Drosophila melanogaster male somatic cells feminized solely by TraF can collaborate with female germ cells to make functional eggs

Abstract

Female differentiation of Drosophila germ cells is induced by cell-nonautonomous signals generated in the gonadal soma that work with germ-cell-autonomous signals determined by germ-cell X chromosome dose. Generation of the nonautonomous feminizing signals was known to involve female-specific protein encoded by the master sex-determination gene Sex-lethal (Sxl) acting on its switch-gene target transformer (tra) to produce Tra(F) protein. However, it was not known whether Sxl's action on tra alone would suffice to trigger a fully feminizing nonautonomous signal. We developed a constitutively feminizing tra transgene that allowed us to answer this question. In gynanders (XX//XO mosaics) feminized by this Tra(F) transgene, functionally Sxl- haplo-X (chromosomally male) somatic cells collaborated successfully with diplo-X (chromosomally female) germ cells to make functional eggs. The fertility of such gynanders shows not only that Tra(F) is sufficient to elicit a fully feminizing nonautonomous signal, but also that haplo-X somatic cells can execute all other somatic functions required for oogenesis, despite the fact that their genome is not expected to be dosage compensated for such diplo-X-specific functions. The unexpected observation that some Tra(F)-feminized gynanders failed to lay their eggs showed there to be diplo-X cells outside the gonad for which Tra(F)-feminized haplo-X cells cannot substitute.

Figure 1.—

Four distinct ovariole genotypes from the gonads of XX//XO mosaics (gynanders) feminized by constitutive expression of Tra^F. All XX cells fluoresce green due to expression of a Ubi-GFP transgene on their maternal X chromosome, while all XO cells have lost this maternal X and are therefore nonfluorescent (from the cross in Table 2). All cells carry the feminizing U2af-tra^F transgene. (A–D) Nomarski images. (E–H) Corresponding GFP fluorescence images. (A and E) Germarium with XX soma and XX germ cells. All cells fluoresce and oogenesis is normal. (B and F) Germarium with nonfluorescent tra^F-feminized XO soma and fluorescent XX germ cells (longer exposure than E). The feminized XO cells support normal oogenesis for XX germ cells. (C and G) Abnormal germarium with fluorescent XX somatic cells and nonfluorescent tumorous XO germ cells. XO germ cells fail to develop normally even in a bona fide female somatic environment. (D and H) Abnormal tumorous cyst from an ovariole with feminized XO somatic cells and XO germ cells. No cells fluoresce. Consistent with G, XO germ cells fail to develop normally in a feminized somatic environment. Bar, 10 μm for A, B, E, and F and 20 μm for C, D, G, and H (anterior to the left).

Figure 2.—

Sxl remains in its male expression state in Tra^F-feminized XO somatic cells that are engaged with XX germ cells in oogenesis. (A–D) Confocal micrographs of stages 4–6 oogenic cysts from the gonads of two different XX//XO mosaics (gynanders) feminized by U2af-tra^F (see Table 2). Green is anti-GFP staining that distinguishes XX cells from the nonstaining XO cells (A and C), red is anti-Sxl^F staining that indicates female Sxl expression (B and D), and blue is DAPI staining of DNA. (A and B) A control gynander with XX somatic and germ cells. All cells express Sxl^F proteins. (C and D) A gynander with Tra^F-feminized XO somatic cells engaged in oogenesis with XX germ cells. Note the absence of Sxl^F proteins in the XO somatic cells that surround germ cells producing Sxl^F. Bar, 20 μm.

Figure 3.—

Only very rarely do germ cells marked as XO reach unambiguously female late stages of development when growing in a female somatic environment. Ovarioles and eggs are from U2af-tra^F-feminized gynanders from the cross described in the footnote to Table 2. (A, C, D, and F) Nomarski images. (B and E) GFP fluorescence images. (A–C) The one atypical ovariole of the only gynander ovary with apparently XO germ cells (nonfluorescent) and XX soma (fluorescent green) that showed unambiguously female differentiation. The white arrow indicates nurse-cell-like (female) differentiation, while the white arrowhead highlights a degenerating cyst. A nearly mature egg from this ovariole (C) had grossly abnormal dorsal appendages (inset) and was abnormally small. (D–F) A developmentally wild-type ovariole and egg from a nonmosaic XX gynander ovary for comparison. Bar, 25 μm for A, B, D, and E (anterior up) and 100 μm for C and F (anterior to left).