Novel genes regulated by Sonic Hedgehog in pluripotent mesenchymal cells


Sonic Hedgehog is a secreted morphogen involved in patterning a wide range of structures in the developing embryo. Disruption of the Hedgehog signalling cascade leads to a number of developmental disorders and plays a key role in the formation of a range of human cancers. The identification of genes regulated by Hedgehog is crucial to understanding how disruption of this pathway leads to neoplastic transformation. We have used a Sonic Hedgehog (Shh) responsive mouse cell line, C3H/10T1/2, to provide a model system for hedgehog target gene discovery. Following activation of cell cultures with Shh, RNA was used to interrogate microarrays to investigate downstream transcriptional consequences of hedgehog stimulation. As a result 11 target genes have been identified, seven of which are induced (Thrombomodulin, GILZ, BF-2, Nr4a1, IGF2, PMP22, LASP1) and four of which are repressed (SFRP-1, SFRP-2, Mip1-γ, Amh) by Shh. These targets have a diverse range of putative functions and include transcriptional regulators and molecules known to be involved in regulating cell growth or apoptosis. The corroboration of genes previously implicated in hedgehog signalling, along with the finding of novel targets, demonstrates both the validity and power of the C3H/10T1/2 system for Shh target gene discovery.


Members of the hedgehog (Hh) protein family are potent secreted morphogens, involved in regulating a diverse range of developmental processes in the mammalian embryo. From the formation of organs such as lung and gut, to the development of bone and cartilage, control of CNS cell specification, patterning of the limbs, development of hair follicles and decisions of left-right asymmetry, signals from hedgehog molecules are vital (reviewed by Ingham and Mcmahon, 2001). In mammals the family is represented by three proteins, Desert (Dhh), Indian (Ihh) and Sonic Hedgehog (Shh), the latter of which appears to play the most widespread developmental role. Shh undergoes auto-cleavage and post-transcriptional modifications, including the addition of a cholesterol moiety, before the N-terminal active form (Shh-N) is released from secreting cells (Porter et al., 1996). The Hh signal is received by the transmembrane receptor Patched, and transmitted through a regulatory cascade. Upon binding Hh, Patched releases its inhibition of Smoothened (Smo), a transmembrane protein reminiscent of the frizzled family of Wnt receptors. Smo is then free to transmit the signal downstream, a major consequence being control of the Gli family of transcription factors, through which many of the effects of Hh are thought to be mediated.

Disruption of the Hh signalling pathway is a major determinant of tumour formation, particularly for the common skin cancer basal cell carcinoma (BCC). This was established from the discovery that Patched is mutated in the familial cancer predisposition disorder nevoid basal cell carcinoma syndrome (NBCCS), as well as in many sporadic BCCs (Hahn et al., 1996; Johnson et al., 1996). In addition to the role of Patched as a tumour suppressor, other members of the Hh pathway, including Smo and Gli1, have been implicated as oncogenes in a range of tumour types (reviewed by Wicking et al., 1999). Consistent with its pivotal role in embryonic development, aberrant hedgehog signalling is also associated with a range of human developmental anomalies (reviewed by Villavicencio et al., 2000).

The pluripotent mesenchymal mouse embryonic cell line C3H/10T1/2 (hereafter abbreviated to 10T1/2) is responsive to Shh stimulation, with pathway activation leading to cellular differentiation. A large proportion of Shh stimulated 10T1/2 cells enter the osteoblastic lineage, leading to a dramatic increase in alkaline phosphatase activity which can be readily assayed (Kinto et al., 1997; Nakamura et al., 1997; Spinella-Jaegle et al., 2001). Studies in 10T1/2 cells have lead to a greater understanding of the functions of Shh (Pepinsky et al., 1998, 2000; Williams et al., 1999; Saeki et al., 2000), Smo (Murone et al., 1999) and Gli family members (Ruiz i Altaba, 1999), as well as providing a means to investigate comparative effects of vertebrate Hh proteins (Pathi et al., 2001). In this study we have extended the use of 10T1/2 cells to provide a useful model system for the discovery of novel downstream target genes regulated by the Hh pathway.

