Sex-specific DoublesexM expression in subsets of Drosophila somatic gonad cells - PubMed (original) (raw)

Sex-specific DoublesexM expression in subsets of Drosophila somatic gonad cells

Leonie U Hempel et al. BMC Dev Biol. 2007.

Abstract

Background: In Drosophila melanogaster, a pre-mRNA splicing hierarchy controls sexual identity and ultimately leads to sex-specific Doublesex (DSX) transcription factor isoforms. The male-specific DSXM represses genes involved in female development and activates genes involved in male development. Spatial and temporal control of dsx during embryogenesis is not well documented.

Results: Here we show that DSX(M) is specifically expressed in subsets of male somatic gonad cells during embryogenesis. Following testis formation, germ cells remain in contact with DSX(M)-expressing cells, including hub cells and premeiotic somatic cyst cells that surround germ cells during spermatogenesis in larval and adult testes.

Conclusion: We show that dsx is transcriptionally regulated in addition to being regulated at the pre-mRNA splicing level by the sex determination hierarchy. The dsx locus is spatially controlled by somatic gonad identity. The continuous expression of DSX(M) in cells contacting the germline suggests an ongoing short-range influence of the somatic sex determination pathway on germ cell development.

PubMed Disclaimer

Figures

Figure 1

Figure 1

dsx m and DSXM in embryos. (A) RT-PCR of dsx m from sex sorted embryos of ages 3–10 hours; 10–16 hours and 16–22 hours. (B) β3-tubulin amplification control. (C) Cartoon of dsx transcripts. The positions of the primers used for the amplification of the male-specific dsx m products are marked (arrows). (D, F, H) Anti-DSXM and (E, G, I) anti-VASA immunofluorescence in (D, E) female, (F, G) male, and (H, I) _dsx_- embryos (_Df(3R)dsx_15/_In(3R)dsx_23). Images in each row are from the same confocal section of an embryo. Because we used GFP to distinguish homozygous _dsx_- from balancer and heterozygous control embryos, embryos in H, I were not sex sorted. However, we never observed DSXM staining in _dsx_- embryos, 50% of which were male. Secondary antibody for anti-DSXM was biotin-coupled goat anti-rat with tyramide signal amplification, and secondary for anti-VASA was Cy5 goat anti-rabbit. Scale bar = 10 μm.

Figure 2

Figure 2

Gonad development. Foregut and hindgut (dark-gray); anterior and posterior midgut (light-gray); somatic gonadal precursors (purple); germ cells (yellow); male-specific somatic gonadal precursors (orange); somatic gonadal precursors of the hub (red); and a previously undescribed group of cells (green) are indicated. (A) Stage 12 embryo. (B) Higher magnification view of the outlined area in A. (C) Stage 13 embryo. (D) Higher magnification view of the outlined area in C. (E) Stage 17 male embryo. (F) Higher magnification view of the outlined area in E. Cartoons of embryonic gonad development were adapted from Hartenstein [52]. During gonad formation (A, B) the germ cells and the associated somatic gonad precursors co-migrate towards abdominal segment 5, where they begin to coalesce to form the gonads [53, 54]. During and after gonad coalescence (C, D), the germ cells are intermingled with the somatic gonad cells [41]. Prior to gonad coalescence male-specific somatic gonadal precursor cells, specified in parasegment 13 in both males and females, are located posterior and ventral to non-sex-specific somatic gonad precursor cells. During stage 13 these cells move toward the gonad in both sexes, but only in males do these cells join the posterior of the coalescing gonad. In females these cell die, making the surviving ones "male-specific" [21]. The anterior somatic gonad also becomes sexually dimorphic early during gonad development (E, F). The hub, a cluster of somatic cells required for germline stem cell maintenance in the adult testis, forms anteriorly in the male embryonic gonad [29]. Later in stage 17, we saw another group of cells envelop the embryonic testis (E, F). The identity of these cells is uncertain, but they may be the precursors of the testis sheath [17].

Figure 3

Figure 3

DSXM and TJ expression in the male somatic gonad. (A-H) Stage 13 male embryo immunofluorescence using: (A) anti-DSXM, (B) anti-TJ, and (C) anti-VASA. (D) Merged images A-C. (E-H) Magnified view of the gonad in A-D. Somatic nuclei expressing DSXM but not TJ are indicated (arrows). (I-P) Stage 15 male embryo immunofluorescence using: (I) anti-DSXM, (J) anti-TJ, and (K) anti-VASA. (L) Merged images I-K. (M-P) Magnified view of the gonad in I-L. The scale bars = 50 μm in A-D; I-L and 10 μm in E-H; M-P. Anterior is to the left. Secondary antibodies were: (A, I) biotin-coupled goat anti-rat with TSA, (B, J) Alexa 488 goat anti-guinea pig, and (C, K) Cy5 goat anti-rabbit.

Figure 4

Figure 4

DSXM but not TJ is expressed in male-specific somatic gonadal precursors. (A-D) Stage 13 testis immunofluorescence using (A) anti-DSXM, (B) anti-EYA, and (C) anti-VASA. (D) Merged images A-C. A DSXM and EYA positive cluster of cell nuclei is located posterior and ventral to the other cells of the somatic gonad (arrows). (E-H) Stage 13 male testis immunofluorescence using (E) anti-DSXM, (F) anti-SOX100B and (G) anti-VASA. (H) Merged images E-G. (I-L) Stage 15 testis immunofluorescence using (I) anti-DSXM, (J) anti-SOX100B, and (K) anti-VASA antibody. (L) Merged images I-K. (M-P) Stage 15 testis immunofluorescence using (M) anti-TJ, (N) anti-SOX100B and (O) anti-VASA. (P) Merged images M-O. The scale bars = 10 μm. Anterior is to the left. Secondary antibodies were: (A, E, I) biotin-coupled goat anti-rat and TSA, (B) Alexa 488 goat anti-mouse, (C) Cy5 goat anti-rabbit), (F, J) Alexa 488 goat anti-rabbit, (G, K, O) Alexa 647 goat anti-chicken, (M) Alexa 488 goat anti-guinea pig, (N) biotin-coupled goat anti-rabbit and TSA.

