Introduction

Top Abstract Introduction Results Discussion Materials & Methods References

Secreted proteins of the Hedgehog family act as key inducing agents in both vertebrates and invertebrates, often influencing position-dependent cell fate over a large field of cells (Ingham 1995). In some cases, including the Drosophila ventral embryonic ectoderm and larval imaginal discs, Hedgehog (Hh) acts locally to induce the synthesis of other secreted molecules, of the Wnt or transforming growth factor- (TGF-) families, that relay the influence of Hh to more distant cells (Lawrence and Struhl 1996). In other cases, Hh may act directly over several cell diameters, inducing concentration-dependent responses (Heemskerk and DiNardo 1994; Roelink et al. 1995; Ericson et al. 1996; Struhl et al. 1997). The direct range of action of Hh is limited by post-translational modifications to Hh itself and by binding to the Hh-receptor, Patched (Ptc), the product of a Hh-inducible gene (Chen and Struhl 1996; Porter et al. 1996). Genetic and biochemical interactions among Hh, Ptc, and Smoothened (Smo) suggest that binding of Hh to Ptc leads to activation of Smo, a predicted seven-transmembrane domain protein presumed to interact with a G protein (Alcedo et al. 1996; Chen and Struhl 1996; Marigo et al. 1996; Stone et al. 1996; van den Heuvel and Ingham 1996). Although protein kinases such as Fused (Fu) and protein kinase A (PKA) are genetically implicated in Hh signaling, there is no evidence at present that their activities are altered by Hh signaling (Préat et al. 1990; Forbes et al. 1993; Jiang and Struhl 1995; Kalderon 1995; Lepage et al. 1995; Li et al. 1995; Pan and Rubin 1995; Strutt et al. 1995). Hh can signal normally in imaginal disc cells if PKA is manipulated to be unresponsive to cAMP (Jiang and Struhl 1995; Li et al. 1995) or if animals lack activity of both Fu and another gene product, Suppressor of Fused (Préat 1992; Thérond et al. 1996a).

The zinc finger protein, Cubitus interruptus (Ci) has DNA-binding activity that is essential to transduction of a Hh signal, suggesting that it acts as a key transcriptional activator (Forbes et al. 1993; Alexandre et al. 1996; Von Ohlen et al. 1997). Ci staining with a monoclonal antibody, 2A1, that recognizes an epitope in the carboxy-terminal half of Ci, increases in response to Hh, loss of Ptc activity, or loss of PKA activity in anterior imaginal disc cells, without any change in ci mRNA levels (Johnson et al. 1995; Slusarski et al. 1995; Domínguez et al. 1996; Aza-Blanc et al. 1997). It has been shown recently that increased 2A1 antibody staining due to Hh signaling corresponds to a change in Ci processing rather than total Ci protein concentration, as assumed previously (Aza-Blanc et al. 1997). Thus, full-length Ci (Ci-155) can be cleaved to an amino-terminal fragment (Ci-75) that is not recognized by the "carboxy-terminal" antibody 2A1, henceforth denoted as Ci-CT antibody (Aza-Blanc et al. 1997). Hh inhibits the cleavage of Ci-155 and consequently leads to increased Ci-CT antibody staining while total Ci protein concentration, assayed by an "amino-terminal" (Ci-NT) antibody remains constant (Aza-Blanc et al. 1997). Because increased Ci-CT antibody staining generally accompanies induction of Hh target genes, whether elicited by Hh, PKA inhibition, or overexpression of a ci cDNA, it is thought that Ci-CT antibody staining, corresponding to an increased concentration of full-length Ci protein, is a critical intermediate step in Hh target gene induction (Johnson et al. 1995; Slusarski et al. 1995; Alexandre et al. 1996; Domínguez et al. 1996; Hepker et al. 1997). We have investigated Hh signaling in Drosophila embryos and find that PKA inhibition and Hh signaling increase Ci protein concentration detected by Ci-CT antibody and induce target gene [wingless (wg) and patched (ptc)] expression, as observed previously in imaginal discs. We also demonstrate novel signaling activities by which both PKA hyperactivity and Hh stimulate wg and ptc expression by a mechanism that requires the activities of Smo and Ci but does not involve any detectable changes in Ci protein concentration.

	Results

Top Abstract Introduction Results Discussion Materials & Methods References

PKA inhibition induces Hh target genes in embryos

In Drosophila embryos Hh is made in segmentally repeated stripes of engrailed (en)-expressing cells and is required to maintain expression of the target genes wg and ptc in stripes of immediately adjacent cells. wg is expressed anterior to each en stripe, whereas ptc is expressed on either side of en, forming two stripes per segment (Hidalgo and Ingham 1990) (Fig. 1a,b). ptc expression may serve to restrict diffusion of Hh (Chen and Struhl 1996) and moderate Hh signaling, whereas localized expression of wg is crucial initially to maintain en and hh expression and subsequently for ectodermal patterning within each parasegment (Peifer and Bejsovic 1992). Ubiquitous Hh expression can induce transcription of ptc in all cells that do not express en and that consequently express ci (Eaton and Kornberg 1990), whereas wg expression can be induced by Hh in a subset of those cells (Ingham 1993). Because the PKA catalytic subunit DC0 is required for normal oogenesis and maternally supplied in sufficient quantities for embryogenesis (Lane and Kalderon 1993, 1994) we used a mutant regulatory subunit, R*, expressed from a GAL4-responsive transgene (UAS-R*) to reduce PKA activity in embryos (Li et al. 1995). R* binds cAMP poorly and therefore maintains catalytic subunit as inactive holoenzyme (R*₂DC0₂) at physiological cAMP concentrations. Expression of R* ubiquitously in the ectoderm ("ubi-R* = E22C-GAL4 + UAS-R*) or solely in non-en cells (using ptc-GAL4) caused an anterior expansion of wg expression (Fig. 1c) and expression of ptc in all non-en cells (Fig. 1d), without any accompanying change in en or hh expression (not shown). The alterations in wg, ptc, and ventral cuticle patterns (Fig. 5a,c, below) because of PKA inhibition in ubi-R* embryos resembled those induced by low-level ubiquitous expression of Hh but were less pronounced than those elicited by high levels of Hh or strong ptc mutations (Bejsovic and Wieschaus 1993; Ingham 1993; Tabata and Kornberg 1994). PKA inhibition restricted to en-expressing cells (using en-GAL4) had no effect on wg or ptc expression (not shown). Hence, inhibition of PKA can mimic the inductive effects of Hh in Hh-responsive cells of the embryo, just as in imaginal discs.

View larger version (121K): [in this window] [in a new window]   Figure 1.     Increased or decreased PKA activity results in activation of Hh target genes wg and ptc. (a,c,e,g) Ventral view of stage 11-12 embryos hybridized with wg RNA probe. Anterior is to the left. (b,d,f,h) Lateral view of embryos hybridized with ptc RNA probe. Anterior is left and dorsal is up. (a,b) Wild-type embryos showing the normal one-cell wide stripes of wg RNA and two thin stripes of ptc RNA in each segment of the ectoderm (clearest at the outer edge of the embryo in b). (c,d) ubi-R* embryos in which PKA is inhibited uniformly. wg RNA expression expands 1-4 cells toward the anterior beginning in late stage 11 embryos, and ptc RNA expression is expanded to all non-en-expressing cells. (e,f) ubi-mC* embryos in which PKA activity is uniformly elevated. The expansions of wg RNA expression are more robust than for ubi-R*, reaching a width of 6-8 cells and are visible by early stage 11. ptc RNA expression expands to all non-en cells. (g,h) ubi-R*/mC* embryos expressing both PKA transgenes have almost normal expression of wg and ptc, demonstrating the opposing activities of the two transgenes.

View larger version (119K): [in this window] [in a new window]   Figure 5.     Regulation of PKA by cAMP is not essential for Hh signaling. Ventral cuticles (fifth and sixth abdominal segments) of wild-type larvae (a) and larvae that expressed mC* from an actin-5C promoter but were maternally and zygotically null for DC0 (b) showed the same trapezoidal pattern, number, and spacing of rows, indicating normal Hh signaling. In ubi-R* larvae (PKA inhibition) (c) and ubi-mC* larvae (PKA hyperactivity) (d) the wide posterior rows of each segment are replaced by narrower and often incomplete rows of different denticle morphology and polarity to give a rectangular denticle pattern characteristic of embryos expressing Hh ubiquitously at low levels. They do not show a duplication of the most anterior row in place of the posterior rows that is characteristic of ptc mutations.

Ci protein, detected by monoclonal antibody 2A1, is expressed in all non-en cells but is elevated in response to Hh at the borders of each broad segmental stripe (Motzny and Holmgren 1995) (Fig. 2b,h) and was induced to even higher levels throughout its normal expression domain by PKA inhibition in ubi-R* embryos (not shown). PKA inhibition in alternating segments of "alt-R*" [= paired (prd)-GAL4 + UAS-R*] embryos produced a cell-autonomous increase in Ci-CT antibody staining (Fig. 2a,d) without affecting ci RNA levels (Fig. 2f) or Ci-NT antibody staining (not shown). Thus, PKA inhibition appeared to have similar effects to Hh on Ci staining, presumably corresponding to an increased concentration of full-length Ci protein and could induce ectopic Hh target gene expression in the embryo. However, PKA inhibition induced higher levels of Ci-CT staining than Hh (Figs. 2, b,d, and h; and 3c) but was less potent than Hh at inducing wg and ptc expression in ubi-R* embryos (Fig. 1c,d), and especially in alt-R* embryos (Fig. 3a,b), where only a slight expansion of ptc expression is seen and wg expression appears normal. This discrepancy provided the first hint that PKA or Hh might have additional functions that do not affect Ci protein levels.

View larger version (98K): [in this window] [in a new window]   Figure 2.     Decreased but not increased PKA activity increases Ci-CT antibody staining. (a) Antibody to -galactosidase reveals the expression pattern of GAL4 attributable to the prd-GAL4 transgene in alt-lacZ (= prd-GAL4 + UAS-lacZ) embryos. (b-h) Embryos stained with monoclonal antibody 2A1, which detects an epitope in the carboxy-terminal half of the full-length Ci protein. (d) In alt-R* (= prd-GAL4 + UAS-R*) embryos there is a large increase in Ci-CT antibody staining in roughly alternating segments corresponding to those cells that express the PKA inhibitor (as in a), but ci RNA (f) is uniformly expressed in all non-en cells, as in wild-type embryos. The extent of induction of Ci-CT staining by PKA inhibition is better seen in Fig. 3. Here (d), it is underestimated because Ci staining is saturated to allow direct comparison to other embryos. (e) In alt-mC* (= prd-GAL4 + UAS-mC*) embryos PKA hyperactivity does not alter Ci-CT antibody staining. (b,c,g,h) The focal plane differs from d and e to highlight accentuated Ci staining at the border of each band in wild-type embryos (b) that is absent from ubi-mC* embryos (c) (PKA hyperactivity) and also absent from embryos zygotically mutant for smo (smo11x43) (g) or (hhts2) (h).

View larger version (66K): [in this window] [in a new window]   Figure 3.     Hh and Smo can affect wg and ptc expression without altering Ci protein levels. Stage 10-12 embryos were hybridized with wg RNA (a,d,g), ptc RNA (b,e,h), or double-stained with Ci-CT antibody (brown) and En antibody (blue) (c,f,i). In alt-R* embryos wg expression is close to wild type (a), ptc RNA is slightly expanded (b), but Ci-CT antibody staining is very high in cells of alternating segments that express the PKA inhibitor (c) (as in Fig. 2d). These embryos were stained more lightly for Ci than those in Fig. 2 so that normal levels of Ci protein are barely visible. (d-f) alt-R* embryos also mutant for hh (hhts2 at 29°C) lose wg (d) and En (f) expression in all segments by stage 10 but the levels of Ci-CT antibody staining remain very high (f) and ptc RNA is still expressed in cells of alternating segments that express the PKA inhibitor (e). (g-i) alt-R* embryos lacking maternal and zygotic smo gene activity (smo11x43) lose wg RNA (g), ptc RNA (h), and En expression (i) in all segments by stage 10 without alteration of high Ci-CT antibody staining in alternating segments (i). alt-R* embryos lacking only zygotic smo have a similar phenotype, in which wg, ptc, and En expression decay at the same rate in adjacent segments and are substantially lost by the end of stage 11. wg and ptc expression also decay equally in adjacent segments of alt-R* embryos lacking ci activity [Df(4)M62f or ciDR50; not shown].

Hh and Hh pathway components can induce wg expression independent of Ci protein levels

To test for additional functions of Hh we introduced Hh pathway mutations into alt-R* embryos, in an attempt to circumvent the need for Hh to alter Ci protein levels. In alt-R* embryos the induction of high levels of Ci-CT antibody staining (brown, Fig. 3c) anterior to the odd-numbered En stripes (blue, Fig. 3c) by PKA inhibition was not affected by hh or smo mutations (Fig. 3f,i). These embryos were double-stained with En antibody to identify the hh and smo mutant embryos among their siblings and were deliberately understained to show that the levels of immunoreactive Ci were not affected by hh or smo mutations (cf. Figs. 3c and 3f and i). In these understained embryos normal levels of Ci anterior to even-numbered En stripes are barely visible. Although they did not alter the effects of PKA inhibition on Ci staining, hh and smo mutations each eliminated expression of wg in alt-R* embryos, even in those cells with high levels of Ci-CT antibody staining (Fig. 3a,d,g). Hence, Hh and Smo each provides an essential contribution to wg expression without detectably altering Ci protein levels, indicating a bifurcating pathway of Hh signaling downstream of Smo (Fig. 6, below). This is particularly clear in alt-R* embryos lacking Smo activity, in which adjacent wg stripes decay at identical rates despite a huge difference in Ci-CT antibody staining between adjacent segments (Fig. 3g,i).

View larger version (26K): [in this window] [in a new window]   Figure 6.     Hh signal transduction in Drosophila embryos. (Right) The familiar antagonistic effects of Hh and PKA on Ci protein levels detectable by monoclonal antibody 2A1, which we assume here to reflect the concentration of full-length Ci protein. High levels of full-length Ci protein are normally associated with activation of Hh target genes. (Left) Novel additional actions of Hh, Smo, and PKA that stimulate Hh target gene expression in embryos without discernibly altering the concentration of Ci proteins. Induction of wg and ptc expression by PKA hyperactivity required smo but not hh, implying that Smo either mediates the response to PKA (B and A) or has a basal activity that acts in concert with PKA (C and A). In embryos with high levels of Ci-CT antibody staining attributable to PKA inhibition, Smo activity was required to activate wg and ptc expression, whereas Hh activity was required only for wg expression. This suggests that Smo has a basal (Hh-independent) activity sufficient to induce ptc expression (A, solid arrow) that can be enhanced by Hh (A, open arrow) to stimulate wg expression, in each case without affecting Ci protein concentration. Thus, basal activities of PKA and Smo (solid vertical lines) poise the cell to respond by two distinct mechanisms to Hh (open arrows).

Expression of ptc RNA in response to PKA inhibition in alternating segments was still observed in hh mutants (Fig. 3e), suggesting a greater dependence of wg expression than ptc expression on the pathway of Hh signaling that does not alter Ci levels (Fig. 6, A, open arrow, below). In contrast, smo mutations eliminated expression of ptc in response to PKA inhibition (Fig. 3h), suggesting that Smo has a significant basal activity in the absence of Hh (Fig. 6, A, solid arrow, below). Expression of wg and ptc was not affected in alt-R* or ubi-R* embryos by loss of one active ci allele but was eliminated by complete loss of Ci activity (not shown).

Increased PKA activity also induces Hh target genes in embryos

A constitutively active mouse PKA catalytic subunit transgene, mC*, was shown previously to oppose the elevated expression of Ptc protein in response to Hh in imaginal discs (Li et al. 1995). In stark contrast, expression of mC* in embryos ubiquitously (ubi-mC*) or under the influence of ptc-GAL4, but not en-GAL4, caused ectopic expression of wg and ptc (Fig. 1e,f) without altering en or hh expression (not shown). The anterior expansion of wg stripes was more robust in ubi-mC* than ubi-R* embryos but was also only apparent after stage 11 and associated with ventral cuticles resembling those due to low-level ectopic Hh, rather than strong ptc mutations (Fig. 5d, below).

In embryos expressing both mC* and R* (ubi-mC*/R*) wg and ptc expression patterns were close to wild type (Fig. 1g,h), as was Ci-CT antibody staining (not shown). This confirmed the opposing actions of the two transgenes on PKA activity, as was also verified biochemically (Li et al. 1995). In contrast to all other documented manipulations of Hh pathway components, PKA hyperactivity in alt-mC* embryos caused ectopic expression of wg and ptc (Fig. 4a,b) without any increase in the level of ci RNA (not shown) or Ci protein detected by Ci-CT antibody (Fig. 2e) or Ci-NT antibody (not shown). PKA must therefore have a second target relevant to Hh signaling that does not influence Ci protein levels.

View larger version (114K): [in this window] [in a new window]   Figure 4.     Induction of wg and ptc by elevated PKA activity requires Smo and Ci but not Hh. (a,b) alt-mC* (= prd-GAL4 + UAS-mC*) embryos show expanded wg RNA (a) and ptc RNA (b) expression in the even parasegments. (c,d) alt-mC* embryos carrying the hhts2 mutation lose wild-type stripes of wg RNA (c) and ptc RNA (d) in odd-numbered parasegments by stage 10, but expanded wg and ptc expression in cells with increased PKA activity is unaffected by the hh mutation. In alt-mC* embryos that also lack maternal and zygotic smo (smo11x43) (e,f) or zygotic ci activity [Df(4)M62f or ciDR50] (g,h), wg RNA (e,g), and ptc RNA (f,h) expression are lost in all parasegments by stage 10, indicating that the responses to elevated PKA activity require smo and ci activity. In alt-mC* embryos deficient only for zygotic smo (not shown), wg and ptc RNA expression decay at equal rates in adjacent segments and are lost by the end of stage 11.

Although PKA hyperactivity did not discernibly affect Ci staining in cells in the center of each Ci stripe that expressed wg and ptc ectopically, it did suppress the elevation of Ci-CT antibody staining normally elicited by Hh at the borders of each Ci stripe (Fig. 2c). Thus, in the absence of Hh, Ci-CT staining responds dramatically to reductions in PKA activity (Figs. 2d and 3c) but is insensitive to increased PKA activity.

Responses to elevated PKA activity require Smo and Ci but not Hh

To explore the positive actions of PKA on Hh target gene expression further we examined the effects of various Hh pathway mutations. Loss of Hh activity did not impair the ectopic induction of wg and ptc by elevated PKA activity in alternating segments of alt-mC* embryos (Fig. 4a-d). In contrast, Smo activity was required to observe any response of wg or ptc to elevated PKA even in embryos deficient only for zygotic smo (Fig. 4e,f). The essential activity of Smo, as opposed to Hh, cannot be explained by effects on Ci protein levels, as zygotic smo mutations and hh mutations have equivalent effects, reducing Ci-CT antibody staining only at the borders of each Ci stripe (Fig. 2b,g,h). The absolute requirement for Smo to observe transcriptional induction by PKA hyperactivity is consistent with two mechanisms. Either PKA acts on Smo, directly or indirectly, perhaps to uncouple it from the inhibitory influence of Ptc (Fig. 6, B and A, below) or, alternatively, Smo has Hh-independent activity that acts in parallel with PKA to stimulate wg and ptc expression (Fig. 6, C and A, below). Although the response to PKA hyperactivity did not involve changes in Ci staining it did absolutely require Ci activity (Fig. 4g,h), indicating a mechanism that either involves modification of Ci or requires Ci to act as a cofactor for wg and ptc expression.

Hh signaling does not require cAMP-mediated regulation of PKA activity

The structure of Smo has invited speculation that it couples to a G protein and might therefore regulate cAMP concentration (Alcedo et al. 1996; van den Heuvel and Ingham 1996). To examine this idea we tested whether low-level expression of mC* from an actin-5C promoter could substitute for Drosophila PKA catalytic subunit DC0 activity in embryos. The mC* catalytic subunit binds poorly to regulatory subunit and is therefore active regardless of cAMP concentration (Orellana and McKnight 1992). Expression of mC* in the female germ line rescued the functions of DC0 in oogenesis and allowed a small proportion of eggs to be fertilized and to develop (Y. Zhang, W. Li, and D. Kalderon, unpubl.). Many of these embryos hatched even if maternally and zygotically null for DC0 and exhibited ventral cuticle patterns similar to wild type (Fig. 5a,b), implying normal Hh signaling. Hence, as observed previously in imaginal discs (Jiang and Struhl 1995; Li et al. 1995), neither transcriptional nor cAMP-dependent regulation of PKA activity appear to be essential for Hh signaling.

	Discussion

Top Abstract Introduction Results Discussion Materials & Methods References

Previous models of Hh signaling in Drosophila have emphasized changes in the concentration of Ci proteins, detected by Ci-CT antibody, as the goal of signal transduction. Also, the antagonistic effects of PKA on Hh target gene expression and involvement of the serpentine transmembrane protein Smo, predicted to couple to a G-protein, have sometimes been rationalized as indicative of cAMP acting as a key intermediate. We have shown that regulation of Ci protein levels is a common focus for the antagonistic actions of Hh and PKA in Drosophila embryos but have also obtained evidence for novel positive actions of both PKA and Hh in inducing Hh target genes without affecting Ci protein levels. In addition, we have shown that, as in imaginal discs, regulation of PKA activity by cAMP is not essential to Hh signal transduction in embryos.

Repression of Hh target genes by PKA; possible targets

PKA inhibition in Drosophila embryos induced ectopic expression of the Hh target genes wg and ptc (Fig. 1) and was accompanied by increased Ci-CT antibody staining, without affecting Ci-NT antibody staining or ci mRNA levels (Fig. 2). Because Hh has similar effects and has been shown to inhibit the cleavage of full-length Ci protein (Ci-155), it is very likely that PKA inhibition also reduces cleavage of Ci-155. This hypothesis must be tested directly in the future but is consistent with our observation that increased PKA activity suppresses the effects of Hh on Ci-CT staining (Fig. 2c). It is believed that Ci-155, or an uncharacterized derivative, acts as a transcriptional activator, whereas Ci-75 is believed to act as a transcriptional repressor (Aza-Blanc et al. 1997) so that an increase in the proportion of Ci protein that is full length is expected to stimulate Hh target gene expression. Most likely, therefore, the altered levels of Ci proteins detected by Ci-CT antibody are responsible for the ectopic expression of wg and ptc induced by PKA inhibition in embryos. The effect of PKA on Ci protein levels is unchanged in hh or smo mutant embryos and might therefore be mediated by direct phosphorylation of Ci, which includes several consensus PKA sites (Orenic et al. 1990).

Positive action of PKA in Hh signaling; possible PKA targets

Previously PKA has been shown only to repress Hh target gene expression in Drosophila and in vertebrates (Fan et al. 1995; Kalderon 1995; Concordet et al. 1996; Epstein et al. 1996; Hammerschmidt et al. 1996; Ungar and Moon 1996). We have demonstrated that PKA can also stimulate Hh target gene expression in Drosophila embryos by using an altered mouse catalytic subunit, mC*, as a source of hyperactive PKA. The response to mC* was suppressed by R*, which can inhibit endogenous Drosophila PKA but not mC*, implying that Drosophila PKA can and normally does phosphorylate the mC* substrate that promotes Hh target gene expression. Because we can reduce but not eliminate PKA activity from early embryos we do not know if the stimulation of Hh target gene expression by PKA is essential in wild-type embryos. The mechanism by which PKA hyperactivity induces Hh target genes does not involve discernible changes in Ci protein levels and is therefore clearly different from the cellular response to reducing PKA activity.

The induction of wg and ptc expression by PKA hyperactivity was eliminated by smo or ci mutations but not by hh mutations. Hence, Smo and Ci but not Hh are potential obligatory mediators of the positive effects of PKA on Hh target genes. It is possible that PKA acts directly on Smo, rendering it fully or partially active even in the absence of Hh (Fig. 6, B). Smo contains several potential PKA phosphorylation sites (Alcedo et al. 1996; van den Heuvel and Ingham 1996), and there is considerable evidence that phosphorylation can alter the activity of G protein-coupled receptors (Freedman and Lefkowitz 1996). Also, in this scenario a single mechanism downstream of Smo (Fig. 6, A) would account for the actions of both PKA and Hh that stimulate Hh target gene expression without altering Ci protein levels. Alternatively, PKA and Smo may contribute independently to Hh target gene expression (Fig. 6, C and A). In this case, Ci could be a relevant PKA target. It is even possible that both positive and negative effects of PKA on wg and ptc expression are mediated by phosphorylation of Ci, so long as distinct sites of different affinity and regulatory consequence are used.

Dual actions of PKA

Two PKA targets with opposing actions on Hh target gene expression (Fig. 6) can account for the initially surprising observation that both PKA inhibition and PKA hyperactivity induced wg and ptc expression in embryos. The negative target, relevant to regulating Ci protein levels, was sensitive almost exclusively to reduction of PKA activity (Fig. 2d,e). Thus, only the positive target (Fig. 6, left) responds to PKA hyperactivity, leading to ectopic expression of wg and ptc in ubi-mC* (Fig. 1e,f) and alt-mC* embryos (Fig. 4a,b). When PKA activity was decreased in ubi-R* embryos the conflicting influences of a large increase in Ci-CT antibody staining, presumably reflecting full-length Ci protein concentration, and a diminished contribution of the positive PKA target (of unknown magnitude) combined to produce a more modest ectopic induction of wg and ptc (Fig. 1c,d). In alt-R* embryos the combination of these two influences produced only a slight induction of ectopic ptc expression and no ectopic induction of wg expression (Fig. 3a,b).

Despite the potent effects of PKA on Hh target gene expression, changes in cAMP concentration cannot account for Hh signaling. First, in embryos, as in imaginal discs, replacement of the major Drosophila PKA catalytic subunit DC0 with mC*, which is insensitive to cAMP, did not prevent Hh signaling (Jiang and Struhl 1995; Li et al. 1995). Second, neither inhibition nor activation of PKA in embryos stimulated Ci-CT antibody staining and wg RNA in the same proportion as normally induced by Hh. These observations do not exclude the possibility that modulation of PKA activity might make some contribution to Hh signaling, but they are not consistent with a central role for cAMP as a mediator of Hh signaling.

Dual actions of Hh signaling pathway

Prior to this study the only significant indicator that Hh signaling in Drosophila may involve more than simply regulating Ci protein levels were phenotypes attributable to fu mutations in which Hh target gene expression was attenuated or eliminated despite extremely high levels of Ci-CT antibody staining (Motzny and Holmgren 1995; Slusarski et al. 1995). Whether this activity of Fu reflected an activity of Hh was unclear, however, as Fu, although phosphorylated in response to Hh (Thérond et al. 1996b), is not required for Hh signaling in the absence of Su(fu) (Thérond et al. 1996a). In this study, we used PKA inhibition as a means of substituting for the effect of Hh on Ci protein processing. The increase in Ci-CT antibody staining because of PKA inhibition was at least as great as induced during normal Hh signaling and was not accompanied by any change in Ci-NT antibody staining, suggesting that PKA inhibition does phenocopy the effects of Hh on Ci cleavage. Under these circumstances, we found that both Hh and Smo were essential to maintain wg expression despite unchanged Ci staining. Thus, both Hh and Smo, an established mediator of Hh signals, provided a positive input to wg expression that was independent of detectable changes in Ci protein levels (Fig. 6A).

It was shown recently that in embryos with normal PKA activity wg can be induced by overexpression of a ci cDNA even in the absence of Hh activity (Alexandre et al. 1996). It is likely that we observed stricter requirements for Hh activity to induce wg expression because we simultaneously reduced the positive input from PKA toward Hh target gene expression (Fig. 6, B or C).

Functional implications of dual actions of PKA and Hh

We believe that the novel mechanisms depicted in Figure 6, namely a positive action of PKA and an action of Hh that is not related to alteration of Ci protein levels, are also operative in imaginal discs. In wing discs most patterning responses to Hh and the expression of ptc reveal only an antagonistic effect of PKA (Jiang and Struhl 1995; Lepage et al. 1995; Li et al. 1995; Pan and Rubin 1995). However, we have found that formation of non-neuronal bristles at the wing margin in response to either Hh or loss of ptc function requires PKA activity (J.T. Ohlmeyer, W. Li, and D. Kalderon, unpubl.), suggesting conservation of the positive function of PKA in Hh signaling. A similar defect in patterning of wing margin bristles is seen as a consequence of reduced Fu activity even though Ci-CT antibody staining is high in fu mutant cells responding to Hh (Slusarski et al. 1995; Sánchez-Herrero et al. 1996; J.T. Ohlmeyer, W. Li, and D. Kalderon, unpubl.). These observations suggest that the novel activities of Hh and PKA that we have described are general features of Hh signaling in Drosophila that are particularly obvious in embryos. It is possible that in vertebrates, as in Drosophila imaginal discs, a positive contribution of PKA to a subset of Hh responses is also present but not easily discerned in the background of the more prominent repressive effect of PKA on Hh target gene expression (Fan et al. 1995; Concordet et al. 1996; Epstein et al. 1996; Hammerschmidt et al. 1996; Ungar and Moon 1996).

The target specificity of the two arms of Hh signaling (Fig. 6) need not be identical even though the common requirement for Ci activity suggests that a Ci-binding site will always be present in target genes. In embryos we observed a differential dependence of wg and ptc expression on the novel arm of Hh signaling (Fig. 6A) by assaying the response to Hh at constant Ci protein levels (Fig. 3e,h). Similarly, in imaginal discs fu mutations allow Hh-dependent changes in Ci protein levels but have a greater inhibitory effect on the induction of anterior en expression than on the induction of decapentaplegic (dpp) expression by Hh (Slusarski et al. 1995; Sánchez-Herrero et al. 1996; J.T. Ohlmeyer, W. Li, and D. Kalderon, unpubl.). Also, productive signaling in each branch of the pathway might require a different threshold concentration of Hh. Hence, dual arms of Hh signaling provide a mechanism that may underlie the ability of Hh family molecules to produce different transcriptional responses in similar cells according to Hh concentration (Heemskerk and DiNardo 1994; Roelink et al. 1995; Ericson et al. 1996; Struhl et al. 1997) or the presence of other signaling molecules (Hooper 1994; Yoffe et al. 1995).

