Phosphoinositide 3-kinase and Akt are essential for Sonic Hedgehog signaling

Abstract

Hedgehogs (Hhs) are key signaling regulators of stem cell maintenance and tissue patterning in embryos, and activating mutations in the pathway that increase Gli transcriptional activity are causal in a diversity of cancers. Here, we report that phosphoinositide 3-kinase (PI3-kinase)-dependent Akt activation is essential for Sonic Hedgehog (Shh) signaling in the specification of neuronal fates in chicken neural explants, chondrogenic differentiation of 10T1/2 cells, and Gli activation in NIH 3T3 cells. Stimulation of PI3-kinase/Akt by insulin-like growth factor I potentiates Gli activation induced by low levels of Shh; however, insulin-like growth factor I alone is insufficient to induce Gli-dependent transcription. Protein kinase A (PKA) and glycogen synthase kinase 3β sequentially phosphorylate Gli2 at multiple sites, identified by mutagenesis, thus resulting in a reduction of its transcriptional activity. Gli2 mutant proteins in which the major PKA and glycogen synthase kinase 3β phosphorylation sites were mutated to alanine remain fully transcriptionally active; however, PKA-mutant Gli2 functions independently of Akt signaling, indicating that Akt positively regulates Shh signaling by controlling PKA-mediated Gli inactivation. Our findings provide a basis for the synergistic role of PI3-kinase/Akt in Hh signaling in embryonic development and Hh-dependent tumors.

Hedgehog (Hh) signaling regulates tissue patterning and stem cell maintenance in vertebrate and invertebrate embryos (1). Hh, by binding to its receptor Patched (Ptc), releases Ptc inhibition of Smoothened (Smo), a membrane protein related to G protein-coupled receptors, which then transduces a signal for activation and nuclear translocation of a family of transcription factors, Ci in Drosophila and Glis (Gli1, Gli2, and Gli3) in vertebrates. Although Smo signal transduction mechanisms are not well understood, Smo is known to control Ci phosphorylation. In the absence of Hh, Smo is localized mostly in vesicles (2) and Ci is phosphorylated by protein kinase A (PKA) at multiple sites, which prime additional phosphorylation at interspersed sites by the glycogen synthase kinase 3 (GSK-3β) homologue shaggy and casein kinase-I (3, 4). Hyperphosphorylated Ci is targeted for proteasomal degradation to generate a repressor form. During pathway activation, Smo is enriched at the plasma membrane, and Ci phosphorylation is prevented, leading to stabilization and nuclear translocation of full-length Ci. The role of phosphorylation in the regulation of vertebrate Gli proteins has not yet been defined, although PKA is a known inhibitor of vertebrate Hh signaling (5, 6).

A diversity of human cancers are caused by mutations that lead to inappropriate Hh pathway activation, including loss-of-function mutations in Ptc, gain-of-function mutations in Smo, or Gli gene amplification and overproduction of Hh ligand (7). Genetic studies in mice reveal that the insulin-like growth factor (IGF)/phosphoinositide 3-kinase (PI3-kinase)/Akt pathway provides a synergistic signal for Hh tumor formation (8, 9). Interestingly, there is a high incidence of loss-of-function mutations of PTEN, a negative regulator of PI3-kinase activity, in ≈39% of Hh-dependent human pancreatic cancers (10), which apparently strictly depend on PI3-kinase for proliferation (11). Thus, we hypothesized that the Hh pathway and the PI3-kinase pathway could be part of a cross-regulatory signaling network. Our results establish that PI3-kinase and Akt are indeed essential for Hh signaling and provide a basis for the increased tumor formation potential when the Hh pathway is activated in tissues with enhanced PI3-kinase signaling.

Results

To investigate the role of the PI3-kinase/Akt pathway in the control of Hh signaling, we tested the effect of IGF-I on Sonic Hedgehog (Shh) signaling in LIGHT cells. Recombinant Shh (N-Shh) induces a dose-dependent activation of Gli-luciferase, and this response is enhanced ≈3-fold by IGF-I at submaximal N-Shh concentrations (Fig. 1A). IGF-I strongly induces Akt phosphorylation, an effect that is sensitive to the specific PI3-kinase inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). However, IGF-I is not sufficient to drive Gli-luciferase activation (Fig. 1B). N-Shh alone also induces the activation (≈2-fold) of PI3-kinase-dependent Akt phosphorylation in LIGHT cells within 5 min (Fig. 1C), in agreement with previous findings in endothelial cells (12). Notably, LY294002 treatment inhibits Shh-activated Gli-luciferase expression to the same extent as cyclopamine, a potent Smo inhibitor (Fig. 1D). LY294002 also blocks activation of the Gli-luciferase reporter elicited by SmoM2, an oncogenic mutant of Smo (Fig. 5, which is published as supporting information on the PNAS web site) (13). These data indicate that PI3-kinase activity, stimulated by Shh or by other ligands, is essential for Gli activation.

Fig. 1.

PI3-kinase signaling through Akt is essential for Shh signal transduction. (A) Gli-luciferase activity in LIGHT cells induced for 24 h by increasing concentrations of N-Shh in the absence or presence of 50 nM IGF-I. (B) Gli-luciferase activity in LIGHT cells after 24 h of treatment with IGF-I or N-Shh (5 μg/ml). Phosphorylation of Akt (Ser-473) after 15 min of treatment with increasing concentrations of IGF-I was determined by Western blot (phospho-Akt, 1:1,000; total Akt 1:2,000). (C) Effect of 5 μg/ml N-Shh on Akt phosphorylation at different time points, assessed as in B. Densitometry values representing the increase in P-Akt are indicated at the bottom. (D) Effect of LY294002 (15 μM) and KAAD-cyclopamine (100 nM) on Gli-luciferase induced by Shh-conditioned medium (Shh-CM) or N-Shh in LIGHT cells at 24 h. (E) LIGHT cells were transfected with Akt1 or control siRNA, and 48 h later they were stimulated with N-Shh for 24 h. (F) Effect of dnAkt on Gli-luciferase activity in NIH 3T3 cells transiently transfected with the indicated constructs. (G) LIGHT cells stably transfected with LacZ or myr-Akt were stimulated with 5 μg/ml N-Shh with or without LY294002 (15 μM) for 24 h and assayed for Gli-luciferase activity, normalized to the baseline in the absence of N-Shh. Data represent the mean ± SEM of three experiments. ∗, P < 0.01. RLU, relative luciferase units.

To investigate whether PI3-kinase regulates Shh signaling through Akt, we reduced endogenous Akt levels with an Akt1 small interfering RNA (siRNA) or impaired its activity with a nonphosphorylatable Akt mutant that acts as dominant negative (dnAkt). Akt1 siRNA reduces Shh-induced luciferase activity in LIGHT cells by 60% (Fig. 1E). The ≈20% difference in efficacy of LY294002, which totally blocks PI3-kinase signaling through Akt, and Akt1 siRNA to suppress Shh signaling is likely caused by the coexpression of low levels of Akt2 and Akt3, which likely function redundantly. Moreover, dnAkt potently inhibits Gli-luciferase induced by Shh, SmoM2, Gli1, and Gli2 in NIH 3T3 cells (Fig. 1F), establishing that Akt is essential for Gli function. Similar inhibition was observed with a kinase-dead dnAkt (data not shown). In addition, overexpression of a constitutively active form of Akt (myr-Akt) in LIGHT cells rescues activation of the Gli-luciferase reporter by N-Shh in the presence of LY294002 (Fig. 1G). Rapamycin, a specific inhibitor of mTOR pathway downstream of PI3-kinase, does not affect N-Shh-induced Gli-luciferase (Fig. 6, which is published as supporting information on the PNAS web site). These results support a requirement of Akt for Shh signaling and rule out a contribution of the mTOR pathway.

