Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2009 Sep 1.
Published in final edited form as: Dev Biol. 2008 Jun 18;321(1):188–196. doi: 10.1016/j.ydbio.2008.06.014

The decoupling of Smoothened from Gαi proteins has little effect on Gli3 protein processing and Hedgehog-regulated chick neural tube patterning

Wee-Chuang Low 1,$, Chengbing Wang 1,$, Yong Pan 1,$, Xin-Yun Huang 2, James K Chen 4, Baolin Wang 1,3,*
PMCID: PMC2597282  NIHMSID: NIHMS67575  PMID: 18590719

Abstract

The Hedgehog (Hh) signal is transmitted by two receptor molecules, Patched (Ptc) and Smoothened (Smo). Ptc suppresses Smo activity, while Hh binds Ptc and alleviates the suppression, which results in activation of Hh targets. Smo is a seven-transmembrane protein with a long carboxyl terminal tail. Vertebrate Smo has been previously shown to be coupled to Gαi proteins, but the biological significance of the coupling in Hh signal transduction is not clear. Here we show that although inhibition of Gαi protein activity appears to significantly reduce Hh pathway activity in Ptc−/− mouse embryonic fibroblasts and the NIH3T3-based Shh-light cells, it fails to derepress Shh- or a Smo agonist-induced inhibition of Gli3 protein processing, a known in vivo indicator of Hh signaling activity. The inhibition of Gαi protein activity also cannot block the Sonic Hedgehog (Shh)-dependent specification of neural progenitor cells in the neural tube. Consistent with these results, overexpression of a constitutively active Gαi protein, Gαi2QL, cannot ectopically specify the neural cell types in the spinal cord, whereas an active Smo, SmoM2, can. Thus, our results indicate that the Smo induced Gαi activity plays an insignificant role in the regulation of Gli3 processing and Shh-regulated neural tube patterning.

Keywords: Hedgehog, Smoothened, G protein, Gli3, neural tube

Introduction

The Hedgehog (Hh) family of secreted molecules plays an important role in the patterning of the central nervous system, limb, and many other embryonic structures (Hooper and Scott, 2005). The Hh signal is transmitted by two receptor molecules, Patched (Ptc) and Smoothened (Smo). Ptc is a twelve-pass membrane protein, and Smo is a seven transmembrane protein that resembles G protein-coupled receptors (GPCRs)(Kalderon, 2000). In the absence of the Hh signal, Ptc suppresses Smo activity and hence Hh signaling activity. As a result, most of the full-length Gli3 transcription factor and, to a much lesser extent, Gli2 undergo proteasome-mediated processing into transcriptional repressors (Pan et al., 2006; Wang et al., 2000). When the Hh ligand is present, it binds Ptc and relieves its suppression on Smo so that the processing of Gli2 and Gli3 is inhibited and the full-length Gli2 and Gli3 are activated, which in turn activates their downstream transcriptional targets. The regulation of Gli2 and Gli3 processing is therefore a key step in the Hh signaling pathway, and the levels of the Gli2 and Gli3 repressors are direct indicators of Hh pathway activity.

Because of the structural similarity between Smo and GPCRs, Smo has long been suspected to couple with G proteins. Indeed, several studies using pharmacological treatment and overexpression have suggested that vertebrate Smo can be coupled to different types of G proteins (DeCamp et al., 2000; Hammerschmidt and McMahon, 1998; Kanda et al., 2003; Kasai et al., 2004), and a recent study showed that the stimulated Smo or a constitutively active form of Smo can activate Gαi proteins in isolated cell membranes (Riobo et al., 2006). However, the biological significance of the coupling of Smo to G proteins remains to be determined for the following reasons. First, most of these studies were carried out in cell culture wherein Hh pathway components were over expressed, and/or relied on a synthetic Gli-dependent reporter, non-endogenous or indirect endogenous molecular indicators as readouts for Hh signaling activity. Second, only a minor increase of GTPase activity was detected when the membranes of untransfected mouse embryonic fibroblasts (MEFs) were incubated with the Smo agonist purmorphamine. Third, so far there is no loss-of-function study of the in vivo role of Gαi proteins in Hh signaling.

Besides coupling to G proteins, the C-terminus of Smo is clearly required for Hh signaling. This is evidenced by the fact that a Smo lacking its C-terminus completely loses its ability to elicit Gli proteins to activate a Gli-dependent reporter (Riobo et al., 2006; Varjosalo et al., 2006). Nevertheless, overexpression of a membrane-tethered Smo C-terminus alone is not sufficient to result in an abnormal phenotype in transgenic mice. These studies suggest that both G protein coupling and the C-terminus-dependent activity are necessary for Smo function.

In this study, we used several in vivo Hh signaling indicators—the level of Gli3 processing, neural tube patterning, and expression of the endogenous Hh target gene Gli1—to determine how important the G-protein-coupled Smo activity is in Hh signal transduction. We found that although inhibition of Gαi protein activity can partially suppress expression of reporter genes and Hh target gene Gli1 in mouse embryonic fibroblasts (MEFs) and NIH3T3-based Shh-light cells, it cannot reverse the Hh- and Smo-agonist- stimulated suppression of Gli3 processing. Suppression of Gαi activity has little effect on the specification of ventral neural cell types in developing spinal cord. Consistent with these observations, over expression of a constitutively active form of the Gαi protein failed to induce the development of neuronal cell types in the ventral spinal cord, even though an active Smo was able to do so. Thus, our data indicate that the activation of Gαi proteins by Smo is not essential for the regulation of Gli3 processing and Shh-regulated neural tube patterning.

Materials and Methods

Materials

Cyclopamine (CPM) was obtained from LC Laboratories (Woburn, MA) and Forskolin (FSK) from Calbiochem. [35S]GTPγS [1,300 Ci/mmol (1Ci = 37GBq)] and [32P]-NAD were purchased from PerkinElmer Life Sciences (Boston, MA). Cholera toxin (CTX), pertussis toxin (PTX), and PTX catalytic S1 subunit were obtained from List Biological Laboratories, Inc. The Gli3 antibody and SAG were previously described (Chen et al., 2002b; Wang et al., 2000).

