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First published online 31 March 2004
doi: 10.1242/dev.01077

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A genomic analysis of Drosophila somatic sexual differentiation and its regulation

Michelle N. Arbeitman^1,*,, Alice A. Fleming^1,, Mark L. Siegal¹, Brian H. Null² and Bruce S. Baker^1,

¹ Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA
² Stanford Genome Technology Center, 855 California Avenue, Palo Alto, CA 94306, USA

View larger version (14K): [in a new window]   Fig. 1. Somatic portion of the sex determination hierarchy. In females, Sxl is activated and regulates the splicing of tra pre-mRNA, resulting in the production of TRA. TRA together with TRA2 regulates the female-specific splicing of dsx and fru pre-mRNAs. In males, Sxl is not activated and no functional TRA is produced, resulting in default splicing of dsx and fru pre-mRNAs. dsx produces both female- and male-specific isoforms. fru produces a male-specific isoform.

View larger version (80K): [in a new window]   Fig. 2. Hierarchical cluster of female and male somatic genes. Genes were identified by statistical analysis, clustered hierarchically using Cluster and visualized with Treeview (Eisen et al., 1998). Compromised ESTs (chimeric or from a contaminated source) were removed for this analysis (see Table 2). The cluster includes whole animal data from wild-type animals at all stages of Drosophila development, including embryos (E), larva (L), pupae (P) and adults (A) (Arbeitman et al., 2002), and from the following mutants: tud female (FTud) and male (MTud); XX tra (tra); XX DsxD pseudomales (dsxD); fru; and dsx intersexual XY (dsxXY) and XX (dsxXX). Expression relative to the reference RNA is shown; yellow, blue and black indicate high, low and median levels of expression, respectively (see scale bar). Results of RNA blot analyses are shown (RNA blot), with key below. Gene names are shown on the right; red indicates genes chosen for further analysis.

View larger version (104K): [in a new window]   Fig. 3. (A-E) Characteristic examples of RNA blot analyses. Each blot consists of 3 µg polyA+ RNA per lane from female (F) or male (M) 5-day-old adult animals: wild type (WT) and tud. Microarray values for each gene (see Materials and methods) are included below the respective lanes. Probes were derived from ESTs for female genes (A-C) or male genes (D-E). (A) CG17012, (D) CG2858 and (E) CG8708 agree with microarray data. (B) CG7777 agrees with microarray data in that there is somatic expression (slight elevation in expression of female over male tud lanes). Germline expression is also apparent (much greater intensity of wild-type female over tud female bands). (C) CG7899 expression is sex-specific, but is due to germline rather than somatic expression. (F) Internal controls. Two genes, each with similar levels of female and male expression according to the microarray data, were used as internal control probes: CG4659 (Srp54K) (upper band) and CG10664 (cytochrome c oxidase subunit IV). Expression levels in the wild-type female lane of CG4659 and the tud male lane of CG10664 are typically slightly elevated.

View larger version (93K): [in a new window]   Fig. 4. Temporal and tissue-specific expression in female internal genitalia. (A) Schematic of the internal female reproductive system. Modified, with permission, from Miller (Miller, 1950). (B) Expression profiles of two female genes (see Fig. 2 legend). (C, parts I-IV) In situ hybridization of wild-type and tud 5-day adult female reproductive tissues using probes derived from female (I,II) and male (III,IV) genes. I part a, II part a, and III parts a, h and i, are frozen tissue frontal sections of whole animals; all other images are from whole-mount in situ hybridization analysis. (I) Expression is seen in spermathecae (arrows) and parovaria (arrowheads) in wild-type (a-d) and tud (e) tissues. (II) Expression in nurse cells of stage 8-10 egg chambers (a-c); in follicle cells covering an early stage 14 egg chamber, including the dorsal appendage (arrowhead; inset is at a closer focal plane; d); in oviducts (e); in spermathecae of wild-type (f) and tud (g) tissues; and in sheath of the immature ovaries found in tud animals (g). (III) Expression of male-enriched mfas in female tissues (a-f): in follicle cells of stage 9-10 egg chambers (b,c); in sloughed off follicle cells of a later stage egg chamber (d); in parovaria (e); in fat tissue (f); and, sex-nonspecifically, in CNS (g,h). (IV) Expression of male-enriched prd in female fat tissue (a-c). Insets are digital magnifications unless otherwise noted.

View larger version (81K): [in a new window]   Fig. 5. Temporal and tissue-specific expression in male internal genitalia (A) Schematic of the internal male reproductive system. Modified, with permission, from Miller (Miller, 1950). (B) Expression profiles of male genes (see Fig. 2 legend) (C) In situ hybridization of wild-type 5-day-old (I-VI), or 0-24 hour (VII), adult and mutant tud male reproductive tissues using probes derived from male genes. I-V part a, VII part a, and II parts e and f are frozen tissue frontal sections of whole animals; all other images are of whole-mount tissues. (I) Expression in the accessory glands: CG18284 (a-c), CG17022 (d) and CG17843 (e). (II) Expression of CG8708 in the anterior ejaculatory duct (a-d; c and d are different focal planes), including the papilla (d), and, sex-nonspecifically, in adult and embryonic salivary glands (e,f). (III) Expression of CG2858 in the ejaculatory bulb (a-e), showing individual differences in expression (c-e). (IV) Expression in accessory glands and the ejaculatory bulb: CG12558 (a-d) and CG9519 (e). (V) mfas expression in testes (a-c) and on the surface of seminal vesicles where the testes attach (c); in accessory glands (a,b,d); and in the ejaculatory bulb (b,e). (VI) prd expression in testes (T) and accessory glands (A). (VII) Expression of CG6788 in 0-24 hour ejaculatory bulb (a-c). Insets are digital magnifications.

View larger version (75K): [in a new window]   Fig. 6. Characteristic examples of RNA blot analyses for tra2 temperature-shift experiments. Total RNA (20 mg) was derived from tra2ts1/tra2ts2 (lanes 1-4) animals and tra2ts/Cyo controls (lanes 5-8); RNA in lanes 1-6 was from animals that were chromosomally XX, whereas and RNA in lanes 7 and 8 was from animals that were chromosomally XY. Animals were either maintained at one temperature, or raised at one temperature then shifted as 2-day adults to the other temperature, as indicated. (A) CG18284, a male gene encoding two transcripts; (B) CG7777, a female gene; and (C) Yp1.

View larger version (72K): [in a new window]   Fig. 7. Expression of CG17012 and prd in wild-type and mutant tissues from 5-day-old flies. (A-D) Whole-mount in situ analysis of CG17012 expression in female internal genitalia in: (A) wild type, (B) dsx intersexual animals, (C) virgin wild type, and (D) tud females. In A and B, the region within the box has been digitally magnified and shown on the right. Staining is observed in cells of the spermathecae and in the fat cells that surround the spermathecae in A, but only in the fat cells in B. Fat tissue is not visible in C and D. (E) CG17012 sense probe (negative control). Low level signal was observed in fat tissue, a common occurrence with both sense and anti-sense probes. Arrows indicate cells of the spermathecae and arrowheads indicate fat cells. (F) Whole animal frozen section in situ analysis of prd expression in dsx intersexual and wild-type animals. Arrowheads indicate expression regions