To test whether PI3-kinase and Akt play a role in Shh-regulated developmental signaling, we analyzed the effects of LY294002 on Shh-dependent dorsal-ventral patterning in embryonic neural tube explants cocultured with its associated, Shh-producing notochord. LY294002 treatment of neural tube-notochord explants reduces phospho-Akt levels and causes neural patterning defects consistent with Shh loss of function. These patterning defects include a 50% decrease in Islet1-expressing motor neuron progenitors, which normally form in the ventral neural tube in response to high concentrations of Shh from the floor plate and notochord, and a reciprocal ≈300% increase in dorsally localized Pax7-expressing progenitors (Fig. 2A and B), which are normally repressed by Shh (14, 15). Cyclopamine causes identical defects in Shh-induced neural tube patterning in this experimental model (16). LY294002 treatment of neural tube explants does not disrupt Shh expression in the floor plate (Fig. 2A) and does not affect cell proliferation or apoptosis in the neural tube, as determined by BrdUrd incorporation and caspase-3 cleavage (Fig. 2B), suggesting a specific effect of LY294002 on Shh-induced neural tube patterning.

Fig. 2.

PI3-kinase signaling is required for Shh-induced neural patterning and chondrogenesis. (A Upper) Treatment of neural tube/notochord explants for 48 h with LY294002 (30 μM) blocked Akt phosphorylation, as assayed by Western blotting, and blocked Shh signaling, as assayed by whole-mount immunostaining for Pax7 and Islet1 expression. (A Lower) Explants were photographed dorsal side up for Pax7 and ventral side up for Islet1. High-magnification insets show nuclear localization. Lowest panel shows staining of Shh in the floor plate (fp). (Scale bar: 100 μm.) (B) Quantification of Pax7 and Islet1 expression, BrdUrd incorporation, and activated caspase-3 in control and LY294002-treated neural tube explants. (C) Effects of LY294002 (15 μM) on AP induction by 5 μg/ml N-Shh in 10T1/2 cells after 4 days. RU, relative units. (D) 10T1/2 cells stably transfected with empty vector or p-myr-Akt treated as in C. (E) Effect of dnAkt on Gli-luciferase activity in 10T1/2 cells transiently transfected with the indicated constructs. Data are the mean ± SEM of three experiments performed in triplicate. ∗, P < 0.01.

In 10T1/2 mesodermal cells, Shh induces the late expression of alkaline phosphatase (AP), a marker of chondrogenic differentiation (17). Using this model, we found that LY294002 completely inhibits AP induction by N-Shh (Fig. 2C), which is rescued by ectopic expression of myr-Akt (Fig. 2D), further demonstrating the essential function of PI3-kinase and Akt in Hh developmental signaling. Finally, dnAkt also reduces the activation of a Gli-luciferase reporter by Shh, SmoM2, Gli1, and Gli2 in 10T1/2 cells (Fig. 2E), demonstrating a broad requirement of Akt signaling in Gli-dependent Hh signaling.

As Akt is known to phosphorylate and inhibit GSK-3β (18), we investigated the role of PI3-kinase/Akt in the regulation of Gli2 transcriptional activity by PKA and GSK-3β phosphorylation. PKA represses Gli2 transcriptional activity in NIH 3T3 cells, which is further reduced by GSK-3β (Fig. 3A), thus establishing that PKA and GSK-3β are negative regulators of Gli2, as shown for Drosophila Ci (3, 4). However, GSK-3β alone is insufficient to inhibit Gli2-mediated activation of the Gli-luciferase reporter, consistent with an obligatory priming role of PKA for GSK-3β phosphorylation (3). Immunoprecipitated Gli2 is phosphorylated directly by PKA, and indirectly by GSK-3β, after PKA priming (Fig. 3B). Comparison of Ci and Gli2 sequences reveals four clusters of consensus PKA phosphorylation sites interspersed with GSK3-β and casein kinase I sites within conserved domains (Fig. 3C), as well as a single PKA site located N-terminal of cluster I. The functionality of each putative PKA and GSK-3β phosphorylation site was tested directly by in vitro phosphorylation by using 23-mer peptides corresponding to the four WT clusters and modified peptides containing Ser/Thr to Ala substitutions in each site. These experiments reveal that S805, S817, and S956 in clusters I, II, and IV are functional PKA sites. However, only S801 and S813 in clusters I and II, and not T952, appear to be functional GSK-3β phosphorylation sites in the peptide assay. Phosphorylation of the GSK-3β sites strictly depends on the phosphorylation of the neighboring PKA sites (Fig. 3D). To test the functionality of those candidate sites, we generated Gli2 mutants with triple PKA site mutations (PSM) in clusters I, II, and IV or triple GSK-3β site mutations (GSM). After expression in 293 cells and immunoprecipitation, the Gli2 PSM and Gli2 GSM mutants show a significant reduction in PKA- and GSK-3β-dependent phosphorylation, respectively (Fig. 3E). Mutation of all five putative PKA sites results in total lack of phosphorylation by PKA (data not shown). We then sought to determine whether Gli2 is phosphorylated in intact cells by endogenous PKA at those sites. Indeed, PKA stimulation with forskolin in [³²P]orthophosphate-labeled 293 cells induces a significant increase in ³²P incorporation in WT Gli2, whereas phosphorylation of the PSM mutant is markedly decreased (Fig. 3F).

Fig. 3.

Gli2 has functional sites for PKA and GSK-3β phosphorylation. (A) NIH 3T3 cells were cotransfected with Gli2 and empty vector, PKA, GSK-3β or both and assayed for Gli-luciferase activity 48 h later. (B Upper) Immunoprecipitated myc-Gli2 was incubated in 40 mM Tris·HCl (pH 7.4), 20 mM magnesium acetate, and 0.2 mM [γ-³²P]ATP (1 μCi/μmol) (control), with 10 units of PKA catalytic subunit or with PKA and 10 units of protein kinase inhibitor (PKI) for 30 min at 30°C. Beads were washed with 50 mM Tris·HCl (pH 7.5) and subjected to 5% SDS/PAGE and autoradiography. (B Lower) myc-Gli2 was incubated in 20 mM Tris·HCl (pH 7.5), 10 mM MgCl₂, 5 mM DTT, and 200 μM [γ-³²P]ATP (1 μCi/μmol) without (control) or with 500 units of GSK-3β for 30 min at 30°C, or Gli2 was phosphorylated first with PKA and cold ATP, as described above, followed by 10 units of PKI, 500 units of GSK-3β, and 200 μM [γ-³²P]ATP and incubated for 30 min. Beads were processed as before. (C) Sequence of the Gli2 peptides corresponding to the four clusters with the conserved PKA and GSK-3β phosphorylation sites highlighted in boxes. (D) Phosphorylation of peptides shown in C and variant peptides in which each candidate residue was substituted by alanine. Peptides (1 mM) were treated with PKA and GSK-3β or both enzymes sequentially, as described for Gli2 in B. Reactions then were stopped by spotting onto P81 paper and washed with 50 mM H₃PO₄, and ³²P incorporation was determined by liquid scintillation. PKA phosphorylation of kemptide was taken as 100%. (E) Gli2 WT, Gli2 PSM, and Gli2 GSM were expressed in 293 cells, immunoprecipated with Xpress antibody, and used as substrates for phosphorylation by PKA and GSK-3β as described in B. Phosphorylation and expression were assayed by autoradiography (Autorad.) and Western blot (WB) (Xpress Ab 1:5,000). Densitometric analysis of three experiments is shown in the graph. (F) Intact 293 cells were transfected with Gli2 WT or Gli2 PSM, labeled with [³²P]orthophosphate, and stimulated with 10 μM forskolin for 2 h. Gli2 was immunoprecipitated with Xpress Ab, and ³²P incorporation was determined by autoradiography. Quantification by densitometry of three experiments was performed by using the basal level of phosphorylation of Gli2 WT as 100%.

