2012). The interaction of microRNAs with the 39-UTRs of transcribed mRNAs affects both a transcript’s stability and its translation. Each miRNA can target several different mRNAs and each mRNA can be targeted by multiple miRNAs, generating an intricate network of gene expression regulation. As miRNAs could provide a rapid and tissue-specific means to alter gene expression, they represent ideal candidates for the regulation of spatial and temporal expression patterns of sex-determination genes, their cofactors, and downstream targets. Ultimately, the sex-biased expression of miRNAs could control directly the differential expression of many genes contributing to sexually dimorphic traits at a given time and place during development. Sexually dimorphic miRNA profiles have been reported in mouse and chicken gonads, and in whole adult Caenorhabditis elegans (Mishima et al. 2008; Kato et al. 2009; Baley and Li 2012). In Drosophila, probing miRNA populations in whole animals during development has revealed widespread developmental regulation (Aravin et al. 2003; Ruby et al. 2007). However, the small RNA libraries generated in these studies came from either mixed-sex samples or single-sex but nonhomogenous tissues, which may mask important sexand tissue-specific variability in miRNA expression and function. To date, Drosophila lacks a critical examination of miRNA expression in two important contexts: sex-biased expression that may lead to sexually dimorphic function or spatial and temporal heterogeneity in expression that may drive tissuespecific functions. Both are critical to understanding the role of miRNAs across development. To investigate these issues, we first established the profiles of miRNAs in several male and female adult parts and organs, larval dissected tissues, and embryonic cells. Their comparison reveals, in each tissue, sets of maleand female-biased miRNAs, increasing in number and extent with the complexity and sexual dimorphism of each tissue. We further address two aspects of miRNA functions in the 648 D. Fagegaltier et al. context of sexual identity: first, we test whether X-linked miRNAs are regulated by dosage compensation in males and, second, we explore the role of the steroid-induced miRNA let-7 in regulating sexually dimorphic traits and how its male-biased expression in the gonads affects germline differentiation programs. Materials and Methods Fly strains and genetics Oregon-R flies were used for miRNA profiling. Msl3p, mle1, and pr mle12.17 mutants are described in Fagegaltier and Baker (2004). All chromosomes but the mutant-bearing allele were exchanged to create isogenized lines by back crossing to a w1118; MKRS/TM6B stock for .10 generations. Wandering non-Tbmutant male and female larvae were identified by their gonads. Overexpression of miRNAs was performed using a dsx–GAL4 driver (Robinett et al. 2010). UAS–NLS–GFP flies are from Bloomington (BL4776); UAS–let-7, UAS-mir-100, UAS–mir-125, and UAS–let-7-C constructs are described in Bejarano et al. (2012). In let-7-C and ecd mutant studies, flies were raised on standard cornmeal–yeast–agar-medium at 25 and fattened on wet yeast paste 1 day before dissection unless otherwise stated. The two knockout alleles let-7-CGK1 and let-7-CKO1 lack the whole let-7-C cluster; let-7-CGK1 contains the transcriptional activator GAL4 under the control of the let-7-C promoter; let-7-CGK1/let-7-CKO1 are referred to as Dlet-7-C (Sokol et al. 2008). Flies with a transgene rescuing the let-7-C cluster (P{W8, let-7-C}; let-7-CGK1/let-7-CKO1) are referred to as let-7-C Rescue. The P{W8, let-7-C Dlet-7} construct restores all let-7-C miRNA members except for let-7. For miRNA loss of function, let-7-CGK1/let-7-CKO1; P{W8, let-7-C Dlet-7}/+ flies referred to as Dlet-7 were used. The following additional fly stocks were used: FRT40A let-7 mir-125/CyO and UAS–let-7-C; Sco/CyO (Caygill and Johnston 2008), UAS–let-7/TM6 (Sokol et al. 2008), Ubi–GFP FRT40A/ CyO; bab1–Gal4:UAS–Flp/TM2 (a gift from A. González-Reyes), UAS–CD8GFP:UAS–nuc lacZ (a gift from F. Hirth), Oregon-R, w1118, and ecd1ts (BL4210). Sample collections for miRNA–Seq and validation To ensure that miRNA–Seq samples are not contaminated by other tissues, 120 Oregon-R heads were individually separated with a scalpel from the rest of the body of 24-hr-old males and females, collected on ice and quickly frozen. Salivary glands were dissected from 130 wandering late L3 larvae of each sex identified by their gonads. For qPCR validations of miRNA–Seq data sets, at least two additional independent collections were performed as above. We also collected ovaries and testes from 0to 2and 2to 4-day-old Oregon-R individuals, S2 (Invitrogen), and Kc-167 cells (DGRC) washed in 13 PBS. All dissected tissues and cells were quickly snap frozen in liquid nitrogen and RNA preparations enriched for small RNAs using an adapted Trizol protocol. miRNA–Seq 30-100mg of total RNAs were subject to 2S rRNA depletion and DNAse treated. Size selected 18-29nt sRNAs were cloned according to (Malone et al. 2012). Libraries were clustered and sequenced on the Illumina GAIIx platform. Cuticle preparations Threeto 4-day-old flies were placed in ethanol and incubated in 10% NaOH for 1 hr at 70 . Adult abdominal cuticles were mounted and flattened in 30% glycerol. Pictures were taken at the same magnification using a Nikon SMZ150 microscope and Nikon DS-RiI camera. Perturbation of ecdysone levels The ecd1ts temperature-sensitive mutation is known to reduce ecdysone levels at the nonpermissive temperature (Garen et al. 1977). Oregon-R and ecd1ts flies were kept at the permissive temperature (18 ) and 2to 4-day-old adults were shifted to the restrictive temperature (29 ) for 5–11 days to block ecdysone synthesis. Control Oregon-R and ecd1ts flies were kept at 18 for the same time.