Sox100B, a Drosophila group E Sox-domain gene, is required for somatic testis differentiation

Abstract

Sex determination mechanisms are thought to evolve rapidly and show little conservation among different animal species. For example, the critical gene on the Y chromosome, SRY, that determines sex in most mammals, is not found in other animals. However, a related Sox domain transcription factor, SOX9, is also required for testis development in mammals and exhibits male-specific gonad expression in other vertebrate species. Previously, we found that the Drosophila orthologue of SOX9, Sox100B, is expressed male-specifically during gonad development. We now investigate the function of Sox100B and find, strikingly, that Sox100B is essential for testis development in Drosophila. In Sox100B mutants, the adult testis is severely reduced and fails to interact with other parts of the reproductive tract, which are themselves unaffected. While a testis initially forms in Sox100B mutants, it fails to undergo proper morphogenesis during pupal stages, likely due to defects in the pigment cells. In contrast, no substantive defects are observed in ovary development in Sox100B mutant females. Thus, as is observed in mammals, a Sox9 homolog is essential for sex-specific gonad development in Drosophila, suggesting that the molecular mechanisms regulating sexually dimorphic gonad development may be more conserved than previously suspected.

Fig. 1

Expression of Sox100B in the testis. In this and subsequent figures, the colors for each antibody staining are indicated by the colored text in each panel. A Wild type stage 15 embryonic testis stained with anti-Vasa to mark the germline and anti-Sox100B revealing the male-specific somatic gonadal precursors (msSGPs). B In eve3 + 7-Gal4;UAS-Gal4;UAS-GFP stage 15 gonads from male embryos, GFP and Sox100B mostly co-localize although some Sox100B expressing cells are negative for GFP (arrows) indicating they originate outside parasegment 13. C In the adult testis the GFP from the eve3 + 7-Gal4;UAS-Gal4;UAS-GFP reporter labels terminal epithelium (te) in addition to seminal vesicle (sv), which is also labeled with Abd-B, and other genital disc-derived structures. D Wild type embryonic testis at stage 17 revealing the germ line and the Sox100B positive superficial cells coating the gonad, the pigment cell precursors. E Third larval instar testis stained to reveal the germ line, DNA and Sox100B. Note the staining in the superficial pigment cells with their characteristic large nuclei (arrowhead). F–J Anti-Sox100B staining during testis morphogenesis: day 1 pupal testis (F), day 2 pupal testis (G), day 3 pupal testis (H), day 4 pupal testis (I), adult testis (J) showing the continued expression of Sox100B in pigment cells (inserts in F and I, and arrows in J focus on pigment cell nuclei). K The accumulation of Sox100B expressing cells at the basal end of the testis marks the terminal filament as revealed by staining with anti-FasIII, the insert shows an optical section demonstrating that the pigment cells lie over the terminal epithelium. L Sox100B does not co-localize with the somatic cyst cell marker Eya. Developmental stages of selected testes are indicated. E15, E17 = stage 15 or 17 embryos; L3 = late third instar larva; P1–4 = days of pupal development; Ad = adult.

Fig. 2

Sox100B is expressed in the developing ovary. A Ovary from 3rd instar larva stained for Sox100B, the germline, and DNA. Sox100B expression is strongest in the anterior region, and is also present in posterior cells that may derive from adjacent fat (arrow). B Ovary from a Day 3 pupae (∼ 76 h after puparium formation) stained for Sox100B and Eya, showing that they are expressed in distinct populations of somatic cells in the filaments and that Sox100B is cytoplasmic in the ovariole. C, D Adult wild-type ovarioles stained to reveal Sox100B, the germline, and DNA. Sox100B is observed in terminal filaments, interfollicular stalk cells (arrows) and cap cells, although staining is mostly cytoplasmic (inset in D).

Fig. 3

Isolation of Sox100B mutants.A Diagram of part of the 100B region on chromosome arm 3R with simplified gene models derived from FlyBase; arrowheads on the last exon indicate the direction of transcription. The insertion site of the j3B9P-element is indicated by the triangle. The solid lines indicate the extent of the 4 deletions. Sequence coordinates are according to release 4 of the genome sequence. B–F Dissected male reproductive tracts from adults or pharate adults. B In the wild type the testes (Te) are long and coiled, the accessory glands are labeled (Ag). C In Sox100B mutants, testis development is severely disrupted but occasionally a rudimentary lobe is visible. D Most frequently there is little testicular tissue evident, somatic derivatives of the genital disc such as the accessory glands are normal. Color micrographs of wild type (E) and Sox100B (F) mutant testes; the seminal vesicle (arrows) is not pigmented in the mutant. G Degenerated testis from a Sox100B mutant stained with anti-Vasa indicating the presence of very few germ cells and anti-Eya indicating the presence of rudimentary terminal epithelia. H Degenerated testis from Sox100B mutant stained with anti-Abd-B to reveal the testis sheath and with anti-Vasa to label the germline.I, J Staining for muscle myosin in wild type (I) and Sox100B (J) testes indicates that in rudimentary testis lobes the muscle sheath appears normal. DNA staining shows that the large nuclei characteristic of the pigment cells (arrows and insert in I) are absent from Sox100B mutant testes.

Fig. 4

Normal aspects of Sox100B mutant testes. A, B Stage 15 male embryonic gonads. Wild type (A) and Sox100B mutants (B) stained to reveal Abd-B expression in male-specific somatic gonadal precursors (msSGPs) and the germ cells. C–F Stage 17 embryonic gonads. Wild type (C) and Sox100B mutant (D) stained for the germline and anti-β-Gal to reveal the LacZ[842] enhancer trap expressed in somatic gonad precursors, including those giving rise to cyst and hub cells (arrows, hub cells). Wild type (E) and Sox100B mutant gonads (F) stained to reveal pigment cell precursors (Ems) showing that these cells are present in the mutants. In all cases, wild type and mutant are very similar. Note the similar Vasa staining in all cases indicating a normal germ line. The stripe of Ems expression to the right of the testis in F is from the CNS and unrelated to the gonad expression. G, H 16-cell spermatocyte cysts from wild type (G) and Sox100B mutant (H) 3rd larval instar testes. I–L Third larval instar testes from wild type (I and K) and Sox100B mutants (J and L) stained for the germline and for Eya indicating the presence of the msSGP-derived terminal body in Sox100B mutants (arrowheads). Anti-EMS staining marks the pigment cells (arrows in K and L).

Fig. 5

Sox100B mutant testes fail to elongate during metamorphosis. A Diagram showing stages of male reproductive tract development during metamorphosis (after Bodenstein, 1950). Testis is shaded in dark grey and genital disc derivatives in light grey. At puparium formation (left) the larval testis and genital disc are distinct. At 16 h of pupal development (middle), the testes begin to elongate towards the outgrowing genital disc. The adult reproductive tract (right), showing the characteristic coiling of the testes. B–G Pupal testes: In the wild type day 1 pupae (B) the testis has begun to elongate whereas in Sox100B mutants (C) it remains in a larval-like state. In 2 day pupae the testis of wild type (D) continues to elongate while the Sox100B mutant still remains in a larval-like state (E). In wild type day 1 pupal testis, staining with anti-Ems reveals the pigment cells (F). In the Sox100B mutants, the number of Ems-positive pigment cells is considerably reduced and staining appears weaker (arrows) than in wild type (G).