Genes downstream of Shh are thought to provide the messages that tell cells how to differentiate or when to divide. Little is known about how dysregulation of these target genes is involved in neoplastic transformation. In order to answer this question we need to identify genes controlled through Hh signalling, and determine how their expression is affected by pathway activation. To date only a small number of target genes have been described in vertebrate tissues. These include HNF-3β (Roelink et al., 1995) and COUP-TFII (Krishnan et al., 1997) in the developing neural tube, SWiP-1 (Vasiliauskas et al., 1999) and SFRP-2 (Lee et al., 2000) in somitic mesoderm, and angiopoietin2 in developing vasculature (Pola et al., 2001). Other tissue-specific targets include members of the BMP (Bitgood and Mcmahon, 1995), PAX (Ericson et al., 1996), SOX (Hargrave et al., 2000) and TBX (Gibson-Brown et al., 1998; Garg et al., 2001) families. Hedgehog-interacting protein (HIP) is a target in a number of cell types adjacent to Hh expressing regions, and is thought to act as an antagonist of Hh signalling (Chuang and Mcmahon, 1999). In all systems studied to date the Patched gene is a target of its own repression, and its transcription is elevated upon stimulation with Hh. This up-regulation may antagonise further Hh stimulation, or may play a direct role in cell cycle control as Patched is reported to interact with Cyclin B1 (Barnes et al., 2001).

A model system ideally suited to screening for expression changes in as yet unidentified downstream genes has been established by activating the Hh pathway in 10T1/2 cells and using known target genes as markers of the response. This approach has proved successful, with a number of novel Shh target genes identified, and targets previously reported in other cell types being corroborated, using cDNA microarrays. The newly discovered Shh target genes encode molecules involved in nerve formation, transcriptional regulators, putative Wnt signalling antagonists, and various genes with roles in growth or apoptosis. Our finding of novel Shh regulated genes provides candidates whose abnormal expression may be decisive in initiating tumour formation in a range of human cancer types.


Cell-to-cell contact is required before 10T1/2 cells become fully responsive to Shh

Initial experiments involving transient transfection of a Shh-N expression construct (pShh-N-PMT21) and analysis of the response of known target genes suggested that 10T1/2 cells may not become fully responsive to Shh until the cells reach confluence (2 days post-transfection). We have previously observed that cells treated with Shh conditioned media show more rapid response times with several markers if the cells are already confluent at the time of Shh addition (data not shown). Experiments were conducted to investigate a possible relationship between cell density and the ability of these cells to respond to Shh. Using alkaline phosphatase (AP) activity as a marker of Shh induced osteoblastic differentiation, cells were found to respond more strongly as initial seeding density was increased prior to stimulation with Shh conditioned media (Figure 1). To show that this effect was due to cell density, and did not simply reflect a higher percentage of cells being stimulated from the outset, a second experiment was conducted where the level of cell contact was limited. This was achieved by continuously stimulating pools of cells, all with the same initial seeding level, and trypsinising every third day in order to control cell density. Cells which were allowed to reach confluence displayed a strong AP response, whilst cells in which cell contact was limited throughout the 9 day stimulation period had no detectable increase in AP activity (Figure 1).

Figure 1

Effect of cell contact on Shh response in 10T1/2 cells. Error bars represent +/−1 standard deviation. (a) Alkaline phosphatase (AP) assays performed on cells at a range of initial densities and stimulated for 7 days with Shh conditioned media show a correlation between increasing starting cell density and AP activity. Each data point represents four replicate wells. (b) AP assays performed on pools of cells with equal starting densities but differing degrees of allowed density show cell contact is required for a full response to Shh. Cell pools were trypsinized every 3 days. Cells permitted to reach confluence were allowed to re-adhere at current density, whilst cells for which contact was limited were split over a number of dishes. Stimulation with Shh conditioned media was maintained for 9 days prior to assay. Cells were counted prior to assay to ensure equivalent numbers were harvested at end of growth period. Each data point represents three replicate pools of cells

Establishing timing of response to Shh using known downstream target genes

In order to determine the most appropriate timepoints to harvest cells for microarray analysis, responses of a number of known Shh target genes were investigated by Northern blotting following Shh transfection. 10T1/2 cultures were transiently transfected with either wild-type Shh-N (pShh-N-PMT21) or a truncated functionally null (as shown by Figures 1,2,3,4 and 6, and other data not shown) mutant construct (pΔ64-Shh-N-PMT21). Cells were harvested at intervals from 3 h to 5 days post-transfection, and RNA used for Northern analysis. The first observed responses were the up-regulation of Patched and Gli1, initially detected 2 days post-transfection, and continuing to increase over following days (Figure 2). Up-regulation of Hedgehog interacting protein (HIP) was observed later, 4 days post-transfection. Patched2 up-regulation was also observed in this late timeframe (Figure 3). Induction of Angiopoietin2 and HNF-3β transcription was observed in an intermediate timeframe, as was an increase in alkaline phosphatase enzyme activity (data not shown). As a result of these investigations, timepoints from 24–96 h were chosen for microarray analysis. This range was chosen to maximize the chance of finding both early target genes and genes responding in a similar fashion to Patched2 and HIP.

