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First published online November 3, 2003
doi: 10.1242/10.1242/dev.00865

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A positive role for Patched in Hedgehog signaling revealed by the intracellular trafficking of Sex-lethal, the Drosophila sex determination master switch

¹ Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, 1918 University Boulevard, Birmingham, Alabama 35294, USA
² Department of Biological Sciences, Columbia University, New York, New York 10027, USA

View larger version (44K): [in a new window]   Fig. 1. Sxl is in the same complex as Ci. Extracts from wild-type 0-8 hour embryos treated with either anti-Sxl (A), anti-Ci rat monoclonal (B,C) or anti-Su(fu) antibodies (D), and the immunoprecipitates (IPs) tested for specific proteins by western blot analysis. (A) Sxl immunoprecipitates probed for Cos2, full-length Ci, Fu and Sxl. BicD was tested as a negative control for specificity of the immunoprecipitates. (B,C) Ci immunoprecipitates from OreR and Su(fu)LP embryos, respectively, probed for Sxl and Fu. (Note the same result as in B was obtained using an anti-Ci amino-terminal polyclonal.) (D) Wild-type extract treated with Su(fu) antibodies probed for Sxl. Extract (E) shows the migration position of the relevant protein in an extract from OreR females on the same blot.

View larger version (35K): [in a new window]   Fig. 2. Cos2 is necessary for the maintenance of Sxl expression. Embryos from cos2w1/CyO, wg-lacZ stock triple stained for ß-gal (A), Sxl (B), and Msl-2 (C). Lack of wg-lacZ stripes identifies the homozygous mutant embryo which has very low levels of Sxl (compare to the wild-type female at the top) and expresses significant levels of Msl-2 but not equivalent to a wild-type male (lower left embryo).

View larger version (133K): [in a new window]   Fig. 3. Sxl shows graded nuclear entry along the AP axis of wild-type wing discs. (A-C) Wing pouch of disc stained for Sxl (A) and with propidium iodide (B). (C) Merged image of A and B. Sxl is mostly cytoplasmic and the nuclear stain is primarily unaltered in C in most of the cells. Insets are enlargements of region from the top right of image. Scale bar: 20 µm. (D-F) Disc treated with 100 ng/ml LMB for 3 hours stained for Sxl (D) and full-length Ci (E). (F) Merged image of D and E. Arrowheads show that besides the posterior compartment, there is more nuclear Sxl (brighter signal) at the AP boundary where the signals for Sxl and Ci overlap. Inset in D is of a region at the AP boundary. There are no distinct nuclei visible as the protein is not entirely nuclear but punctate regions of more intense staining. Scale bar: 40 µm. (G-I) Disc treated with high levels of LMB (250 ng/ml for 3 hours) stained for Sxl (G) and with propidium iodide (H). (I) Merged image of G and H. Insets are enlargements of the region in the middle of the disc at the AP boundary. Although there is more Sxl in the nuclei (note the overlap in the two signals and change in color, in contrast to C), there still is some protein in the cytoplasm; green signal around orange nuclei and inset. Bar is 20 µm. All are confocal images; anterior is to the left, ventral at the top.

View larger version (106K): [in a new window]   Fig. 4. Hh signaling promotes Sxl nuclear entry but does not require Smo. All discs were treated with 75-200 ng/ml LMB in DS2 medium for 3 hours before fixing. (A-C) hhMRT disc stained for full-length Ci (A), Sxl (B), and merged image of A and B (C). Full-length Ci identifies areas in the disc with ectopic Hh expression. Sxl levels are highest where Ci is highest (e.g. arrow) and where Ci is lower (arrowhead) nuclear Sxl levels are also lower. Under these conditions the posterior compartment shows lower levels of nuclear Sxl (signal less bright) relative to the anterior compartment. (D-F) Enlargement of region that spans across high and low nuclear Sxl levels as well as full-length Ci, near the arrow in A-C. In F the region of higher Sxl signal has brighter green spots, which most probably reflect more nuclear Sxl than Ci. (G-I) hhts2 disc shifted to 29°C for 12 hours stained for Ci (G), Sxl (H) and merged image (I). The arrowhead in G marks the remnants of full-length Ci at the AP boundary and in H where the nuclear Sxl in the wing pouch is absent and the signal appears very diffuse. After 36 hours at 29°C, the low levels of nuclear Sxl in (H) appear reduced (not shown). (J-L) smo2 clones in wing disc pouch at room temperature stained for Sxl (green), ß-gal (red) and (L) merged image of J and K. smo2 clones in wing disc at 18°C stained for (M) ß-gal (red), (N) Sxl (green) and (O) with Hoechst (blue). Mutant clones marked by loss of ß-gal signal. There is no correspondence between the clones that mark the loss of Smo with the nuclear levels of Sxl (N). Clones (M), Sxl signal (N) and merged image (P). In N,O and Q where the optical section does not go through nuclei the Sxl signal is present but weaker as there is protein in the cytoplasm (marked by arrowhead). When the Sxl signal overlaps with the nuclear stain, a change in color to a lighter shade of blue is observed (Q). Line in J-Q marks the AP border. All are confocal images; anterior is to the left, ventral at the top. Scale bar: 40 µm in A-C; 20 µm in J-Q.