	Materials and methods

Top Abstract Introduction Results Discussion Materials & Methods References

Fly stocks and crosses

Flies homozygous for UAS-R* and UAS-mC* transgenes (Li et al. 1995) were crossed to flies homozygous for the "E22C" enhancer-trap line (A. Brand and N. Perrimon, unpubl.) or a prd-GAL4 transgene, "RG1" (Yoffe et al. 1995) to generate embryos with reduced or excess PKA activity, respectively (confirmed by measurements in extracts in the presence of 1 µM cAMP as described previously (Li et al. 1995). In all cases, embryos were collected for 3 hr at 25°C and incubated at 29°C for an additional 5 hr to obtain stage 10-13 embryos and to maximize expression of GAL4-responsive transgenes. hh mutations were introduced by crossing UAS-R* or UAS-mC*; hh^ts2/TM6B flies to hh^ts2 RG1/TM6B flies. Homozygous hh^ts2 progeny were identified by loss of wg, ptc, or En (in double stains with Ci) expression in alternate segments and were found in the expected proportions. hh^ts2 is a hypomorph reported to have no discernible activity at 29°C (Ma et al. 1993). Embryos zygotically mutant for smo^11x43 were similarly identified among progeny of the crosses smo^11x43 UAS-mC*/CyO or smo^11x43/CyO; UAS-R*/UAS-R* × smo^11x43/CyO; RG1/RG1 (or E22C smo^11x43/CyO). Embryos also maternally deficient for smo were generated by the FLP-dominant female sterile (DFS) technique (Chou et al. 1993), inducing germ-line clones in females that were smo^11x43 FRT40A/P[ovo^D1] FRT40A; UAS-R*/+ or smo^11x43 FRT40A UAS-mC*/P[ovo^D1] FRT40A and crossing to smo^11x43/CyO; RG1/RG1 males. smo^11x43 is amorphic (van den Heuvel and Ingham 1996). The effects of ci mutations were tested by crosses between UAS-R*/UAS-R* or UAS-mC*/UAS-mC*; Df(4)M62f/ey^D or ci^DR50/M63a females and E22C/E22C or RG1/RG1; Df(4)M62f/ey^D or ci^DR50/M63a males. M62f is a deficiency that is null for ci and ci^DR50 is a loss-of-function mutation (Slusarski et al. 1995); identical results were found with each allele. DC0 null germ-line clones (Lane and Kalderon 1994) were generated at the same time as activating mC* expression from an actin-5C promoter by inducing FLP recombinase in females that were DC0^H2 FRT40A actin < y⁺ < mC*/P[ovo^D1] FRT40A (where < denotes an FRT) to produce the germ-line genotype DC0^H2 FRT40A actin < mC*/DC0^H2 FRT40A and crossing to Df(2L)15/P[y⁺] CyO males heterozygous for a DC0 deficiency. Cuticles of hatching y larvae were examined.

Examination of embryos

In situ hybridizations on whole-mount embryos used digoxigenin-labeled RNA probes synthesized by T7 RNA polymerase for wg, plasmid wgPx4 (J. Mohler, Barnard College, New York, NY) cut by PstI; ptc, plasmid Dra2Nptc (S. DiNardo, Rockefeller University, New York, NY) cut by HincII; hh, plasmid 4.1/8B#6 (J. Mohler) cut by NdeI; and ci, plasmid pGEM7Zci (R. Holmgren, Northwestern University, Evanston, IL) cut by BamHI and XbaI. Antibody stainings were performed using monoclonal En/Inv antibody [4D9; (Patel et al. 1989)], monoclonal Ci -CT antibody [2A1; (Motzny and Holmgren 1995)], polyclonal Ci-NT antibody (Aza-Blanc et al. 1997), or polyclonal anti--galactosidase (Cappel). Larval cuticles were prepared by a modification (S. DiNardo) of a published procedure (Heemskerk and DiNardo 1994).