Research article. Summary The sex determination master switch, Sex-lethal (Sxl), Introduction

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1 6101 A positive role for Patched in Hedgehog signaling revealed by the intracellular trafficking of Sex-lethal, the Drosophila sex determination master switch Jamila I. Horabin 1, *, Sabrina Walthall 1, Cynthia Vied 1,2 and Michelle Moses 1 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 *Author for correspondence ( jhorabin@uab.edu) Accepted 11 September 2003 Development 130, The Company of Biologists Ltd doi: /dev Summary The sex determination master switch, Sex-lethal (Sxl), controls sexual development as a splicing and translational regulator. Hedgehog (Hh) is a secreted protein that specifies cell fate during development. We show that Sxl is in a complex that contains all of the known Hh cytoplasmic components, including Cubitus interruptus (Ci) the only known target of Hh signaling. Hh promotes the entry of Sxl into the nucleus in the wing disc. In the anterior compartment, the Hh receptor Patched (Ptc) is required for this effect, revealing Ptc as a positive effector of Hh. Some of the downstream components of the Hh signaling pathway also alter the rate of Sxl nuclear entry. Mutations in Suppressor of Fused or Fused with altered ability to anchor Ci are also impaired in anchoring Sxl in the cytoplasm. The levels, and consequently, the ability of Sxl to translationally repress downstream targets in the sex determination pathway, can also be adversely affected by mutations in Hh signaling genes. Conversely, overexpression of Sxl in the domain that Hh patterns negatively affects wing patterning. These data suggest that the Hh pathway impacts on the sex determination process and vice versa and that the pathway may serve more functions than the regulation of Ci. Key words: Hedgehog, Sex-lethal, Patched, Signaling, Drosophila melanogaster Introduction Sex-lethal (Sxl) is the sex determination master switch in Drosophila. Early in development, it is activated in females where the X chromosome to autosome (X:A) ratio is 1. In males, where the X:A ratio is 0.5, Sxl remains off. These two modes of Sxl expression are maintained through the rest of the life cycle (Sanchez and Nothiger, 1983; Cline, 1984). Sxl controls sexual development as a splicing and translational regulator. It regulates dosage compensation (Lucchesi, 1978), turning off the system by splicing regulation and translational repression (Bashaw and Baker, 1997; Kelley et al., 1997). In regulating somatic sexual differentiation, Sxl promotes female differentiation by controlling the female-specific splicing of the transformer gene (Boggs et al., 1987; McKeown et al., 1987). Hedgehog (Hh) is a secreted protein that patterns and specifies cell fate during the development of several different tissues. Binding to its receptor Patched (Ptc), relieves Smoothened (Smo) from the inhibition of Ptc and enables Smo to activate the transcription factor Cubitus interruptus (Ci). In cells that have not been exposed to Hh, the predominant form of Ci is the 75 kda isoform (Aza-Blanc et al., 1997), which acts as a transcriptional repressor. Phosphorylation of Ci by protein kinase A (PKA) promotes processing of Ci to the 75 kda isoform. This processing is dependent on Supernumerary limbs (Slmb) protein (Jiang and Struhl, 1998). Hh reduces the phosphorylation of Ci and generates the full-length 155 kda Ci isoform (Chen et al., 1999) that activates transcription of wingless (wg), decapentaplegic (dpp) and ptc (reviewed by Ingham, 1998). This regulated processing and nuclear import of Ci is achieved through a complex of Ci with the cytoplasmic members of the Hh signaling pathway, the known members of which are Costal 2 [Cos2; also known as Costa (Cos)], Fused (Fu) and Suppressor of Fused [Su(fu)] (Robbins et al., 1997; Sisson et al., 1997; Stegman et al., 2000). Fu appears to be a serine threonine kinase (Therond et al., 1996; Nybakken et al., 2002); Cos2 has sequence similarity to the motor domain of kinesin (Sisson et al., 1997), while Su(fu) shows no homology to any known protein (Preat et al., 1993). The complex is tethered to microtubules by Cos2, and on Hh signaling is released from microtubules resulting in full-length Ci in the nucleus (Robbins et al., 1997; Sisson et al., 1997). Previously, we showed that the stem cells and early cystoblasts of Drosophila ovaries use the Hh signaling pathway to regulate the degradation and trafficking of Sxl into the nucleus (Vied and Horabin, 2001; Vied et al., 2003). As Ci is not expressed in germ cells, the suggestion was raised that Sxl may replace Ci in the Hh cytoplasmic complex. We show here that in somatic cells, Sxl is in a complex that contains all of the known Hh cytoplasmic components, including Ci. Hh promotes the nuclear entry of Sxl in the wing disc and, surprisingly, in the anterior compartment Ptc appears to be a

