QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Vied, C. Articles by Horabin, J. I. Search for Related Content PubMed PubMed Citation Articles by Vied, C. Articles by Horabin, J. I.

The sex determination master switch, Sex-lethal, responds to Hedgehog signaling in the Drosophila germline

Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, 1918 University Boulevard, Birmingham, Alabama 35294, USA

View larger version (53K): [in a new window]   Fig. 1. Wild-type germaria stained for Sxl, -Spectrin and Orb proteins. All germaria are positioned with the anterior to the left and posterior to the right. (A) An EM section through the germarium, modified from Bopp et al. (Bopp et al., 1993). 2c, two-cell cluster; 16c, 16-cell cluster; cb, cystoblast; fc, follicle cells; s, stem cells; tf, terminal filament. (B) Germarium stained for Sxl (red). Note the Sxl distribution from strongly cytoplasmic in early germ cells to low cytoplasmic levels with nuclear foci in later germ cells (arrow). (C) Germarium stained for -Spectrin (red) and DNA (green). The spectrosome (unbranched fusome) is indicated by arrows and the fusome by arrowheads. (D) Germarium stained for Orb (red) and DNA (green). It localizes to the future oocyte in region 2 (arrow).

View larger version (100K): [in a new window]   Fig. 2. Distribution of Sxl, Fu, Cos2, Ptc and -tubulin proteins in the germarium. (A) Sxl in a wild-type germarium. (B) Fu in same germarium as in A. (C) Merged image of A,B, showing cytoplasmic co-localization (yellow) in early region 1 and the downregulation (shown by line) in the same cells of mid-region 1. Arrow indicates a nuclear focus of Sxl. (D) Wild-type germarium stained with anti-Ptc antibody. (E) Wild-type germarium stained for Cos2. (F) Germarium of a wild-type ovary stained for -tubulin. Cytoplasmic -tub23 is only detected in early germ cells, where Sxl is strongly cytoplasmic. The bright spots of -tubulin mark the centrosomes (arrow, Wilson et al., 1997). (G-I) Germarium from female with a fu ts allele stained for Sxl (green, G) and Fu (red, H). (I) Merged image of G,H showing almost complete overlap (yellow). All ovarioles contain tumorous chambers (n>50). (J-M) Tumorous chambers from Sxlf4 ovaries (all Sxlf4 egg chambers are tumorous and have many small germ cells with cytoplasmic Sxl): (J) double stained with -Spectrin (red) and DNA (green; arrow indicates the spectrosome); (K,L) double stained for Sxl (red, K) and Fu (green, L); (M) merged image of K,L. There is high overlap in distribution of the two proteins (yellow). All panels are laser confocal images.

View larger version (77K): [in a new window]   Fig. 3. Loss of Hedgehog produces ovaries with predominantly cytoplasmic Sxl. (A-F) ovaries from hhts/hhAC females kept at 29°C for 8-9 days. (A) Germarium showing mostly cytoplasmic Sxl. 98.9% of hhts/hhAC ovarioles contain multi chambered cysts (n>50). (B) Germarium from a hhts/hhAC ovary stained with anti-Ptc antibody. The level of Ptc is high and the normal decrease in expression is absent (82%). (C) Germarium stained for -Spectrin. Branching of the fusome (arrow) occurs closer to the anterior end of the germarium than in wild type (Fig. 1C). (D) Germarium stained for Orb. (E) Germarium in D stained for Orb (red) and DNA (green). Orb accumulation shown with arrowhead. (F) Later stage egg chamber that lacks Sxl. This chamber is adjacent to the germarium in A; note brighter staining cells on left of panel. (G) Egg chamber from hhts/hhAC females with a constitutive allele of Sxl (SxlM4) stained for Sxl. Sxl expression is maintained in the later stage germ cells and remains cytoplasmic throughout. 87% of ovarioles contained multi chambered cysts (n>50). The nuclei in the chambers (F,G) are polyploid. (H) RT-PCR of RNA from ovaries of hhts/hhAC females, females with a transgene of the hedgehog cDNA under a heat-shock promoter (hs-hhM11), Sxlf4 and wild-type females. Significant levels of male Sxl RNA are detected in Sxlf4, hhts2/hhAC and hs-hhM11 ovaries.

View larger version (39K): [in a new window]   Fig. 4. Overexpression of Hh leads to fewer cells with cytoplasmic Sxl. All hs-hhM11 females heat shocked for 3 days. Germaria positioned with anterior (stem cells) to the left and are laser confocal images. (A) Germarium stained with anti-Sxl antibody. Only one or two cells show significant cytoplasmic Sxl (inset). Note that the germ cells show an overall decrease in Sxl (arrow) and signal is detected mostly from the surrounding follicle cells. 85% of germaria show a decrease of early germ cells with cytoplasmic Sxl. (B) Germarium stained with anti-Fu antibody. Fewer cells at the anterior end show significant Fu (85.5% germaria show decreased cells with Fu). Note that late germ cells show a significant decrease in Fu (arrow). (C) Germarium stained for Orb (green) and DNA (red). Orb expression begins in early to mid region 1 (arrow), as opposed to wild type, which begins in region 2 (see Fig. 1D). (D) Germarium stained for Ptc (red). (E) Ptc and nucleic acid (green). Ptc expression in the early germ cells (bar in D) is very reduced relative to wild type (82% of germaria show reduced levels of Ptc in the early germ cells). Somatic cells and later chambers are normal. (F) Germarium stained for -Spectrin. No spectrosome is visible in the early germ cells (arrow) and the fusome is also not detectable (87% of germaria have no visible fusome). Where reported, the percent for phenotype frequency is from a count of greater than 50 ovarioles.