Figure 5

Figure 5

DSXM is not expressed in all somatic testis cells. (A-D) stage 17 testis immunofluorescence using (A) anti-DSXM, (B) anti-SOX100B, and (C) anti-VASA. (D) Merged images A-C. (E-H) stage 17 testis immunofluorescence (imaged in a focal plane with the hub) using (E) anti-DSXM, (F) anti-FAS III, and (G) anti-VASA. (H) Merged images E-G. The scale bars = 10 μm. Secondary antibodies were: (A, E) biotin-coupled goat anti-rat and TSA, (B) Alexa 488 goat anti-rabbit, (C) Alexa 647 goat anti-chicken, (F) Alexa 647 goat anti-mouse, and (G) Cy5 goat anti-rabbit.

Figure 6

Figure 6

DSXM in hub cells. (A-D) Larval (3rd instar) testis immunofluorescence using (A) anti-DSXM, (B) anti-TJ, and (C) anti-FAS III. (D) Merged images A-C. (E-F) Adult testis immunofluorescence using (E) anti-DSXM, (F) anti-TJ, and (G) anti-FAS III. (H) Merged images E-G. The hub is outlined (white dashes). The scale bars = 10 μm. Anterior is up and out of the plane toward the viewer (the outlined hub is most anterior). Secondary antibodies were: (A, E) biotin-coupled goat anti-rat and TSA, (B, F) Alexa 488 goat anti-guinea pig, and (C, G) Alexa 647 goat anti-mouse.

Figure 7

Figure 7

DSXM expression during spermatogenesis. (A-D) Larval testis immunofluorescence using (A) anti-DSXM, (B) anti-TJ, and (C) anti-EYA. (D) Merged images A-C. (E-H) Higher magnification view of A-D. (I-L) Adult apical testis immunofluorescence using (I) anti-DSXM, (J) anti-TJ and (K) anti-EYA. (L) Merged images I-K. (M-P) Higher magnification view of I-L. The scale bar = 50 μm in A-D, 20 μm in E-L, and 5 μm in M-P. Anterior is up. Secondary antibodies were: (A, I) biotin-coupled goat anti-rat and TSA, (B, J) Alexa 488 goat anti-guinea pig, (C, K) Alexa 647 goat anti-mouse.

Figure 8

Figure 8

DSXM expression does not require germ cells nor EYA or TJ. (A-D) Agametic testis of stage 15 males from gs(1)N26 mothers. Immunofluorescence using (A) anti-DSXM, (B) anti-TJ, and (C) anti-VASA. (D) Merged images A-C. (E-H) Isolated somatic gonadal precursors and germ cells formed in homozygous eya mutants. Immunofluorescence using (E) anti-DSXM, (F) anti-TJ, and (G) anti-VASA. (H) Merged images E-G. (I-L) Expression in tj eo mutant testis revealed with immunofluorescence using (I) anti-DSXM, (J) anti-TJ, and (K) anti-VASA. (L) Merged images I-K. Expression of the non-functional truncated TJ protein in tj eo mutant embryos is diffuse within the somatic gonadal precursors (J). The scale bars = 10 μm. Anterior is to the left. Secondary antibodies were: (A, E, I) biotin-coupled goat anti-rat and TSA, (B) Alexa 647 goat anti-guinea pig, (C) Alexa 488 goat anti-rabbit, (F, J) Alexa 488 goat anti-guinea pig, and (G, K) Cy5 goat anti-rabbit.

Figure 9

Figure 9

DSXM regulation. DSXM is regulated by the intersection of the sex-determination alternative pre-mRNA splicing hierarchy and spatial/temporal regulation (A). Positive (green arrows) and negative interactions (red) are indicated. See text for details. Cellular markers are differentially expressed in somatic cell types of the male gonad during embryonic and adult stages (B). Cell cartoons and expression indicators are color-coded. Germ cells (yellow), somatic gonad precursors and cyst-cells (purple), male-specific somatic gonadal precursors (orange), hub cells (red), and a novel layer of embryonic testis cells (green) are shown.

Similar articles

Cited by

References

    1. Marin I, Baker BS. The evolutionary dynamics of sex determination. Science. 1998;281:1990–1994. doi: 10.1126/science.281.5385.1990. - DOI - PubMed
    1. Burtis KC. The regulation of sex determination and sexually dimorphic differentiation in Drosophila. Curr Opin Cell Biol. 1993;5:1006–1014. doi: 10.1016/0955-0674(93)90085-5. - DOI - PubMed
    1. Cline TW, Meyer BJ. Vive la difference: males vs females in flies vs worms. Annu Rev Genet. 1996;30:637–702. doi: 10.1146/annurev.genet.30.1.637. - DOI - PubMed
    1. Zarkower D. Invertebrates may not be so different after all. Novartis Found Symp. 2002;244:115–135. - PubMed
    1. Burtis KC, Baker BS. Drosophila doublesex gene controls somatic sexual differentiation by producing alternatively spliced mRNAs encoding related sex-specific polypeptides. Cell. 1989;56:997–1010. doi: 10.1016/0092-8674(89)90633-8. - DOI - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources