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First published online 5 October 2005
doi: 10.1242/dev.02054

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Splitting the Hedgehog signal: sex and patterning in Drosophila

Jamila I. Horabin^*,

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

View larger version (60K): [in a new window]   Fig. 1. HhN does not influence Sxl nuclear entry. A wing disc stained for Ci (red; A,C,D), Sxl (green; B,C,E) and ß-gal (blue; D,E). ß-Gal marks the actin>GAL4 clones expressing HhN (Hh with only the palmitoyl modification). The disc was treated with 100 ng/ml LMB for 3 hours. Full-length Ci is detected anterior to the AP boundary (e.g. arrow in A). In the ß-gal-positive region where HhN levels are maximal (arrowheads) there is a reduction of full-length Ci, but no effect on Sxl nuclear entry (B,E; brighter, more punctate signal) (Horabin et al., 2003). Insets in A,B enlarge the areas indicated by arrowheads. (C) Overlay of Sxl and Ci; (D) overlay of Ci and ß-gal; (E) overlay of Sxl and ß-gal. Scale bar: 40 µm. Anterior towards the left, ventral is towards the top. Confocal images throughout.

View larger version (155K): [in a new window]   Fig. 2. Sxl allows Hh with only cholesterol modification to stabilize Ci. (A-F) Discs expressing Hh with only the cholesterol modification, C84S-Hh, with dpp-GAL4 driver stained for Ci (red) and Sxl (green). dpp is expressed in a broad band in the anterior compartment, a few cells away from the AP boundary – see dpp-lacZ staining in Fig. 6A. Both male and female discs were treated with 50 ng/ml LMB for 3 hours, and scanned at about the same laser intensity. (A-C) Male disc; (D-F) female disc. In males, as previously reported (Lee et al., 2001), the levels of Ci fall (arrowhead in A for example). For females, the amount of LMB is about one-third of that needed in wild-type discs to detect nuclear, full-length Ci in region around dpp expression zone (e.g. arrowhead in D); Sxl is also nuclear (arrows in E, F). Insets in A,C enlarge the area indicated by arrowheads; inset in E enlarges the area indicated by an arrow. (G-L) Discs expressing Sxl and C84S-Hh with dpp-GAL4 driver stained for Ci (red) and En (green). (G-I) Male disc; (J-L) female disc. Ci signal is punctate (e.g. G) and En-positive cells extend towards the anterior compartment (arrow in H), which was seen in all male discs (n>12) of this genotype (non-Tubby larvae). Inset in G enlarges the area indicated by an arrowhead. Male discs were treated with 50 ng/ml LMB for 3 hours; female discs were treated with half the amount of LMB. The size of the nuclei and scale bars in I,L both illustrate that the female disc is larger than the male. Punctate signal is indicative of nuclear Ci and there is a relative increase in size of the anterior compartment (bracket in L). Scale bars: in C, 20 µm for A-C; in F, 20 µm for D-F; in I, 20 µm for G-I; in L, 20 µm for J-L. For all discs, anterior is towards the left, ventral is towards the top. Confocal images throughout.

View larger version (120K): [in a new window]   Fig. 3. Sxl enhances Hh signaling. Discs were treated with 150 ng/ml LMB for 3 hours and antibody incubations were carried out in the same dish (sexed using Sxl signal). (A-F) Wild-type male (A-C) and female (D-F) discs stained for Ci (red) and Sxl (green). The Ci signal in males is relatively diffuse in contrast to females where it is brighter and more discrete, indicative of nuclear localization. (G-I) Male discs expressing Sxl in dorsal compartment by ap-GAL4. The level of Sxl protein is low (arrowhead in H) compared with endogenous signal of females; Ci levels are increased in the dorsal half (arrowhead in G) relative to the ventral half (arrow in G) of the disc. (J-L) Female disc expressing Sxl in dorsal compartment by ap-GAL4. The Ci signal shows an enhancement even in females (arrowhead marks the dorsoventral boundary at the AP boundary). Bracket in I,L indicates expansion of the dorsal half of the disc. Scale bars: 20 µm. Anterior is towards the left, ventral is towards the top. Confocal images throughout.

View larger version (19K): [in a new window]   Fig. 4. Sxl increases Ci levels. Western blot of adult male (M) and female (F) flies stained for Ci and ß-tubulin (ß-tub). Left pair shows relative loading difference of protein between the sexes (females less) when the Ci signal is approximately the same; right pair has the ß-tubulin levels approximately the same between the sexes, females have more Ci. Lanes taken from the same blot; for each protein, the time of exposure for male versus female lane is the same.

View larger version (25K): [in a new window]   Fig. 5. ptc expression is enhanced by Sxl. Discs stained for ß-gal which reports ptc transcription from a ptc-lacZ transgene. (A) Wild type, (B-E) discs expressing Sxl in dorsal compartment by ap-GAL4 driver; (B,D) male, (C,E) female. In both sexes, ptc expression is more intense in the dorsal compartment (arrowhead marks dorsoventral boundary at the AP boundary for all panels). Arrow in B indicates broadening of ptc-lacZ zone and beginning of wing pouch expansion in dorsal half; in D, the fold from overgrowth is larger and the ptc-lacZ forms a U (arrow). The width of ptc-lacZ expression narrows in females (C), more so in E (which also has a larger dorsal half). Normal width of ptc-lacZ in E indicated by arrows. Scale bars: 20 µm. Anterior is towards the left, ventral is towards the top. Confocal images throughout.

View larger version (104K): [in a new window]   Fig. 6. Dpp signaling is enhanced by Sxl. (A-C) Wild type. (D-O) Male (D-F,M-O) and female (G-L) discs expressing Sxl in the dorsal compartment by ap-GAL4 driver, stained for ß-gal (green; reports Dpp through a dpp-lacZ transgene) and Sal (red; induced by Dpp). (C,F,I,L,O) Merged images. In all panels, the arrowheads mark the dorsoventral compartment boundary. The zone of Dpp expression widens in the dorsal compartment in males (D and M), less so in females (G and J). Below the dorsoventral boundary of the wing pouch is a widened zone of Dpp expression and corresponding widening/intensity of Sal expression compared with the ventral half. Arrow in I indicates slightly distorted dorsal half; (J-O) dorsal wing pouch halves have an extra fold from the increase in size (arrows). Arrow in M indicates the wing pouch–notum boundary and arrow in N indicates the edge of fold in the distended wing pouch. Scale bars: 20 µm in A-L; 40 µm in M-O. Anterior is towards the left, ventral is towards the top. Confocal images throughout.

View larger version (90K): [in a new window]   Fig. 7. Dpp signaling target pMad is enhanced by Sxl. (A-C) Wild type; (D-F) male and female (G-I) discs expressing Sxl in the dorsal compartment stained for ß-gal (red; reports dpp expression though dpp-lacZ) and pMad (green; reports activation of Dpp signaling). (C,F,I) Merged images. Below the dorsoventral boundary of the wing pouch (arrowheads) is a widened zone of Dpp expression (D-I) and corresponding widening of pMad compared with the ventral half. Scale bars: in C, 20 µm for A-C; in F, 20 µm for D-F; in I, 20 µm for G-I. Anterior is towards the left, ventral is towards the top. Confocal images throughout.

View larger version (104K): [in a new window]   Fig. 8. Sxl-null clones in females show reduced Hh signaling. (A-C) Small Sxl-null clone near AP border (arrowhead) identified by loss of GFP signal (A), shows a reduction in Ci levels (B); (C) merged image. (D-G) Larger Sxl-null clone in notum region of wing disc (arrowhead) identified by loss of GFP (D) and Sxl signal (E), showing a reduction in Ci levels (F); (G) merge of D-F. Sxl and Ci are primarily cytoplasmic as no LMB was used. (H-K) Sxl-null clones (arrowheads), identified by loss of GFP (H), stained for ß-gal (I) and Sal (J); (K) merged image. ß-gal, which reflects dpp transcription, and Sal levels show a slight reduction. Arrow indicates slightly higher ß-gal in non-clone region. The few nuclei with slightly brighter ß-gal signal near bottom clone are wild type for Sxl (small arrowhead in (I)). Scale bars: in G, 20 µm for D-G; in K, 20 µm. Anterior towards the left, ventral is towards the top. Confocal images throughout.

View larger version (104K): [in a new window]   Fig. 9. Sxl raises mitotic rate. Wild-type (A-C); male (D-F) and female (G-I) discs expressing ectopic Sxl in the dorsal compartment by ap-GAL4 driver stained for BrdU incorporation (red) and Ci (green). (E-H) Expansion of Ci in the dorsal compartment identifies discs expressing ectopic Sxl (arrowhead). (D,G) The density of BrdU positive nuclei increases in the dorsal compartment in both males and females (arrow). Scale bars: in C, 20 µm for A-C; in F, 20 µm for D-F; in I, 40 µm for G-I. Wild type (J-L); male (M-O) and female (P-R) discs expressing ectopic Sxl in the dorsal compartment by ap-GAL4 driver stained for PH3 (green) and ß-gal (red; reports dpp expression through dpp-lacZ). Arrowheads in J-R indicate dorsoventral boundary at AP boundary. (M,P) A larger number of PH3 positive cells are found below the dorsoventral boundary relative to J. (N,Q) dpp expression changes in the dorsal half of the disc, which is a little larger than the ventral. Scale bars: in L, 20 µm for J-L; in O, 40 µm for M-O; in R, 40 µm for P-R. Anterior is towards the left, ventral is towards the top. Confocal images throughout.

View larger version (128K): [in a new window]   Fig. 10. Sxl induces disc growth which can be non autonomous. (A) Wild-type disc; (B-C) cross sections of wild-type discs (B) and male (C) and female (D) discs expressing Sxl in the dorsal compartment stained for Ci (red) and Wg (green). Broken line in A indicates the position of the cross section. Arrowheads mark the Wg stripe at the D/V boundary of the wing pouch and at the dorsal end. Arrows indicate the folds in the dorsal wing pouch caused by disc overgrowth. (C) In the male disc, the fold near the dorsoventral border is over the ventral half of the disc and an additional fold is over the dorsal half. (D) The female disc cross-section is at a slight angle to show the folds and thickening in the dorsal half. Ventral is towards the left. Confocal image stacks taken with a 40x objective (female with zoom at 0.9). (E,F) Male (E) and female (F) discs expressing Sxl in only the dpp zone of anterior compartment stained for Ci (red) and Wg (green). Arrows indicate more severe curve of Wg stripe caused by bulge from growth. Compare with the gentle curve of Wg stripe in the wing pouch of the wild-type disc in A, which normally is relatively flat. Arrowheads in E,F indicate folds in wing pouch in posterior compartment caused by increased growth.

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