Figure 2

Northern blots showing early responses of known target genes to Shh in 10T1/2 cells. RNA obtained from cells transfected with pShh-N-PMT21 (Shh-N) or pΔ64-Shh-N-PMT21 (mutant) plasmids, or treated with conditioned media collected from cells expressing the same constructs. (a) Up-regulation of full length Patched mRNA (transcript 1) and alternate transcripts (2 and 3) in response to Shh. Transcript 4 is not regulated by Shh. (b) Up-regulation of Gli1 transcripts in response to Shh stimulation by transfection and conditioned media treatment. GAPDH probe provides loading control

Figure 3

Northern blots showing late responses of known target genes to Shh. (a) Multiple transcripts of Hedgehog interacting protein (HIP) are up-regulated by pShh-N-PMT21 (Shh-N) or pN-Myc-hGli-PRK7 (Glil) expression in 10T1/2 cells. GAPDH probe provides loading control. (b) Poly(A)+ blot of pShh-N-PMT21 (Shh-N) or pΔ64-Shh-N-PMT21 (mutant) transfected RNA at 4 days post-transfection. The full length Patched2 mRNA (transcript 1) is significantly up-regulated by Shh when assessed by GAPDH normalized densitometry

Figure 4

Northern blots showing responses of a selection of Shh target genes identified by microarray. RNA obtained from 10T1/2 cells transfected with pShh-N-PMT21 (Shh-N) or pΔ64-Shh-N-PMT21 (mutant) plasmids, or treated with conditioned media collected from cells expressing the same constructs. GAPDH probe provides loading control. (a) Peripheral myelin protein 22 (PMP22) and Brain factor 2 (BF-2) show up-regulation, whilst Secreted frizzled related protein 2 (SFRP-2) shows an inhibition in the increased expression level that is observed in control cells over time. (b) Thrombomodulin (Thbd) is up-regulated by Shh. (c) Nuclear receptor subfamily 4, group A, member 1 (Nr4a1) is up-regulated by Shh

Figure 6

Regulation of members of the TSC-22 family by Shh. Northern blots of RNA from 10T1/2 cells transfected with pShh-N-PMT21 (Shh-N) or pΔ64-Shh-N-PMT21 (mutant) plasmids, or treated with conditioned media collected from cells expressing the same constructs. GAPDH probe provides loading control. Glucocorticoid induced leucine zipper (GILZ) was identified by microarray as a downstream target of Shh, and shows up-regulation by Northern blot when stimulated by transfection or conditioned media treatment. In contrast, two other known members of this gene family, TGFβ-stimulated clone 22 (TSC-22) and TSC-22 homologue 1 (Thg1), do not show regulation by Shh in 10T1/2 cells (using GAPDH normalized densitometry) at the timepoints investigated

Microarrays yield novel target genes regulated by Sonic Hedgehog

Microarrays containing 3936 clones from a normalized mouse embryonic branchial arch (NMEBA) library, the mouse UniGene set, and a number of other known genes were screened. Pair-matched experiments were conducted with Shh-N and the null-mutant control to obtain RNA for microarray analysis at 24, 48, 72 and 96 h timepoints for cells stimulated by transient Shh transfection, and at 72 and 96 h for cells treated with Shh conditioned media. Conditioned media experiments complemented the transfection studies to maximize the chances of target gene discovery and provide a further level of confirmation for Shh responsive genes. Twelve microarray experiments were performed, with two duplicate RNA samples hybridized to slides at each timepoint for both conditioned media and transfection studies. Each slide contained two duplicate sets of the spotted clones, positioned in separate blocks. The null-mutant control construct was designed so that the cell would produce a near full length Shh-N mRNA but a highly truncated protein. This was used rather than an empty vector control to ensure the general transcription and translation mechanisms of the cells were stimulated in both the reference and test RNA populations, minimizing false positive results.

Microarray spots were considered for further investigation if the geometric mean of normalized ratios for induction or repression was above twofold in any one time/treatment combination, or above 1.5-fold in multiple time/treatment combinations. Putative target genes were used to probe a set of blots containing RNA from each investigated timepoint for both conditioned media and transient transfection. RNA for blots was isolated from transfections independent of those used to perform microarray analysis. Genes that showed obvious regulation by Shh were used to probe a further set of RNA blots as a second validation (where independent plasmid preparations were used for transfection). Those which showed altered expression in all stimulation experiments were considered genuine Shh target genes and are presented in Tables 1 and 2.