View larger version (60K): [in a new window]   Fig. 5. Sxl expression in PKA, cos2 and Su(fu) mutant ovaries. (A-F) Germline clones are marked by the absence of ß-galactosidase (see Materials and Methods). (A-C) Germarium with cos2 clones stained for ß-galactosidase (A) and Sxl (B). (C) Merged image of (A,B). One of the early germ cells is positive for ß-galactosidase (cos2+), while the other germ cells are negative for ß-galactosidase (cos2–). Expression of Sxl is normal in the cos2+ cell and very reduced in the cos2– cells. All germarial clones counted appeared similar. (D-F) Germline clone of PKA null allele stained for ß-galactosidase (D) and Sxl (E). (F) Merged image of D,E. Two of the early germ cells are PKA–, and the accumulation of Sxl in the nucleus is stronger (arrow in F). (G) Su(fu)LP ovary stained for Sxl. Two chambers appear necrotic with degenerating follicle and germ cells. (H) Ovariole from doubly homozygous Sxlf4 and Su(fu)LP female stained for Sxl. Germ cells appear to be detaching from the follicle cell layer and degenerating.

View larger version (36K): [in a new window]   Fig. 6. Sxl Co-immunoprecipitates with Fu, Cos2 and -tubulin. (A) Sxl was immunoprecipitated from wild-type ovaries, Sxlf4 ovaries and Sxl null (SxlfP7BO) males. 10% of the post-immunoprecipitation lysate and all of the immunoprecipitates were analyzed by Western blot with anti-Fu, anti-Cos2 or anti--tubulin antibodies. Fu, Cos2 and -tubulin co-immunoprecipitates with Sxl in Sxlf4 and wild-type ovaries. The percent of protein that immunoprecipitated with Sxl in wild type and Sxlf4 ovaries was, respectively, 13.6 and 15.1% for Cos2, 8.8 and 10.6% for Fu, and 10.6 and 10.2% for -tubulin. The same blots were probed with Fu, Cos2, -tubulin and Sxl antibodies but the Cos2 signal in the Sxlf4 immunoprecipitates is a repeat immunoprecipitate for a cleaner signal. Immunoprecipitates were performed at least twice. (B) BicD was immunoprecipitated from wild-type ovaries and probed with Fu and -tubulin antibodies. -tubulin was immunoprecipitated from Sxlf4 ovaries and probed with anti-Sxl antibodies.

View larger version (42K): [in a new window]   Fig. 7. Proteasome inhibitors and colchicine counteract Hh overexpression on Sxl. (A-C) Germarium from wild-type ovaries dissected and incubated at 25°C for 3 hours in the presence of DMSO (A), MG132 (proteosomal inhibitor) (B), or colchicine (C), stained for Sxl. (B) The level of Sxl in early germ cells has greatly increased. Inset is a lower laser intensity scan of early germ cells. All germaria scanned showed a range from wild type levels to greatly increased levels of Sxl. (C) Colchicine produces the same effect as proteasome inhibitors, with greatly increased levels of Sxl in early germ cells. (D-F) Germaria from hs-hhM11 females dissected after heat shock and incubation at 25°C for 3 hours in the presence of DMSO (D), MG132 (E) or colchicine (F), stained for Sxl. Fewer early germ cells with strong cytoplasmic expression of Sxl can be seen in hs-hhM11 DMSO-treated ovaries, as in Fig. 4A, whereas MG132 treatment (E) increases the levels and numbers of early germ cells with cytoplasmic Sxl (also in follicle cells). Colchicine also increases the levels and numbers of early germ cells with cytoplasmic Sxl (F). Images in D-F were scanned at the same settings. 69% of hs-hhM11 germaria treated with MG132 show an increase of early germ cells with cytoplasmic Sxl (E).

View larger version (19K): [in a new window]   Fig. 8. Model for the regulation of Sxl in early germ cells by the Hedgehog pathway. In the absence of Hh, Sxl is retained in the cytoplasm bound to microtubules in a complex with Fu, -tubulin and Cos2. The Hh signal is proposed to release the complex from microtubules, but in germ cells may be transduced through a homolog of Ptc or Smo (see Discussion). Some of the Sxl moves into the nucleus (PKA reduces this) while the majority of Sxl, -tubulin and Fu, are degraded.