2 6102 Development 130 (24) positive effector of this Hh promoted nuclear entry. Our data show a cross talk between the sex determination and the Hh signaling pathways suggestive of a common functional origin of some of the Hh cytoplasmic components. Materials and methods Fly stocks and generation of disc clones OreR was used as the wild-type control fly stock. The genes used in this study are described in FlyBase ( Unless otherwise stated, flies were raised at 25 C. For embryo analysis the following strains were used: cos2 w1 /CyO, wg-lacz, smo 2 /CyO, wg-lacz, DCO H2 /CyO wg-lacz, ptc 16 /CyO, wg-lacz, Su(fu) LP, slmb 2 /TM3, ftz-lacz, hh AC /TM3, ftz-lacz, and P[fu + ]/CyO; fu 94 /FM6, which are described in FlyBase. hh ts2 discs were from a hh ts2 /TM3 stock shifted to 29 C for 12 or 36 hours. The tissue was gently rocked at 29 C in leptomycin B (LMB) before fixing, as described below. Clones were generated by the FLP-FRT mitotic recombination system (Chou and Perrimon, 1992). Recombination was induced in 1st to 2nd instar larvae with a 1 hour heat shock at 37 C to induce hs-flp. Clones were generated in the genotypes: ptc S2 (hsp70-flp; FRT42D ptc S2 /FRT42D arm-lacz); ptc 16 (hsp70-flp; FRT42D ptc 16 en-lacz/frt42d); PKA (hsp70-flp; FRT40A DCO H2 /FRT40A arm-lacz); smo (hsp70-flp; FRT40A smo 2 /FRT40A arm-lacz); cos2 (hsp70-flp;frtg13 cos2 w1 /FRTG13 arm-lacz); slmb (hsp70-flp; FRT82E slmb 2 /FRT82E hs-gfp); ptc x ; hh MRT (hsp70- flp/+; FRT42D ptc x /FRT42D; hh MRT /+). Recovery after heat shock was at room temperature for the ptc clones in the hh MRT background. For fu homozygous discs (fu 94 and fu mh63 ), fu ; P[fu +, y + ]/CyO males were mated to fu /FM6 females. For expression of the PtcD584N protein, UAS-PtcD584N flies were mated to apterousgal4/cyo flies. For expression of Sxl protein, UAS-Sxl flies were mated to dppgal4/cyo flies. Immunofluorescence, immunoprecipitations and western blots Antibodies used have been described by Vied and Horabin (Vied and Horabin, 2001) except for anti-su(fu) (1:100; D. Robbins), anti-msl- 2 (1:250; B. Baker), anti-ci (1:3; R. Holmgren), anti-en (1:1; Developmental Studies Hybridoma Bank) and anti-β-gal (1:1000; Promega). Biotinylated donkey anti-rat antibody followed by Cy 3-conjugated streptavidin (Jackson ImmunoResearch Laboratories, Inc), goat anti-rabbit Alexa 488, goat anti-mouse Alexa 594, goat anti-mouse Alexa 647 (Molecular Probes) and propidium iodide or Hoechst were the fluorescent probes. Embryos and discs were mounted in aquapolymount (Polysciences, Inc.). Stainings, immunoprecipitations from 50 µl of 0- to 8-hour OreR embryo extracts, and western blots were performed as described by Vied and Horabin (Vied and Horabin, 2001). For the Ci immunoprecipitations from Su(fu) LP embryos, 100 µl of whole embryo extracts were used, as Ci levels are reduced in this background. Each antibody used in the immunoprecipitation was crosslinked to the protein A beads to minimize the signal from the heavy and light chains in the western blot. LMB incubations and staining of discs Two to 3 days after heat shock, female wandering larvae were bisected and the head portion turned inside out in 1 PBS. The tissue was washed in DS2 medium (Mediatech, Inc.) and then incubated, while gently rocking, for 3 hours in ng/ml LMB in DS2 medium. After 3 hours the tissue was fixed in 4% paraformaldehyde and stained as previously described for ovaries (Vied and Horabin, 2001). Su(fu) LP control discs were treated identically except that LMB was left out of the incubation. Mounting of forelegs and wings Females in which the forelegs had transformed sex combs were desiccated in ethanol, the legs were removed and mounted in Permount (Fisher). They were photographed with an Olympus AX70 using the Zeiss Axiovision program. Results Sxl is in the same complex as Ci Sxl co-immunoprecipitates with Cos2 and Fu in the female germline. As Ci is not expressed in germ cells, it is probable that a different Hh cytoplasmic complex might exist in germ cells. In somatic cells, Sxl is expressed in all