To test whether PKA and GSK-3β regulate Gli2 transcriptional activity by phosphorylation, mutants and WT Gli2 were coexpressed with PKA and GSK-3β. Single or double mutations of the PKA phosphorylation sites in all clusters did not alter Gli2 activity or its sensitivity to repression by PKA or GSK-3β (data not shown). However, Gli2 PSM is completely resistant to repression by PKA or PKA plus GSK-3β (Fig. 4A), demonstrating the functionality of the PKA kinase sites identified above. Both Gli2 PSM and Gli2 GSM are expressed at higher levels than WT Gli2 (Fig. 4A), because of reduced degradation (Fig. 7, which is published as supporting information on the PNAS web site), establishing that PKA and GSK-3β phosphorylation function cooperatively to control Gli2 turnover. Moreover, WT Gli2 and Gli2 PSM are similarly localized primarily in the cytoplasm (Fig. 4C). These results suggest that the apparent elevated transcriptional activity of the mutants is likely a result of increased stability rather than of differential subcellular localization. In addition, Gli2 with mutations of the GSK-3β sites in clusters I and II (GSM I and II) or all four clusters (GSM I–IV) are inhibited by PKA, but are resistant to additional repression by coexpressed GSK-3β (Fig. 4A), demonstrating the functionality of those sites in intact cells.

Fig. 4.

Mechanism of Akt-mediated Shh signaling. (A) Gli-luciferase activity in NIH 3T3 cells transiently transfected with WT Gli2 (WT), Gli2 GSM (clusters I–IV), and Gli2 PSM (clusters I, II, and IV), either alone or in combination with PKA or both PKA and GSK-3β expression vectors. Expression of the Gli2 variants was determined by Western blot. (B) Gli2 WT, Gli2 PSM, or Gli GSM were cotransfected with empty vector or dnAkt in NIH 3T3 cells and assayed for Gli-luciferase activity after 48 h. Expression levels of Gli2 variants in the presence of dnAkt were determined by Western blot. (C) Subcellular localization of Gli2 WT (Left) and Gli2 PSM (Right) in NIH 3T3 cells in the absence (Top) and the presence (Middle and Bottom) of dnAkt. Gli2 was detected with Xpress Ab (1:250)/Alexa Fluor 546 (red) and dnAkt with Akt Ab (1:120)/Alexa Fluor 488 (green). DAPI staining of the nuclei is in blue. Asterisks indicate cells coexpressing dnAkt and Gli2. (D) Model of Shh signaling showing the requirement PI3-kinase/Akt activation by Smo or other proliferative pathways in the regulation of Gli phosphorylation by PKA. All phosphorylation events are represented by ○p. Details are described in the text.

Mutation of S230 in Gli2, a putative Akt phosphorylation site (RXRXXS, ref. 19), does not prevent dnAkt repression (Fig. 8, which is published as supporting information on the PNAS web site). We then tested whether inhibition of Gli2 by dnAkt is exerted through the control of PKA- or GSK-3β-dependent Gli2 phosphorylation and/or degradation. Specifically, we tested the effect of dnAkt on the transcriptional activity of the Gli2 phosphorylation mutants. Gli2 PSM, which is resistant to phosphorylation and repression by PKA, is also fully resistant to inhibition by dnAkt (Fig. 4B). In contrast, dnAkt still represses Gli2 GSM (I–IV) activity (Fig. 4B). The reduction of WT Gli2 activity by dnAkt is associated to a significant decrease in Gli2 expression levels, likely caused by increased degradation, whereas Gli2 PSM and Gli2 GSM levels are less affected by dnAkt (Fig. 4 B and C and Fig. 9, which is published as supporting information on the PNAS web site), thus indicating that Akt does not regulate Gli2 turnover directly, but mediates its positive function through control of PKA phosphorylation. Also, mutation of all conserved putative casein kinase I sites does not prevent repression by dnAkt (Fig. 8). In addition, dnAkt does not change the subcellular localization of WT or PSM Gli2 (Fig. 4C). Altogether, these studies establish that Gli2 turnover and its transcriptional activity are regulated by phosphorylation by PKA and GSK-3β, and that PI3-kinase, acting through Akt, interferes either with PKA phosphorylation of Gli2 or PKA phosphorylation-dependent events.