View larger version (147K): [in a new window]   Fig. 5. Ptc is required for Hh-promoted Sxl nuclear entry. (A-F) Sxl nuclear levels are reduced in ptc– clones: (A-C) ptc16 clones as marked by En expression (red, A) show decreased nuclear Sxl (green, B); merged image (C). Arrowhead marks where Sxl is nuclear between ptc negative clones. Scale bar: 20 µm. (D-F) ptcS2 clones marked by lack of ß-gal expression (red, D) show decreased nuclear Sxl (green, panel E); merged image (F). Arrowhead marks where nuclear Sxl is absent between ptc negative clones with this amorphic allele that is capable of binding to Hh. (G-I) PtcD584N expressed in dorsal compartment of wing disc using the apterousGAL4 driver. Full-length Ci (red, G) and Sxl (green, H) and merged image (I). Note the weak signal of Sxl in the anterior dorsal compartment in both the wing pouch as well as notum because of decreased nuclear levels of the protein. Arrowhead points to the AP and dorsal/ventral boundary meeting point. The arrow indicates the overgrowth of disc in the anterior compartment as a result of the dominant negative effect of the PtcD584N protein. Scale bar: 40 µm. (J-L) ptc16 clones in a hhMRT background stained for Ci (J), Sxl (K). (L) Merged image of J and K. Image is of the anterior region distant from the AP boundary of an overgrown disc. Large arrowhead marks region of lower Ci levels more posterior to region that has ectopic Hh, which is reported by the presence of full-length Ci (arrow). Note the brighter punctate signal for Sxl marking higher nuclear levels where there is an increase in levels of full-length Ci. ptc– clones have lowered levels of Ci (some of the clones, which are round, are marked by small arrowheads) because of the induction of en which inhibits Ci transcription. As seen for ptc16-only clones, the levels of nuclear Sxl within the clones is lowered. Scale bar: 20 µm. All are confocal images; anterior is to the left, ventral at the top.

View larger version (126K): [in a new window]   Fig. 6. Effect of Fu and Su(fu) on Sxl and the expression of Su(fu) in wild-type wing discs. (A-C) fumH63 disc stained for full-length Ci (red, A), Sxl (green, B) and merged image (C). The Sxl distribution is unaffected, but Ci shows the reported widened band at the AP border. (D,E) fu94 discs stained for Sxl. Both show predominantly nuclear Sxl across the disc. The anterior compartment of the disc in D has a remnant of the nuclear gradient; lower levels of nuclear Sxl marked with an arrowhead. Line in D marks the approximate position of the AP border. (F) Su(fu)LP disc stained for Sxl also shows the protein as nuclear across the entire disc. The disc was treated with only 5 ng/ml LMB for 2 hours. The fu94 and fumH63 discs were treated with 50 ng/ml and 100 ng/ml LMB for 3 hours, respectively. (G-I). Su(fu) appears cytoplasmic and uniform across the disc. Ci protein (red, G) Su(fu) protein (green, H), merged image (I). As a control, Su(fu)LP discs were simultaneously stained and these did not show any signal. Scale bar: 40 µm. All are confocal images; anterior is to the left, ventral at the top.

View larger version (87K): [in a new window]   Fig. 7. Sex transformations caused by mutating Hh pathway components and inhibition of Hh signaling by overexpression of Sxl. (A-E) Forelegs of wild-type male (A) and female (B) and of females with PKAH2 (C), cos2w1 (D) and ptcS2 (E) clones. Arrows indicate slightly thickened bristles in legs with mutant clones, particularly cos2w1 (females normally lack thickened bristles). (F-H) Wing from wild-type female (F) and female expressing Sxl in dpp expression zone (G,H). Note difference in spacing between wing veins L3 and L4 (arrows in G) as well as the hairs at the wing margin (arrowheads), which are characteristic of the anterior compartment.