female cells while Ci is expressed in only a subset. To test whether the Hh pathway differentiates between the two proteins in somatic cells, Sxl was immunoprecipitated from embryonic extracts and the immunoprecipitates probed for the various Hh cytoplasmic components. The immunoprecipitates showed that Cos2, Fu and Ci are complexed with Sxl (Fig. 1A). As a negative control, we also probed for the presence of Bicaudal D protein (BicD) as it has sequence similarity to motor proteins such as Cos2. BicD did not co-immunoprecipitate with Sxl. The specificity of this association of Sxl with the Hh pathway components was verified using antibodies to either Ci or Su(fu), and testing the immunoprecipitates for the presence of Sxl. Both co-immunoprecipitated with Sxl (Fig. 1B,D). The Ci immunoprecipitate was also tested for another Hh cytoplasmic component, Fu, which was present as expected. These interactions are maintained in a Su(fu) LP background (protein null allele). An IP of Ci from Su(fu) LP embryos brought down Sxl, as well as Fu and Cos2 (the former two shown in Fig. 1C). Taken together, these data suggest that cells that express Ci and Sxl have both proteins in the same complex with the known cytoplasmic components of the Hh signaling pathway. 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.

3 Positive role for Ptc in Hh signaling 6103 Fig. 2. Cos2 is necessary for the maintenance of Sxl expression. Embryos from cos2 w1 /CyO, wglacz stock triple stained for β-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). cos2 affects Sxl To determine whether the Hh pathway affects Sxl function in somatic cells, we analyzed the expression of Sxl in embryos mutant for each of the known members in the pathway. In all cases, a strong hypomorphic or null allele (see Materials and methods) was used to maximize detection. Eliminating PKA, smo, ptc, fu, Su(fu) or Hh had no clear effect on the levels or intracellular localization of Sxl, in contrast to cos2. cos2 embryos consistently showed lower levels of Sxl protein than their wild-type, heterozygous siblings. The effect was more readily detected in older embryos, most probably reflecting the decay of maternally deposited cos2 mrna. Female cos2 mutant embryos were positive for both Sxl and Male specific lethal 2 [Msl-2; the immediate target of Sxl in the dosage compensation pathway (Bashaw and Baker, 1997; Kelley et al., 1997)] (Fig. 2). The level of Msl-2 was not as high as in males, consistent with the observation that they still express some, albeit low, levels of Sxl protein. It would appear that, as seen in the germline, Cos2 is necessary for maintaining the expression of Sxl. intensity is a function of increased levels of nuclear Sxl, as incubating discs in higher concentrations of LMB abolishes the compartmental difference and the signal becomes uniformly bright (Fig. 3G). The difference in the Sxl signal may also be due to its epitope becoming more accessible when the protein is in the nucleus. In either case, the LMB data indicate that Sxl shuttles between the cytoplasm and nucleus but the rate of shuttling is not homogeneous, and occurs more rapidly in one half of the disc. Staining wild-type female discs for both Sxl and full-length Ci confirmed that it is the posterior compartment with more nuclear Sxl; Ci is found only in the anterior compartment (Fig. An anterior-posterior gradient of Sxl nuclear entry rate Wing discs fixed and stained for Sxl show a diffuse signal of a predominantly cytoplasmic protein, unlike embryos where the protein appears predominantly nuclear. Only low levels of the protein are seen in the nucleus, a curious distribution for a splicing regulator (Fig. 3A-C and inset). This is particularly true for the wing pouch area and less so at the edges of the disc. Incubating the discs in DS2 medium and leptomycin B (LMB) (Nishi et al., 1994) increases the detectable nuclear Sxl. [LMB functions similarly in Drosophila cells as in mammalian and yeast cells, binding to exportin 1/CRM1 preventing the nuclear export of proteins with leucine-rich nuclear export signal (NES) sequences (Fukada et al., 1997; Fasken et al., 2000).] The nuclear Sxl, however, is not uniform. A more concentrated or punctate signal is seen in the posterior compartment where the levels of the protein appear higher (Fig. 3D and inset; see also Fig. 4N where the focal plane excludes nuclei and changes signal intensity). This difference in signal 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.