Discussion

Our findings provide evidence that PI3-kinase and Akt activities are essential for Gli-dependent Shh signaling. Furthermore, stimulation of PI3-kinase/Akt by IGF-I potentiates Gli transcriptional activity in the presence of submaximal amounts of Shh, which, by itself, also stimulates PI3-kinase and Akt signaling at a lower magnitude. Such signaling cross talk between IGF-1 and Hh at the level of Akt provides a mechanism to produce graded Hh signaling responses in embryonic and adult tissues for tissue patterning.

The precise mechanisms by which Akt controls PKA function to regulate Gli-mediated Hh signaling remain unclear. However, our results indicate that Akt is required to antagonize PKA-dependent Gli2 inactivation. It is conceivable that Akt regulates the accessibility of PKA to Gli2, a frequent theme in Hh signaling: Drosophila Hh interferes with the formation of a complex containing Costal-2, Ci, PKA, GSK-3, and casein kinase I (20), and at the same time, PKA phosphorylates Smo C-tail as a requisite for activation (21–23). Vertebrate Hh signaling, however, has significant differences: the key intracellular transducer, Costal-2, is not conserved, and mammalian and fish Smo are phosphorylated by GRK-2 upon activation, not by PKA, and then internalized through a β-arrestin2-dependent mechanism (24, 25). Thus, although a Costal-2-like mechanism for regulation of Gli phosphorylation is unlikely, the target of Akt for Hh signaling could be a protein of the cytoplasmic complex that retains Gli outside of the nucleus and makes it accessible to PKA. Akt-mediated inhibition of this molecule(s) could promote nuclear translocation of Gli, escaping PKA phosphorylation. Alternatively, Akt could target protein(s) that regulate PKA directly, either through control of its activity and/or accessibility to Gli2. A model to explain our findings would be that binding of Shh to Ptc increases surface localization of Smo for activation of PI3-kinase and Akt. Phospho-Akt would then oppose the effects of PKA on Gli2 (and possibly Gli3) required for its proteasomal degradation, followed by Gli2/3 nuclear translocation to activate target genes (Fig. 4D). Cells responding to both Shh and additional PI3-kinase activating signals such as IGF-I would be less dependent on Smo for activation of PI3-kinase, rendering these cells more responsive to Shh. In this way, Shh pathway could integrate a diversity of negative and positive PI3-kinase regulatory signals from surrounding tissues to modulate the final Hh signaling output through Gli function. Interestingly, IGF-2 and IGF binding proteins are themselves transcriptional targets of Shh signaling through Gli (26, 27), suggesting that the activation of Akt by Shh signaling through Smo could become amplified and sustained through an IGF-2 autocrine loop.

Our finding that PI3-kinase/Akt activation is essential for Hh signaling sheds light on the synergy of IGF-2, PI3-kinase, and Akt with Hh in developmental processes (28, 29) and in Hh-dependent cancers (8, 9). Loss of PTEN or overexpression of IGF in Hh cancers would up-regulate PI3-kinase/Akt activity to stimulate even low-level ligand-dependent or ligand-independent Hh signaling caused by mutations in Hh pathway components. The essential role of PI3-kinase/Akt in mammalian Hh signaling suggests their potential use as therapeutic targets in the treatment of Hh-dependent malignancies.

Materials and Methods

Materials.

Rabbit anti-phospho (Ser-473) Akt, rabbit anti-Akt, and rabbit anti-cleaved caspase-3 were from Cell Signaling Technology (Beverly, MA). Mouse anti-BrdUrd was from BD Biosciences (Franklin Lakes, NJ). Mouse anti-Shh 5E1, mouse anti-Islet1 39.4D5, and mouse anti-Pax7 were from Developmental Studies Hybridoma Bank (Iowa City, IA).

Cell Culture and Transfection.

Details are in Supporting Text, which is published as supporting information on the PNAS web site.

Constructs and Mutagenesis.