Constructs, infection, transfection, cell culture, and semi-quantitative RT-PCR

SmoM2 was created by a PCR-based mutagenesis method. SmoWT and SmoM2 were cloned into the EcoRI-HindIII sites of pRK (a CMV-based vector) and the EcoRI-XhoI sites of the pBI-GFP retroviral vector (kindly provided by Jonathan S. Bogan, Yale School of Medicine). Gαi2QL and Gα13QL are constitutively active forms of Gαi2 and Gα13. The retrovirus stock was prepared by the transfection of Phoenix cells (kindly provided by Garry Nolan, Stanford University) with pB-Smo-I-GFP or pB-SmoM2-I-GFP, together with the pEco package viral vector (kindly provided by Robert K. Naviaux, UC San Diego) using a calcium phosphate precipitation method. Shh-light cells and Ptc−/− mouse embryonic fibroblasts (MEFs) were earlier described (Taipale et al., 2000). Chick limb bud micromass cultures were prepared from HH-22-23 chick embryos and grown in F-12 with N2 supplement (Wang et al., 2000). Mouse embryonic fibroblasts were prepared from E10.5-11.5 mouse embryos and grown in DMEM with N2 supplement. HEK293 and Ptc−/− cells were cultured in DMEM with 10% fetal bovine serum (FBS) and penicillin/streptomycin at 37°C with 5% CO2. HEK293 cells were transfected with the pRK-Smo and pRK-SmoM2 constructs with a calcium phosphate precipitation method. For viral infection, Shh-light cells were plated at about 70% confluence, infected with the pBI-GFP, pBI-Smo-GFP, or pBI-SmoM2-GFP virus the next morning. After reaching 100% confluence, the medium was changed to 0.5% calf serum with or without each of the following, or a combination thereof: ShhN-conditioned medium (1/10 v/v), SAG (300 nM), PTX (100 ng/ml), CTX (200 ng/ml), FSK (40 µM), cyclopamine (5 µM), and was left for an additional 24 hrs. Firefly and Renilla luciferase activities were determined in lysates with the Dual Luciferase Reporter Assay system (Promega). After normalized against Renilla luciferase activities, firefly luciferase activities from at least three independent experiments were calculated to obtain the average and standard deviation. Treatment of Ptc−/− cells was similar to that of Shh-light cells. The β-galactosidase activity in Ptc−/− MEFs was assayed using the o-nitro-phenyl-8-D-galactopyranoside (ONPG) substrate.

To determine levels of Gli1 and Ptc RNA expression, total RNA was prepared from cultured cells using Trizol solution (Invitrogen), and 5µg of the total RNA was used to synthesize the first strand cDNA using Superscript II reverse transcriptase (Invitrogen). Semi-quantitative RT-PCR was performed using 1/5 of the cDNA and primers specific for Gli1, Ptc, and NADPH cDNAs.

[35S]GTPγS binding assay

Cell membranes were prepared from HEK293 cells transfected with either an empty vector, Smo, or SmoM2 expression constructs. The [35S]GTPγS binding assay was performed as described(Cussac et al., 2002), using 20 µg membrane per binding reaction.

ADP-ribosyltransferase assays

Shh-light cells or chick limb bud (HH 22–23) micromass culture were treated with PTX (100 ng/ml) for 3 hrs prior to the preparation of cell lysates. The PTX treated cells or chick spinal cords electroporated with CMV-PTX-S1wt or mutant constructs were washed in PBS and lysed in 0.5% Triton X-100 lysis buffer for 30 min on ice. The lysates were cleared by centrifugation and the supernatant was stored at −80°C until the assay. The assay was performed as described (Castro et al., 2001). Briefly, the ADP-ribosylation assay contained, in 25 µl, 0.1 M Tris-HCl (pH7.5), 20 mM dithiothreitol (DTT), 0.1 mM ATP, 0.1 µM [32P]-NAD, 10 ng PTX catalytic S1 subunit, and an aliquot of the protein supernatant. The mixture was incubated for 90 min at 25°C; phosphorylated proteins in the mixture were separated by 12% SDS-PAGE and detected by autoradiography.

In ovo electroporation and immunohistochemistry

For electroporation, 2 µg each of pRK-Smo, pRK-SmoM2, CMV-Gαi2QL, CMV-Gα13GL, CMV-PT-S1 wild-type (wt), or mutant (kindly provided by Nicholas Carbonetti at University of Maryland) (Castro et al., 2001) was mixed with 2 µg of pEGFP for a final concentration of 2 µg/µl, and the DNA mix was electroporated into one side of the neural tube of chick embryos at HH stage 12–14. The electroporation was performed with five 40 V pulses for 50 mseconds at 100-msecond intervals and the two electrodes 0.4 cm apart. Embryos were sacrificed 24 hours later and processed for fixation, embedding, cross sectioning, and immunostaining analysis. Antibodies used were GFP polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and monoclonal antibodies Nkx2.2, Nkx6.1, MNR2, and Pax7 (Developmental Studies Hybridoma Bank, Iowa University, Iowa). All secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA).