4 6104 Development 130 (24) 3D-F). Sxl is also more strongly nuclear in the cells at the anterior-posterior (AP) boundary as the signals for the two proteins overlap (Fig. 3F). Staining discs from wild-type flies that contain a hh-lacz transgene (which expresses β-gal in the posterior compartment) for both Sxl and β-gal also showed that, while the two markers are not completely coincident, most of the cells with the higher levels of nuclear Sxl are in the posterior compartment (not shown). Wing discs uncover differential Hh signaling The differential rate of nuclear entry of Sxl along the wing disc AP axis is suggestive of an involvement of the Hh patterning system. In the wing disc, Hh is produced in the posterior compartment cells, but activates Ci in a graded manner in the cells of the anterior compartment where both its receptor, Ptc, and Ci are expressed. Ptc is thought to limit the range of the effects of Hh, binding to and internalizing the ligand. As a consequence, only a row eight to ten cells deep at the AP border expresses full-length Ci (Chen and Struhl, 1996). Fulllength Ci activates the expression of the Hh target genes, ptc, dpp and wg. In the row of three to four cells right at the AP border where activation of the pathway is at its highest, engrailed (en) expression is also activated by Hh (Strigini and Cohen, 1997). To test the involvement of Hh in Sxl nuclear entry, female wing discs of the hh MRT background were stained for both fulllength Ci and Sxl. hh MRT is a cold sensitive, gain-of-function allele that causes overgrowth and ectopic venation in the anterior distal portions of the wing disc (Tabata and Kornberg, 1994). Full-length Ci was used to report the location of ectopic Hh. As can be seen in Fig. 4A-F, the higher levels of nuclear Sxl (brighter signal) coincide with the cells that have more Ci. Under these conditions and unlike wild-type discs, the cells in the anterior compartment frequently show more nuclear Sxl Fig. 4. Hh signaling promotes Sxl nuclear entry but does not require Smo. All discs were treated with ng/ml LMB in DS2 medium for 3 hours before fixing. (A-C) hh MRT disc stained for full-length Ci (A), Sxl (B), and merged image of A and B (C). Fulllength 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) hh ts2 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) smo 2 clones in wing disc pouch at room temperature stained for Sxl (green), β-gal (red) and (L) merged image of J and K. smo 2 clones in wing disc at 18 C stained for (M) β-gal (red), (N) Sxl (green) and (O) with Hoechst (blue). Mutant clones marked by loss of β-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.