Full-length Xpress-tagged mGli1 and mGli2 expression constructs and the Gli reporter vector p8XGBS-luc were provided by H. Sasaki (Riken Center for Developmental Biology, Kobe, Japan). Myc-tagged mGli1 and mGli2 were constructed by subcloning the XhoI–HindIII and BamHI–SalI fragments of mGli1 and mGli2, respectively, into pAG3-myc. Mutation of mGli2 Ser/Thr residues corresponding to consensus phosphorylation sites to Ala were done by using overlap extension. pRK5-Shh and pGE-SmoM2 were kindly provided by P. Beachy (The Johns Hopkins University, Baltimore). Akt1 (T308A and S473A) and myr-Akt1 were gifts of Y. Mitsuuchi (Fox Chase Cancer Center, Philadelphia). pmyr-Akt-hygro was constructed by subcloning of the HindIII–XbaI myr-Akt1 fragment into pcDNA3.1-hygro. The vector encoding the catalytic subunit of PKA (pMT-CEV) was a gift from S. McKnight (University of Washington, Seattle), and pGSK-3β was kindly provided by P. Klein (University of Pennsylvania).

AP Assay.

10T1/2 cells were cultured in DMEM with 0.5% FBS with 5 μg/ml N-Shh or vehicle and treated with or without 15 μM LY294002. After 5 days, cells were sonicated in 0.1 M Tris·HCl (pH 7.5) and 0.1% Triton X-100. AP activity in lysates was determined by incubation at 37°C in 0.1 M Tris·HCl (pH 9.5), 1 mM MgCl₂, and 1 mg/ml p-nitrophenylphosphate. Phosphate release was determined spectrophotometrically at 405 nm. Results were expressed as AP relative units per mg of protein.

Neural Plate Explant Culture.

Fertilized chick eggs were incubated at 38°C in a humidified incubator until developmental stage 11. Embryos were collected into L15 medium, and a region of the neural plate adjacent to newly formed four somites and presomitic mesoderm was isolated. The dissected neural plate was treated with Dispase (Roche Molecular Biochemicals; 2 mg/ml in L15) at 37°C for 6–10 min to remove somites, presomitic mesoderm, and ectoderm. Neural plate/notochord explants were cultured for 48 h in Neurobasal medium with B-27 (Invitrogen) in 24-well plates precoated with 2 μg/ml fibronectin. For BrdUrd labeling, 1 mg/ml BrdUrd was added to the neural plate culture for 1 h.

Immunostaining of Cultured Neural Plate Explants.

Cultured explants were fixed with 4% paraformaldehyde for 2 h at 4°C and washed three times for 30 min each in PBS (pH 7.4). Explants were blocked in 20% goat serum in PBS, 0.2% Triton X-100, and 0.2% Tween 20 (pH 7.4) for 2 h at room temperature and then incubated with primary and secondary antibodies overnight at 4°C with gentle agitation. Then explants were washed in PBS four times for 1 h at room temperature. After staining, explants were incubated in DAPI (Roche Molecular Biochemicals; 1:10,000 in PBS) for 20 min to visualize nuclei. To quantify cell numbers, explants were embedded in OCT compound at −25°C, cut in serial 10-μm sections, and immunostained with antibodies and DAPI. For BrdUrd staining, sections were washed in PBS, treated with 2 M HCl for 20 min, and incubated with the antibodies. Staining was examined on a Zeiss Axiophot epifluorescense microscope.

Abbreviations

Hh

Hedgehog

Shh

Sonic Hedgehog

N-Shh

recombinant Shh

PI3-kinase

phosphoinositide 3-kinase

PKA

protein kinase A

PSM

PKA site mutations

GSK-3β

glycogen synthase kinase 3β

GSM

GSK-3β site mutations

IGF

insulin-like growth factor

LY294002

2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one

Ptc

Patched

Smo

Smoothened

siRNA

small interfering RNA

dnAkt

dominant negative Akt

AP

alkaline phosphatase

RLU

relative luciferase units.