Results

Overexpressed Smo activates G proteins

A recent study has shown that Smo activates Gαi proteins (Riobo et al., 2006). To confirm this, we performed a [35S]guanosine 5’-(3-O-thio) triphosphate (GTPγS) substrate binding assay, in which the activation of G proteins by Smo was determined in isolated cell membranes by means of guanine nucleotide exchange. In this assay, the amount of GTPγS substrate binding to the Gαi proteins was measured as the levels of G protein activation, since the GTPγS is not hydrolysable (Cussac et al., 2002). The membranes were prepared from HEK293 cells that had been transfected with either an empty vector, a wild type Smo, or a constitutively active Smo (SmoM2) expression construct (Taipale et al., 2000). Overexpression of Smo and SmoM2 alone resulted in about a 1.5- and 3-fold increase, respectively, as compared to the basal level of GTPγS binding. Incubation of a Smo agonist, SAG (Chen et al., 2002b), increased the GTPγS binding capacity to more than twofold in the Smo-expressing membranes (P = 0.0166), but it failed to do so in the SmoM2-expressing membranes, while the addition of cyclopamine, a Smo antagonist (Chen et al., 2002a), resulted in inhibition of GTPγS binding in the membranes containing exogenous Smo (P = 0.0247), but not SmoM2 (Fig. 1). Together, these data indicate that Smo can activate Gαi proteins in vitro and that SmoM2, when over expressed, is not sensitive to SAG.

Figure 1.

Figure 1

Open in a new tab

Smo activates G proteins in vitro. HEK293 cells were separately transfected with either an empty vector, Smo or SmoM2. Two days later, membranes were isolated from the cells and [35S]GTPγS binding was performed in the presence of the indicated chemicals. The results presented were obtained from three independent experiments.

Pertussis toxin (PTX) treatment significantly reduces a Gli-dependent reporter activity in the NIH3T3-based Shh-light cells

PTX catalyzes the ADP-ribosylation of the α subunits of the heterotrimeric guanine nucleotide regulatory proteins, Gαi, Gαo, and Gαt (Bokoch et al., 1983; Codina et al., 1983), and it has been widely used to decouple Gαi proteins from receptors. To ascertain that PTX inhibits Gαi protein activity in the NIH3T3-based Shh-light cells (Taipale et al., 2000), we first performed ADP-ribosylation assay, which has been widely used to determine the efficacy of inhibition of Gαi activity by PTX (Castro et al., 2001; Xu and Barbieri, 1995). In this assay, post-nuclear supernatant (PNS) were prepared from Shh-light cells that were pre-incubated with or without PTX. If PTX entered the cells and catalyzed ADP-ribosylation of Gαi proteins, there would be no or a little Gαi proteins in PNS that were available for in vitro ADP-ribosylation. Thus, when the PNS was incubated with PTX catalytic S1 subunit and 32P-labeled NAD in vitro, there would be no or a little Gαi proteins that were labeled, since they had already been modified by the PTX that was pre-incubated in the culture. As expected, only very small amount of Gαi proteins (41 kD in size) were ribosylated by 32P-NAD in PNS prepared from cells pre-incubated with PTX, but strong labeling was observed in PNS from cells without PTX treatment (Fig. 2A). This result indicates that PTX can indeed inhibit Gαi protein activity in Shh-light cells, thus decoupling the Gαi from receptors.

Figure 2.

Figure 2

Open in a new tab

Smo and SmoM2 induced Gli-dependent luciferase activity was inhibited by pertussis toxin (PTX), cholera toxin (CTX), forskolin (FSK), and cyclopamine (CPM). (A) ADP-ribosylation assay of Shh-light cells pre-incubated with or without PTX. This 32P-labeled band (approximately 41 kD) presumably corresponds to a mixture of Gαi2 and Gαi3 (Castro et al., 2001; Xu and Barbieri, 1995). See Materials and methods for details of the assay. (B) The reporter assay results. Shh-light cells, which contained a stably integrated Gli-dependent firefly luciferase reporter and a Renillar luciferase reporter control, were infected with a retrovirus containing Smo and SmoM2 cDNAs. After being treated with the indicated drugs for 36–48 hrs, the cells were lysed, and firefly and Renilla luciferase activities were measured. The results shown were obtained from three independent experiments.

We next wanted to determine whether the coupling of Smo to Gαi proteins is necessary for Hh signaling. To this end, NIH3T3-based Shh-light cells, which contain a stably integrated Gli-dependent luciferase reporter (Taipale et al., 2000), were first infected with a retrovirus containing either no insert, Smo, or SmoM2 cDNAs. The infected cells were then treated with PTX, and the luciferase activity was measured. Over expression of Smo and SmoM2 activated the reporter to an average of 3.5- and 18.2-fold, respectively, but PTX treatment inhibited the reporter activity to 2.3- (P = 0099) and 11.3-fold (P = 0.0096), respectively (Fig. 2B), indicating that G protein coupling is required for Hh pathway activation in this particular cell line on the basis of this synthetic reporter. To verify that the reporter activity is specifically induced by Smo and SmoM2 expression, the cells were treated with the Smo antagonist cyclopamine (CPM). As expected, CPM treatment brought the reporter activity to a nearly basal level (Fig. 2B).

Protein kinase A (PKA) phosphorylates Gli2 and Gli3 C-termini and induces a proteolytic processing into transcriptional repressors (Pan et al., 2006; Tempe et al., 2006; Wang et al., 2000), which in turn suppress Hh signaling. To determine whether the Smo-induced reporter activity is dependent on Gli activity, the infected cells were incubated with forskolin (FSK) and cholera toxin (CTX), both of which activate PKA by increasing cAMP levels in the cells, although to different extents and through different mechanisms (Seamon and Daly, 1981; Van Heyningen and King, 1975). As predicted, both FSK and CTX treatments effectively inhibited the luciferase reporter activity (Fig. 2B), indicating that the Smo-induced reporter activation is dependent on Gli transcriptional activity.

Although the above results suggest that Smo is coupled to G proteins, overexpression may be forcing such a coupling. To rule out this possibility, the same experiments were repeated using Shh-light cells stimulated with either ShhN-conditioned medium or SAG. Incubation of the cells with ShhN protein and SAG resulted in an activation of the luciferase reporter 10 and 25 times the normal activity, respectively. PTX treatment was still able to significantly suppress the ShhN- and SAG-induced reporter activity—from 10 times to 6 (P = 0.0026) and 25 times to 15 (P = 0.0006), respectively. Similarly, the activation of the luciferase reporter by ShhN and SAG is dependent on endogenous Smo and Gli activity as the reporter activity could be inhibited by CPM, CTX, and FSK (Fig. 3A).

Figure 3.

Figure 3

Open in a new tab

Hh signaling in Shh-light cells was inhibited by PTX, CTX, FSK, and CPM. (A) The Gli-dependent luciferase reporter assay. Shh-light cells were treated with either ShhN protein or SAG together with the indicated agents,; otherwise, the experiment was performed as that in figure 2. (B) Semi-quantitative RT-PCR of Gli1 and Ptc. Total RNA was isolated from Shh-light cells treated with pharmacological agents as indicated above the panel and subjected to semi-quantitative RT-PCR using Gli1, Ptc, and GAPDH (control) specific primers. Histogram shows the relative intensity of bands.

To ascertain that the effect of these pharmacological agents on the reporter activity is dependent on Hh signaling, we also examined expression of Gli1 and Ptc RNAs, two transcriptional targets of Hh signaling. Consistent with reporter assay results, semi-quantitative RT-PCR analysis revealed that SAG-induced Gli1 expression was inhibited partially by PTX, CTX, and FSK, and completely by CPM, and the similar inhibition of Ptc RNA expression was observed with these drugs except PTX (Fig. 3B). Therefore, Smo coupling to the Gαi proteins activates Gli-dependent luciferase reporter in Shh-light cells.

PTX treatment partially inhibited Ptc-LacZ reporter activity in Ptc mutant mouse embryonic fibroblasts (MEFs)

Loss of Ptc gene function leads to full activation of the Hh pathway in the absence of Hh (Goodrich et al., 1997), and Ptc is also a transcriptional target of Hh signaling (Goodrich et al., 1996). To more stringently test whether the Smo-Gαi protein coupling is required for Hh signal transduction, we took advantage of Ptc−/− MEFs that contained a lacZ knock-in reporter gene in place of the Ptc locus. In these cells, the lacZ gene is driven by the endogenous mouse Ptc promoter and is fully activated (Taipale et al., 2000), owing to a loss of Ptc gene function. We initially assayed the β-galactosidase activity in Ptc−/− MEFs treated without or with PTX, CTX, FSK, or CPM. The effect of these pharmacological agents on β-galactosidase activity was less pronounced than that on luciferase reporter in Shh-light cells, but it was still statistically significant (P = 0.0001 for PTX and control) (Fig. 4A). Since these two reporters are not comparable, we also determined Gli1 RNA expression in Ptc−/− MEFs by semi-quantitative RT-PCR analysis. Treatment with PTX, CTX, or FSK reduced Gli1 RNA expression by about a half in these cells, whereas the CPM incubation almost completely suppressed its expression, indicating that it is very potent (Fig. 4B, lanes 3–6). Interestingly, the SAG treatment was still able to induce Gli1 expression nearly two times as the control (compare lane 2 to lane 1), indicating that SAG-induced Smo activity can exceed Smo activity induced by loss of Ptc. Together, these data indicate that, as in Shh-light cells, Smo mediated activation of Ptc-lacZ reporter in Ptc−/− MEFs is partially dependent on Gαi protein activity.

Figure 4.

Figure 4

Open in a new tab

PTX treatment reduced Hh signaling in Ptc−/− MEFs. (A) PTX treatment had little effect on the Ptc-driven lacZ reporter activity in Ptc−/− mutant cells. The β-galactosidase activity in Ptc−/− MEFs was measured, following incubation with SAG, PTX, CTX, FSK, or CPM overnight or for 36–48 hours. The β-galactosidase activity was normalized to the protein amount of cell lysates and derived from three independent experiments. (B) Semi-quantitative RT-PCR of Gli1 and GAPDH (control) in Ptc−/− MEFs treated with the indicated chemicals. Histogram shows the relative intensity of bands. (C) In vitro ADP-ribosylation assay of Ptc−/− MEFs preincubated with or without PTX. See Materials and methods and figure 2A legend for details of the assay.

PTX treatment fails to derepress the Shh-induced suppression of Gli3 processing

Gli3 protein processing is inhibited by Hh signaling and the extent of Gli3 processing is a hallmark of Hh signaling activity in vivo (Wang et al., 2000). We thus wanted to determine whether inhibition of Gli3 processing by Shh signaling is dependent on Smo-Gαi coupling. We first treated Shh-responsive chick limb bud micromass culture with ShhN-conditioned medium alone, or together with either PTX, CTX, or CPM. The extent of Gli3 processing was then evaluated by Western blot with an N-terminal Gli3 antibody, which detected both the full-length and processed forms of Gli3 (Wang et al., 2000). As shown in the left panels of figure 5A and B, ShhN stimulation suppressed Gli3 processing very effectively as measured by the presence of only residual amounts of the Gli3 repressor forms, Gli3-83 and Gli3-75. Addition of PTX did not detectably reverse the ShhN-induced inhibition of Gli3 processing; CTX treatment did, but only slightly, and cyclopamine fully derepressed the suppression. The inability of PTX to derepress the Shh-stimulated suppression of Gli3 processing was not because PTX was not active, since ADP-ribosylation assay revealed that the PTX used was able to modify the Gαi proteins in the chick limb bud micromasses (Fig. 5C).

Figure 5.

Figure 5

Open in a new tab

PTX treatment failed to derepress Shh- or SAG-induced suppression of Gli3 processing. (A) Immunoblots show the full-length and processed forms of Gli3 (Gli3-190, Gli3-83, Gli3-75) in chick limb bud micromass cells (left panel), E11.5 mouse embryonic fibroblasts (MEFs, middle panel), and Ptc−/− MEFs (right panel). Cells were incubated overnight with the medium containing the indicated components above the panels. (B) Histograms show the ratio of the Gli3-83 repressor to Gli3-190 activator from panel A. (C) In vitro ADP-ribosylation assay of chick limb bud micromasses preincubated with or without ShhN and/or PTX. See Materials and methods and figure 2A legend for details of the assay.

We also repeated the same experiment using SAG as a stimulus. Unfortunately, SAG failed to inhibit Gli3 processing, which is most likely due to its inability to bind chicken Smo (data not shown). We thus used primary MEFs prepared from mouse embryos at gestation day 11.5 (E11.5), a stage at which most tissues respond to the Shh signal, to test if PTX treatment would derepress SAG-induced inhibition of Gli3 processing. In these MEFs, Gli3 processing is not as efficient as that in chick limb bud cells (Fig. 5A, compare lane 5 to lane 6). However, as in the chick limb bud micromass culture, PTX treatment was unable to derepress SAG-induced inhibition of Gli3 processing, while CPM could effectively reverse the SAG-elicited suppression of Gli3 processing. The effect of CTX was undetectable, which is likely due to the low level of the Gli3-83 repressor (Fig. 5A and B, middle panels). The inability of PTX treatment to derepress Shh-signaling induced suppression of Gli3 processing was also found in Shh-light cells and Ptc−/− MEFs (Fig. 5A and B, right panels, and data not shown). Together, these data indicate that the activation of Gαi proteins plays a minor or no role in Hh-regulated Gli3 processing in all the cell types examined.

Overexpression of a constitutively active Gαi protein or PTX catalytic S1 subunit had little effect on Hh-regulated neural tube patterning

In the spinal chord, Shh is expressed in the notochord and migrates to the ventral neural tube, where it induces all cell types in a concentration-dependent manner (Jessell, 2000). High concentrations of Shh induce the floor plate at the midline of the neural tube, and progressively lower levels of Shh specify V3 interneurons, motoneurons (MNs), V2, V1, and V0 interneurons in a ventral-to-dorsal order. Each of the cell types is marked by the expression of specific homeodomain (HD)-containing and basic helix-loop-helix (bHLH) transcription factors. Mutants that lack either Shh or Smo gene function fail to specify the floor plate, MNs, and V3-V2 interneurons and to pattern all five types of ventral neurons (Chiang et al., 1996; Wijgerde et al., 2002), while excessive Hh pathway activity leads to an ectopic expansion of these neurons (Cooper et al., 2005; Goodrich et al., 1997; Svard et al., 2006). Therefore, specification of the distinct ventral neurons indicates the levels of Hh signaling activity.

To investigate the potential involvement of Gαi in Hh signaling in vivo, we assayed the expression of ventral HD and bHLH fate determinants in the spinal cord of chick embryos electroporated with Gαi2QL (a constitutively active Gαi2), Gα13QL (a constitutively active Gα13), Smo, or SmoM2 expression constructs. Expression of these constructs was confirmed by immunostaining (Fig. 6Is, Js, and Fig. S1Ds). Electroporation of neither Gαi2QL, Gαl3QL, nor Smo induced ectopic expression of the HD and bHLH markers examined, which included Nkx2.2 (a V3 progenitor neuronal marker), MNR2 (a MN marker), and Nkx6.1 (a V3, MN, and V2 progenitor cell marker) (Fig. 6 As, Bs, Es, and Fig. S1As-Bs). However, electroporation of SmoM2 induced ectopic expression of all these transcription factors (Fig. 6Cs, Ds, and Gs). We also analyzed the expression of the Pax7 transcription factor in the dorsal neural tube that is suppressed by a low concentration of Hh ligand. Again, only SmoM2, but neither Gαi2GL, Gα13QL, nor Smo, was capable of inhibiting Pax7 expression (Fig. 6 Fs, Hs, and Fig. S1Cs). Taken together, these data indicate that activation of Gαi alone is not sufficient to elicit Hh signaling and that Smo needs to be activated to exert its full function.

Figure 6.

Figure 6

Open in a new tab

Misexpression of the active SmoM2, not active Gαi2QL, ectopically activated the expression of ventral neural transcription factors and suppressed a dorsal neural marker. The neural tubes of HH stage 12–14 embryos were electroporated with SmoM2 or Gαi2QL together with GFP expression constructs, and assayed 24h later for the expression of the indicated transcription factors (red) and GFP expression (green). Missexpression of the active SmoM2 resulted in an ectopic expression of Nkx2.2 (a V3 progenitor marker) (Cs), Nkx6.1 (a marker for V3, MNs, and V2 progenitors)(Ds), and MNR2 (a MN marker)(Gs), but it suppressed the expression of the dorsal marker Pax7 (indicated by an arrow in H’). Note that the loss of Pax7 expression corresponded to the GFP expression (compare H with H’ and H”). Missexpression of Gαi2QL failed to do so (As, Bs, Es, and Fs). (Is and Js) Immunostaining showing coexpression of GFP and Gαi2QL or SmoM2 (detected using myc antibody).

To address the question whether Shh-regulated neural tube patterning requires Gαi protein activity, CMV-based expression constructs for wild-type (wt) or mutant (catalytically inactive) PTX catalytic S1 subunit were electroporated into chick neural tube. These two constructs have been successfully used to study Gαi coupled GPCR functions in mammalian cells (Castro et al., 2001). We first determined whether wt S1 subunit was active in the electroporated chick neural tube using the in vitro ADP-ribosylation assay. Since it is technically difficult to separate a small spinal cord into left and right halves, whole spinal cord fragment from neck to hind-limb was arbitrarily collected and used to prepare post-nuclear supernatant (PNS). The results showed that the labeling of Gαi proteins by 32P-NAD was weaker in the PNS prepared from spinal cord fragments transfected with wt S1 construct than that with mutant S1 subunit (Fig. 7G). Since only one side of a small area of chick spinal cord was usually electroporated, this result suggests that wt and mutant S1 subunits were expressed and the former was functional, although the extent of inhibition of Gai activity by overexpression of wt S1 cannot be determined. We next examined the expression of several neural markers in the neural tubes that were electroporated. We found that overexpression of either wt or mutant PTX S1 subunit did not affect the expression and patterning of neuronal markers that were examined, including Nkx2.2 (V3 interneuron marker), Isl1 and MNR2 (motoneuron markers), Lhx3 (V2 interneuron), Nkx6.1 (V1-V3), Pax6, and Pax7 (Fig. 7As-Fs and data not shown). From these results, we conclude that the coupling of Smo to Gαi proteins is not essential for the Shh-dependent specification of V1-V3 neuronal cell types in the neural tube.

Figure 7.

Figure 7

Open in a new tab

Overexpression of PTX catalytic S1 subunit did not inhibit the Shh-regulated specification of neuronal cell types in chick neural tube. Chick neural tubes (see materials and methods for details) were electroporated with PTX-S1 wt or mutant (not shown) together with GFP expression constructs and assayed 24 h later for the expression of the molecular markers (red) and GFP (green) by immunofluorescence (As-Fs). See figure 6 legend for molecular markers. (G) Autoradiogram of the in vitro ADP-ribosylation assay. Expression of wt PTX-S1 reduced the labeling of Gαi proteins by 32P-NAD compared to mutant PTX-S1.

Discussion

Several studies suggested that Smo, when overexpressed, could couple to the Gαi, Gα12, and Gα15 proteins (DeCamp et al., 2000; Hammerschmidt and McMahon, 1998; Kanda et al., 2003; Kasai et al., 2004). In addition, a recent study showed that vertebrate Smo activates Gαi proteins in isolated membranes (Riobo et al., 2006). However, the in vivo physiological significance of the coupling of Smo to the G proteins is not clear. In this study, we examined several known in vivo indicators for Hh signaling activity and investigated the effect of the coupling of Smo to Gαi proteins on Hh-regulated embryonic patterning. We found that when Gli1 expression was assayed for Hh pathway activity, inhibition of Gαi protein activity by PTX significantly reduced Hh pathway activity in Shh-light cells and Ptc−/− MEFs (Fig. 3B and Fig. 4B). However, PTX treatment appeared to have minimal effect, if any, on Ptc RNA expression. This may reflect the differences in responsiveness of these two genes to Gai-dependent Smo activity. PTX treatment could not also derepress the Shh- or SAG-stimulated suppression of Gli3 processing in all the cell types examined in this study (Fig. 5A and B). In addition, although SmoM2 was able to induce ectopic specification of neuronal cell types in the chicken spinal cord, the constitutively active Gαi2QL protein failed to do so (Fig. 6). Furthermore, overexpression of PTX catalytic S1 subunit was also unable to suppress the Shh-regulated specification of neural progenitor cells in the developing chick neural tube (Fig. 7As-Fs). Our data therefore indicate that the coupling of Smo to Gαi proteins is not essential for the Shh-regulated Gli3 processing and neural tube patterning. In this sense, it is similar to Smo in Drosophila, in which knocking down G proteins does not seem to affect Hh signal transduction (Lum et al., 2003a).

Hh signaling is required for both the suppression of Gli3 processing and activation of full-length Gli proteins. A recent study of mutations in the Arl13b gene, which encodes a small GTPase, has demonstrated that the regulation of Gli3 processing and the production of full-length Gli activators are two separable molecular processes (Caspary et al., 2007). Here we show that although inhibition of Gαi protein activity can partially block Hh signaling as measured by the reduction of reporter gene activity and Gli1 RNA expression in Shh-light cells and Ptc−/− MEFs, it fails to reverse the Shh- or SAG-induced inhibition of Gli3 processing. Thus, Gαi protein activity appears to be required for the activation of full-length Gli proteins but not for the suppression of Gli3 processing in these cell types. It would be interesting to determine whether the Arl13b function is dependent on Gαi protein activity that is coupled to Smo.

The inability of PTX treatment to restore Gli3 processing that is inhibited by Shh signaling is likely a general mechanism rather than cell type specific, since this phenomenon is found in all the cell types that we have examined. However, the different requirement of Gαi protein activity for the activation of Hh pathway between MEFs and spinal cord may be attributed to differences in molecular markers. Gli1 and Ptc are often used as markers for Hh signaling activity, but their expression may not necessarily correlate directly with the expression of neuronal markers in spinal cord used to determine the Hh-regulated neural tube patterning. It is also possible that the different effect of PTX treatment on Hh signaling in MEFs and neural tube may simply reflect differences in in vitro and in vivo or cell types. Determining the biological significance of the coupling of Smo to Gαi proteins in Hh signal transduction will ultimately rest on the analysis of Hh-regulated patterning of animals lacking Gαi functions.

Although the role of the coupling of Smo to Gαi proteins in Hh signaling remains enigmatic, the importance of the Smo C-terminal tail in Hh signal transduction has been well documented. Both Drosophila and vertebrate Smo lacking their C-terminal tails are inactive, even though expression of the membrane-tethered C-terminal tail alone is not sufficient to fully activate the Hh pathway (Hooper, 2003; Riobo et al., 2006; Varjosalo et al., 2006). The C-terminus of fly Smo recruits the cytoplasmic Hh signaling complex through its direct interaction with Costal 2 (Cos2), a kinesin-like molecule (Jia et al., 2003; Lum et al., 2003b; Ogden et al., 2003; Ruel et al., 2003). It is also extensively phosphorylated by protein kinase A (PKA) and, subsequently, by casein kinase 1 (CK1), an event required for Hh signaling (Apionishev et al., 2005; Jia et al., 2004; Zhang et al., 2004; Zhou et al., 2006).

However, the mechanisms by which the C-terminus of vertebrate Smo is involved in Hh signaling is less clear since it does not appear to recruit Kif7 and Kif27, two vertebrate homologs of the fly Cos2 (Varjosalo et al., 2006), and lacks the PKA sites present in the fly Smo C-terminus. Nonetheless, vertebrate Smo is phosphorylated by GRK2 (GPCR kinase 2) (Chen et al., 2004; Meloni et al., 2006), presumably in its C-terminus, by analogy to other seven-transmembrane GPCRs. Phosphorylated Smo recruits β-arrestin2, which promotes the internalization of Smo (Chen et al., 2004). It is probably the Smo C-terminal-dependent activity that suppresses Gli3 processing, since the Gαi-dependent Smo activity does not appear to be required for the inhibition of Gli3 processing.

Supplementary Material

01

Figure S1. Misexpression of Gα13QL failed to ectopically activate ventral neural transcription factors and suppressed a dorsal neural marker. Chick neural tubes were electroporated with Gα13QL and GFP expression constructs and assayed for expression of the indicated markers using immunofluorescent staining (As-Cs). (Ds) Coexpression of Gα13QL and GFP detected by myc and GFP antibodies, respectively.

Acknowledgments

We thank Philip Beachy for the Shh-light2 cells, Garry Nolan for the Phoenix cells, Nicholas Carbonetti for PTX S1 constructs, and Jonathan Bogan and Robert K. Naviaux for the retroviral vectors. The Mnr2, Pax7, Nkx2.2, and Nkx6.1 monoclonal antibody were obtained from the Developmental Studies Hybridoma Bank, Department of Biological Sciences, The University of Iowa, under contract NO1-HD-7-3263 from the NICHD. This study was supported by NIH grants to B.W.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Apionishev S, et al. Drosophila Smoothened phosphorylation sites essential for Hedgehog signal transduction. Nat Cell Biol. 2005;7:86–92. doi: 10.1038/ncb1210. [DOI] [PubMed] [Google Scholar]
  2. Bokoch GM, et al. Identification of the predominant substrate for ADP-ribosylation by islet activating protein. J Biol Chem. 1983;258:2072–2075. [PubMed] [Google Scholar]
  3. Caspary T, et al. The graded response to Sonic Hedgehog depends on cilia architecture. Dev Cell. 2007;12:767–778. doi: 10.1016/j.devcel.2007.03.004. [DOI] [PubMed] [Google Scholar]
  4. Castro MG, et al. Expression, activity and cytotoxicity of pertussis toxin S1 subunit in transfected mammalian cells. Cell Microbiol. 2001;3:45–54. doi: 10.1046/j.1462-5822.2001.00092.x. [DOI] [PubMed] [Google Scholar]
  5. Chen JK, et al. Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened. Genes Dev. 2002a;16:2743–2748. doi: 10.1101/gad.1025302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen JK, et al. Small molecule modulation of Smoothened activity. Proc Natl Acad Sci U S A. 2002b;99:14071–14076. doi: 10.1073/pnas.182542899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen W, et al. Activity-dependent internalization of smoothened mediated by beta-arrestin 2 and GRK2. Science. 2004;306:2257–2260. doi: 10.1126/science.1104135. [DOI] [PubMed] [Google Scholar]
  8. Chiang C, et al. Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature. 1996;383:407–413. doi: 10.1038/383407a0. [DOI] [PubMed] [Google Scholar]
  9. Codina J, et al. Pertussis toxin substrate, the putative Ni component of adenylyl cyclases, is an alpha beta heterodimer regulated by guanine nucleotide and magnesium. Proc Natl Acad Sci U S A. 1983;80:4276–4280. doi: 10.1073/pnas.80.14.4276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cooper AF, et al. Cardiac and CNS defects in a mouse with targeted disruption of suppressor of fused. Development. 2005;132:4407–4417. doi: 10.1242/dev.02021. [DOI] [PubMed] [Google Scholar]
  11. Cussac D, et al. Differential activation of Gq/11 and Gi(3) proteins at 5-hydroxytryptamine(2C) receptors revealed by antibody capture assays: influence of receptor reserve and relationship to agonist-directed trafficking. Mol Pharmacol. 2002;62:578–589. doi: 10.1124/mol.62.3.578. [DOI] [PubMed] [Google Scholar]
  12. DeCamp DL, et al. Smoothened activates Galphai-mediated signaling in frog melanophores. J Biol Chem. 2000;275:26322–26327. doi: 10.1074/jbc.M004055200. [DOI] [PubMed] [Google Scholar]
  13. Goodrich LV, et al. Conservation of the hedgehog/patched signaling pathway from flies to mice: induction of a mouse patched gene by Hedgehog. Genes Dev. 1996;10:301–312. doi: 10.1101/gad.10.3.301. [DOI] [PubMed] [Google Scholar]
  14. Goodrich LV, et al. Altered neural cell fates and medulloblastoma in mouse patched mutants. Science. 1997;277:1109–1113. doi: 10.1126/science.277.5329.1109. [DOI] [PubMed] [Google Scholar]
  15. Hammerschmidt M, McMahon AP. The effect of pertussis toxin on zebrafish development: a possible role for inhibitory G-proteins in hedgehog signaling. Dev Biol. 1998;194:166–171. doi: 10.1006/dbio.1997.8796. [DOI] [PubMed] [Google Scholar]
  16. Hooper JE. Smoothened translates Hedgehog levels into distinct responses. Development. 2003;130:3951–3963. doi: 10.1242/dev.00594. [DOI] [PubMed] [Google Scholar]
  17. Hooper JE, Scott MP. Communicating with Hedgehogs. Nat Rev Mol Cell Biol. 2005;6:306–317. doi: 10.1038/nrm1622. [DOI] [PubMed] [Google Scholar]
  18. Jessell TM. Neuronal specification in the spinal cord: inductive signals and transcriptional codes. Nat Rev Genet. 2000;1:20–29. doi: 10.1038/35049541. [DOI] [PubMed] [Google Scholar]