5 Positive role for Ptc in Hh signaling 6105 than the cells in the posterior compartment. Removal of Hh, using hh ts2 animals shifted to the non-permissive temperature of 29 C for 12 or 36 hours, reduces the levels of nuclear Sxl in both the anterior and posterior compartments of the wing pouch (12 hours shown in Fig. 4G-I). These results indicate that, as seen in ovaries (Vied et al., 2003), Hh promotes the entry of Sxl into the nucleus in somatic cells and led us to test whether the other components of the pathway also have a role. Surprisingly, given the effect of Hh on Sxl, removal of Smo had no effect (in either the anterior or posterior compartment). This was observed at room temperature (Fig. 4J-L) as well as at 18 C, the latter a harsher condition for the strong loss-of-function smo 2 allele (Fig. 4M-Q). Control stains indicated that Ci was affected under these conditions and flies with the reported wing defects were recovered (not shown). Conversely, loss of Ptc was found to reduce the rate of Sxl nuclear entry in the anterior compartment (Fig. 5A-F). This was observed with two ptc loss-of-function alleles, ptc S2 and ptc 16. Close examination shows the two ptc alleles are slightly different in their effects. In the case of ptc S2, which binds to and internalizes Hh but fails to relieve Smo inhibition, nuclear Sxl is absent between ptc negative clones that are close together (arrowhead in Fig. 5E). By contrast, in the case of ptc 16, a protein null allele, nuclear Sxl can be found in the cells between ptc clones (arrowhead in Fig. 5B). These observations are consistent with the idea that (a) a Hh gradient affects the rate of Sxl nuclear entry, (b) Ptc S2 protein can sequester Hh from cells in more anterior regions of the wing pouch, an effect the protein null allele does not have, and (c) Ptc acts positively in the Sxl response to the presence of Hh. This disparate outcome of Ptc binding to Hh to facilitate entry of Sxl into the nucleus versus the release of Ptc inhibition of Smo and the subsequent activation of Ci, could be further demonstrated using a UAS transgenic line that expresses the D584N variant form of Ptc. This change is also the cause of the ptc S2 mutation (Martin et al., 2001; Strutt et al., 2001). When expressed in a wild-type wing disc using an apterous GAL4 driver, which expresses GAL4 in only the dorsal compartment of the wing disc, Ptc D584N enlarges the dorsal region of the wing disc since it acts as a dominant negative, producing full-length Ci in the anterior compartment. Staining these wing discs shows that full-length Ci is stabilized in the dorsal but not ventral half of the disc (Fig. 5G). However, this mutant form of Ptc has the opposite effect on Sxl nuclear entry. Nuclear Sxl is not detected in the anterior compartment of dorsal cells while the ventral anterior compartment remains unaffected (Fig. 5H; note brighter Sxl signal in the ventral Fig. 5. Ptc is required for Hh-promoted Sxl nuclear entry. (A-F) Sxl nuclear levels are reduced in ptc clones: (A-C) ptc 16 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) ptc S2 clones marked by lack of β-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) ptc 16 clones in a hh MRT 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 fulllength 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 ptc 16 -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. compartment in contrast to the relatively normal signal for fulllength Ci in the entire wing pouch area and the increased Ci signal in dorsal regions). This result is readily explained by the fact that Ptc D584 is able to sequester Hh but is unable to promote Sxl nuclear entry. Ptc D584 in the dorsal posterior compartment restricts the diffusion of Hh into the anterior compartment, so reducing the nuclear levels of Sxl. Ci, however, responds to the state of Smo.

6 6106 Development 130 (24) In the presence of the dominant negative Ptc D584N protein, Smo is released from the inhibition of the wild-type Ptc in a Hh-independent manner. Ci is thus activated, resulting in the increase in growth. Sxl, in contrast, is dependent on Ptc activation by Hh to accelerate its nuclear entry. To further establish the requirement of Ptc in transmitting the Hh signal to bring about Sxl nuclear entry, clones mutant for Ptc were made in hh MRT /+ wing discs. If Ptc acts downstream of Hh, its absence should negate the effect of the ectopic Hh on Sxl. This was indeed the case. ptc clones (ptc S2 and ptc 16 ) showed the same effect as in discs that have no ectopic Hh (ptc 16 shown in Fig. 5J-L). Full-length Ci and nuclear Sxl were high around the ptc clones because of the ectopic Hh from the MRT allele, but within the ptc clones the levels of nuclear Sxl are reduced. Ci levels were also lower within the clones (induction of en in the clones is antagonistic to high levels of Ci expression, as seen in cells right at the AP border). Altogether, these data indicate that the presence of the Hh ligand can be signaled in a more complex manner than previously thought. Ptc can communicate the presence of Hh Fig. 6. Effect of Fu and Su(fu) on Sxl and the expression of Su(fu) in wild-type wing discs. (A-C) fu mh63 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) fu 94 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 fu 94 and fu mh63 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. to the Hh cytoplasmic complex and the two Hh targets differentially respond to the states of the membrane components. Some of the Hh pathway cytoplasmic components affect Sxl in the wing disc As the Hh membrane components show a differential effect on Sxl and Ci, we examined the cytoplasmic components for their effect on the nuclear entry rate of Sxl. In wing discs of females with clones mutant for PKA H2 or slmb 2, Sxl showed no differences from wild type, suggesting these two genes have little if any effect (data not shown). Unlike in embryos, we found that mutation of cos2 had no obvious effects on the levels or nuclear entry rate of Sxl in either compartment. Homozygous fu and Su(fu) mutant discs were examined. Discs mutant for the kinase specific allele, fu mh63, were identified by the broadening of the expression domain of fulllength Ci protein (Ohlmeyer and Kalderon, 1998). Therefore loss of the Fu kinase had no effect on Sxl (Fig. 6A-C). However, the strong type I fu allele, fu 94, which is truncated for the Fu regulatory domain (Lefers et al., 2001) did have an effect (Fig. 6D,E). In contrast to the wild type AP gradient of nuclear Sxl seen in the anterior compartment of heterozygous discs, Sxl was uniformly nuclear across what we presume are the homozygous mutant discs. In some cases, a very weak anterior gradient of nuclear Sxl could be detected (Fig. 6D). These results suggest that the Fu regulatory domain is normally inhibitory to Sxl nuclear entry in the anterior compartment. The same result was observed for Su(fu) LP (Fig. 6F). Detecting nuclear Sxl in the fu 94 and Su(fu) LP backgrounds required shorter incubation and/or lower concentrations of LMB than in wild-type discs. In fu 94 discs, 10 ng/ml LMB for 3 hours was sufficient to begin to detect nuclear Sxl, but nuclear Sxl is not observed in wild-type discs under these conditions (wild-type discs require ng/ml LMB for 3 hours). In the Su(fu) LP background, 5 ng/ml LMB for 2 hours was sufficient. This low requirement for LMB in the Su(fu) LP background did not reflect constitutively nuclear Sxl in this background, as Su(fu) LP discs that had not been treated with LMB showed predominantly cytoplasmic Sxl. Since Su(fu) has such a strong influence on the nuclear entry rate of Sxl, we determined the distribution of the protein in wild-type discs. An uneven A versus P distribution of Su(fu) would explain the compartmental difference in Sxl localization, and signaling by Hh would account for the nuclear Sxl in the cells at the AP border and its gradation into the anterior compartment. Fig. 6H shows that Su(fu) is uniformly expressed across the wing disc. Its levels, therefore, cannot explain the difference in the rate of nuclear to

7 Positive role for Ptc in Hh signaling 6107 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 PKA H2 (C), cos2 w1 (D) and ptc S2 (E) clones. Arrows indicate slightly thickened bristles in legs with mutant clones, particularly cos2 w1 (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. cytoplasmic shuttling of Sxl along the anterior to posterior axis. Thus, while the effects on Sxl in the anterior compartment show a dependence on the known Hh signaling components, it is not clear what promotes the rapid nuclear entry of Sxl in the posterior compartment. Ptc clones have no effect (and Ptc RNA and protein are not detected in the posterior compartment), but removal of Hh does reduce the nuclear entry rate of Sxl (Fig. 4G-I). Mutations in Hh signaling genes cause weak sex transformations Forelegs of females with clones mutant for the various Hh pathway genes were examined to determine whether the Hh signaling pathway had consequences on the sex determination process. With the exception of slmb which had no effect, almost all of the Hh pathway components, including smo, induced very weak sex transformations represented by a slight thickening of the bristles on the foreleg (Fig. 7A-E). In the case of cos2 clones, occasional females had forelegs with significantly thickened bristles, almost as thick as those in the male sex comb. Overexpressing Sxl at the AP border inhibits Hh signaling To determine whether Sxl could affect the Hh signaling process, the protein was ectopically expressed in the bulk of the region where Hh patterns using a dpp-gal4 driver. No male flies were recovered from this mating, presumably because of the upset in dosage compensation. Eclosed females were found to have varying degrees of leg defects ranging from wild type to a shortening of the more distal regions. All wings showed a reduction in the area between wing veins L3 and L4 (Fig. 7G), the part of the wing that Hh patterns directly, while the rest of the wing appeared normal. Additionally, the hairs at the wing