  19. Jia J, et al. Smoothened transduces Hedgehog signal by physically interacting with Costal2/Fused complex through its C-terminal tail. Genes Dev. 2003;17:2709–2720. doi: 10.1101/gad.1136603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jia J, et al. Hedgehog signalling activity of Smoothened requires phosphorylation by protein kinase A and casein kinase I. Nature. 2004;432:1045–1050. doi: 10.1038/nature03179. [DOI] [PubMed] [Google Scholar]
  21. Kalderon D. Transducing the hedgehog signal. Cell. 2000;103:371–374. doi: 10.1016/s0092-8674(00)00129-x. [DOI] [PubMed] [Google Scholar]
  22. Kanda S, et al. Sonic hedgehog induces capillary morphogenesis by endothelial cells through phosphoinositide 3-kinase. J Biol Chem. 2003;278:8244–8249. doi: 10.1074/jbc.M210635200. [DOI] [PubMed] [Google Scholar]
  23. Kasai K, et al. The G12 family of heterotrimeric G proteins and Rho GTPase mediate Sonic hedgehog signalling. Genes Cells. 2004;9:49–58. doi: 10.1111/j.1356-9597.2004.00701.x. [DOI] [PubMed] [Google Scholar]
  24. Lum L, et al. Identification of Hedgehog pathway components by RNAi in Drosophila cultured cells. Science. 2003a;299:2039–2045. doi: 10.1126/science.1081403. [DOI] [PubMed] [Google Scholar]
  25. Lum L, et al. Hedgehog signal transduction via Smoothened association with a cytoplasmic complex scaffolded by the atypical kinesin, Costal-2. Mol Cell. 2003b;12:1261–1274. doi: 10.1016/s1097-2765(03)00426-x. [DOI] [PubMed] [Google Scholar]
  26. Meloni AR, et al. Smoothened signal transduction is promoted by G protein-coupled receptor kinase 2. Mol Cell Biol. 2006;26:7550–7560. doi: 10.1128/MCB.00546-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ogden SK, et al. Identification of a functional interaction between the transmembrane protein Smoothened and the kinesin-related protein Costal2. Curr Biol. 2003;13:1998–2003. doi: 10.1016/j.cub.2003.10.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pan Y, et al. Sonic hedgehog signaling regulates Gli2 transcriptional activity by suppressing its processing and degradation. Mol Cell Biol. 2006;26:3365–3377. doi: 10.1128/MCB.26.9.3365-3377.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Riobo NA, et al. Activation of heterotrimeric G proteins by Smoothened. Proc Natl Acad Sci U S A. 2006;103:12607–12612. doi: 10.1073/pnas.0600880103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ruel L, et al. Stability and association of Smoothened, Costal2 and Fused with Cubitus interruptus are regulated by Hedgehog. Nat Cell Biol. 2003;5:907–913. doi: 10.1038/ncb1052. [DOI] [PubMed] [Google Scholar]
  31. Seamon KB, Daly JW. Forskolin: a unique diterpene activator of cyclic AMP-generating systems. J Cyclic Nucleotide Res. 1981;7:201–224. [PubMed] [Google Scholar]
  32. Svard J, et al. Genetic elimination of Suppressor of fused reveals an essential repressor function in the mammalian Hedgehog signaling pathway. Dev Cell. 2006;10:187–197. doi: 10.1016/j.devcel.2005.12.013. [DOI] [PubMed] [Google Scholar]
  33. Taipale J, et al. Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine. Nature. 2000;406:1005–1009. doi: 10.1038/35023008. [DOI] [PubMed] [Google Scholar]
  34. Tempe D, et al. Multisite protein kinase A and glycogen synthase kinase 3beta phosphorylation leads to Gli3 ubiquitination by SCFbetaTrCP. Mol Cell Biol. 2006;26:4316–4326. doi: 10.1128/MCB.02183-05. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Van Heyningen S, King CA. Short communications. Subunit A from cholera toxin is an activator of adenylate cyclase in pigeon erythrocytes. Biochem J. 1975;146:269–271. doi: 10.1042/bj1460269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Varjosalo M, et al. Divergence of hedgehog signal transduction mechanism between Drosophila and mammals. Dev Cell. 2006;10:177–186. doi: 10.1016/j.devcel.2005.12.014. [DOI] [PubMed] [Google Scholar]
  37. Wang B, et al. Hedgehog-regulated processing of Gli3 produces an anterior/posterior repressor gradient in the developing vertebrate limb. Cell. 2000;100:423–434. doi: 10.1016/s0092-8674(00)80678-9. [DOI] [PubMed] [Google Scholar]
  38. Wijgerde M, et al. A direct requirement for Hedgehog signaling for normal specification of all ventral progenitor domains in the presumptive mammalian spinal cord. Genes Dev. 2002;16:2849–2864. doi: 10.1101/gad.1025702. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Xu Y, Barbieri JT. Pertussis toxin-mediated ADP-ribosylation of target proteins in Chinese hamster ovary cells involves a vesicle trafficking mechanism. Infect Immun. 1995;63:825–832. doi: 10.1128/iai.63.3.825-832.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zhang C, et al. Extensive phosphorylation of Smoothened in Hedgehog pathway activation. Proc Natl Acad Sci U S A. 2004;101:17900–17907. doi: 10.1073/pnas.0408093101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Zhou Q, et al. The contributions of protein kinase A and smoothened phosphorylation to hedgehog signal transduction in Drosophila melanogaster. Genetics. 2006;173:2049–2062. doi: 10.1534/genetics.106.061036. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01

Figure S1. Misexpression of Gα13QL failed to ectopically activate ventral neural transcription factors and suppressed a dorsal neural marker. Chick neural tubes were electroporated with Gα13QL and GFP expression constructs and assayed for expression of the indicated markers using immunofluorescent staining (As-Cs). (Ds) Coexpression of Gα13QL and GFP detected by myc and GFP antibodies, respectively.

NIHMS67575-supplement-01.pdf (90.8KB, pdf)

RESOURCES