margin between the L3 and L4 vein show the characteristics of the anterior rather than posterior compartment (Fig. 7G,H). Both these phenotypes are indicative of a reduction in the Hh signal, suggesting that an excess of Sxl compromises the ability of the Hh cytoplasmic complex to activate Ci. Discussion Previous work on the germline suggested that the Hh signaling pathway affected the intracellular trafficking of the sex determination master switch, Sxl. We have analyzed the cross talk between these two developmental pathways in tissues where both Hh targets can be present in the same cell. While analysis of embryos only uncovered an effect of Cos2 on Sxl, analysis of wing discs allowed us to uncover several specific effects. At least three new functional aspects of the Hh pathway are suggested: (1) More than one target protein can exist in the Hh cytoplasmic complex Immunoprecipitation experiments using extracts from embryos indicate that Sex-lethal and the known Hh signaling target Ci are in the same complex. The two proteins can coimmunoprecipitate each other as well as other known members of the Hh cytoplasmic complex. Even when Su(fu), the cytoplasmic component that most strongly anchors Sxl in the cytoplasm, is removed we find that Sxl can be coimmunoprecipitate with both Ci and Fu. As a whole, these results suggest that at least some proportion of the two Hh target proteins are in a common complex within the cell. Additionally, the wing defects produced when Sxl is overexpressed in the Hh signaling region suggest that their relative concentrations are important for their normal functioning. (2) The Hh targets can be affected differentially The presence of two targets within the Hh cytoplasmic complex, raises the question of how they can be differentially affected. The data show that the various members of the Hh pathway do not affect Sx1 and Ci similarly. Smo appears to be dispensable for the transmission of the Hh signal in promoting Sx1 nuclear entry, while Smo is critical for the activation of Ci. Conversely, while Ptc is essential for the effect of Hh on Sxl, it is dispensable for the activation of Ci. The Fu kinase (fu mh63 background) also appears to have no role in Hh signaling with respect to Sxl, while it is critical for the activation of Ci. By

8 6108 Development 130 (24) contrast, both Su(fu) and the Fu regulatory domain act similarly on Sxl and Ci, serving to anchor them in the cytoplasm. Taken together, these data suggest that the presence of Hh can be relayed to the cytoplasmic components differentially and, while our data do not address the point, suggest how different outcomes might be achieved. Ptc has been proposed to be a transmembrane transporter protein that functions catalytically in the inhibition of Smo (Taipale et al., 2002) via a diffusible small molecule (Chen et al., 2002; Frank- Kamenetsky et al., 2002). The stimulation of Sxl nuclear entry by the binding of Hh to Ptc might also involve a change in the internal cell milieu, but in this case the Hh cytoplasmic complex may be affected independently, not requiring a change in the activity of Smo or the Fu kinase. (3) Ptc can signal the presence of the Hh ligand in a positive manner Several experiments indicate that Hh bound to Ptc enhances the nuclear entry of Sxl. That Smo has no role in transmitting the Hh signal is most clearly demonstrated by expressing the PtcD584 protein in both the anterior and posterior compartments of the dorsal half of the wing disc. PtcD584 acts as a dominant negative and so activates Ci in the anterior compartment, but it fails to enhance the levels of nuclear Sxl in the anterior because it sequesters Hh in the posterior compartment. The double mutant condition of ptc clones in a hh MRT background clearly places Ptc downstream of Hh, while showing Ptc can act positively in transmitting the Hh signal. A positive role for Ptc, but in this case in conjunction with Smo, in promoting cell proliferation during head development has recently been reported (Shyamala and Bhat, 2002). In this situation, however, Hh acts negatively on both Ptc and Smo in their activation of the Activin type I receptor, suggesting an even greater variance from the canonical Hh signaling process. While the effects on Sxl in the anterior compartment show a dependence on the known Hh signaling components, it is not clear what promotes the rapid nuclear entry of Sxl in the posterior compartment. Su(fu) is expressed uniformly across the disc so it does not appear to be responsible for the AP differences, and ptc clones have no effect (and Ptc RNA and protein are not detected in the posterior compartment). Removal of Hh, however, reduces the nuclear entry rate of Sxl in both compartments. In this regard, the parallel between Hh pathway activation and Sxl nuclear entry in the posterior compartment is worth noting. Ramirez-Weber et al. (Ramirez- Weber et al., 2000) demonstrated that Fu is also activated in the posterior compartment in a Hh-dependent manner, even though Ptc is not present. It is not clear what mediates between Hh and Fu. A changing Hh cytoplasmic complex? The data also suggest that the Hh cytoplasmic complex may have slightly different compositions in different tissues and/or at developmental stages. In the female germline (Vied and Horabin, 2001) and in embryos, the absence of Cos2 leads to a severe reduction in Sxl levels. However, in wing discs when mutant clones are made using the same cos2 allele, there is no effect on Sxl. We suggest that between the third instar larval stage and eclosion, the composition of the Hh cytoplasmic complex may change again to make Sxl more sensitive to Cos2. This would explain why removal of Cos2 can produce sex transformations of the foreleg even though mutant clones in wing discs (and also leg discs; unpublished observations) show no alterations in Sxl levels. A similar argument might apply to the weak sex transformations of forelegs produced by PKA clones. Alternatively, PKA may have a very weak effect but our assay on wing discs is not sufficiently sensitive to allow detection of small effects; PKA was found to have a modest effect on Sxl nuclear entry in the germline (Vied and Horabin, 2001). Sxl is sufficiently small (38-40 kda) to freely diffuse into the nucleus, or the protein may enter the nucleus as a complex with splicing components. This may account for the limited sex transformations caused by removal of Hh pathway components. Removal of several of the Hh pathway components, such as smo, gives the same weak sex transformation phenotype, even though smo had no effect on Sxl nuclear entry. Additionally, there is no correlation between a positive and a negative Hh signaling component and whether there is a resulting phenotype. Changing the dynamics of the activation state of the Hh cytoplasmic complex may perturb the normal functioning of Sxl, since Sxl appears to be in the same complex as Ci. For example, if the Hh pathway is fully activated because of a mutant condition, the relative amounts of Sxl in the cytoplasm versus nucleus at any given time, may be different from the wild-type condition. Perturbing the usual cytoplasmic-nuclear balance could compromise the various processes that Sxl protein regulates. Sxl acts both positively and negatively on its own expression through splicing (Bell et al., 1991) and translation (Yanowitz et al., 1999) control and, additionally, regulates the downstream sex differentiation targets. The latter could also be responsible for the weak sex transformations seen, in view of the recent demonstration that doublesex affects the AP organizer and sex-specific growth in the genital disc (reviewed by Christiansen et al., 2002). A cytoplasmic to nuclear shuttling complex? With the exception of Cos2, which can produce relatively substantial effects on Sxl levels in embryos as well as sex transformations in the foreleg, the effects of removal of any of the other Hh pathway components are generally not large. The strong effects of Cos2 on Sxl could be because it affects the stability of Sxl. However, Sxl depends on an autoregulatory splicing feedback loop for its maintenance making the protein susceptible to a variety of regulatory breakdowns. If Cos2 altered the nuclear entry of Sxl, for example, its removal could compromise the female-specific splicing of Sxl transcripts by reducing the amounts of nuclear Sxl. Splicing of Sxl transcripts would progressively fall into the male mode to eventually result in a loss of Sxl protein. Cos2 and Fu have been reported to shuttle into and out of the nucleus, and their rate of nuclear entry is not dependent on the Hh signal (Méthot and Basler, 2000). That Ci and Sxl are complexed with the same Hh pathway cytoplasmic components, and share and yet have unique intracellular trafficking responses to mutations in the pathway, makes it tempting to speculate that the Hh cytoplasmic components may have had a functional origin related to intracellular trafficking that preceded the two proteins. Whether this reflects a more expanded role in regulated nuclear entry remains to be determined.

9 Positive role for Ptc in Hh signaling 6109 We are indebted to Dr M. Yoshida for the gift of LMB without which these experiments could not be conducted. Our special thanks also go to Dr D. Kalderon for the numerous stocks he sent for making disc clones, Dr R. Holmgren for both the fu 94 stock and the anti-ci full-length monoclonal. We thank Dr D. Robbins for the anti-su(fu) and anti-fu polyclonal antibodies, Dr T. Kornberg for the anti-ci polyclonal, Drs K. Ho and M. Scott for the anti-cos2 antibodies, Dr B. Baker for anti-msl-2. Special thanks to Dr D. Bopp for the UAS- Sxl line. The anti-engrailed antibody developed by Dr Corey Goodman was obtained from the Developmental Studies Hybridoma Bank maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA We would also like to thank Albert Tousson and Shawn Williams from the UAB imaging facility. This work was supported by a grant from NIH to J.I.H. References Aza-Blanc, P., Ramirez-Weber, F. A., Laget, M. 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