Table 1 Downstream targets induced by Sonic Hedgehog in 10T1/2 cells identified by microarray approach and confirmed by Northern blotting
Table 2 Downstream targets repressed by Sonic Hedgehog in 10T1/2 cells identified by microarray approach and confirmed by Northern blotting

Validated targets were identified from within all three sets of genes present on the microarrays, with data for GILZ, BF-2, IGF2 and SFRP-2 obtained from multiple independent clone spots. Clones for GILZ, SFRP2 and BF-2 were present in the NMEBA and UniGene sets. The Shh targets showed detectable expression changes in the range of 3 to 4 days, with the majority of the genes showing a detectable response at 2 days. No validated target responses were identified at 24 h. As with known targets investigated in pilot studies, the earliest observed responses appear to correlate with the time the cells form a confluent monolayer.

The induced genes generally showed increased changes in expression as the timecourse progressed. The two Shh repressed SFRP genes behaved in a similar fashion to each other. In the absence of Shh these genes showed an increase in expression level with increasing cell density or time in a monolayer environment. Upon stimulation with Shh, this increase in expression over time was inhibited (Figure 4). Fold change data from the four day transient transfection and conditioned media treatments has been included in Tables 1 and 2, and generally represents the maximal response for the timepoints investigated. Northern blots of RNA from stimulated and control treated cells for a selection of the novel target genes identified in this work are shown in Figure 4.

IGF2, BF-2, Amh and SFRP-2 are regulated by Gli1

There is evidence to suggest that hedgehog signalling is frequently mediated by the Gli genes, but that this is not exclusively the case. We investigated a number of the newly identified target genes in 10T1/2 cells to find out whether they were regulated by an elevation in Gli1 transcription. Cells were transiently transfected with a Gli1 expression construct, and RNA from these cells and appropriate controls was collected at 12, 24, 48 and 72 h timepoints. Two of the Shh induced genes, IGF2 and BF-2, showed up-regulation in response to Gli1, whilst Shh repressed genes Amh and SFRP-2 were down-regulated by Gli1 (Figure 5). Thrombomodulin, LASP1 and Mip1-γ did not show any detectable response to Gli1 transfection.

Figure 5

Northern blots of 10T1/2 cells transiently transfected with a human Gli1 expression construct (pN-Myc-hGli-PRK7) or empty vector control show newly identified Shh target genes also regulated by Gli1. (a,b) Expression of Insulin-like growth factor 2 (IGF2) and Brain factor 2 (BF-2) is induced in response to Gli1. (c,d) Expression of Secreted frizzled related protein 2 (SFRP-2) and Anti-mullerian hormone (Amh) is repressed by Gli1. All genes shown gave greater than 1.5-fold changes in expression at two or more timepoints when quantified by GAPDH normalized densitometry

GILZ appears to be the only TSC-22 family member regulated by Shh in 10T1/2 cells

The TSC-22 family in mammals has three characteristic members, Transforming growth factor β stimulated Clone-22 (TSC-22), TSC-22 homologue 1 (Thg1; also called Thg-1pit in mouse), and Glucocorticoid Induced Leucine Zipper (GILZ), the latter of which was identified in the present study as a target of Shh signalling. In Drosophila the family is represented by a single gene, bunched/shortsighted, which is implicated in decapentaplegic (dpp) signalling in the developing eye (Treisman et al., 1995; Dobens et al., 1997, 2000). The fact that Hh is responsible for controlling dpp expression in Drosophila, combined with the recent discovery that TSC-22 is down-regulated by stable Gli1 expression in rat kidney cells (Yoon et al., 2002), makes this family of particular interest in the Shh pathway. All three members of this family were investigated in the 10T1/2 system. Whilst the induction of GILZ on microarrays and northerns was strong and reproducible, no evidence was found of detectable change to the basal expression level of TSC-22 or Thg1 on northerns upon Shh stimulation (Figure 6). The absence of a response of TSC-22 to Shh in 10T1/2 cells, even though TSC-22 is a known target of Gli1, highlights the fact that different response profiles are present in different tissue systems.


Microarrays containing 3936 cDNA elements were screened to identify novel targets of Shh signalling in the 10T1/2 embryonic mouse cell line, which has the ability to differentiate into a variety of mesodermal cell lineages. A range of strategically chosen timepoints, based on responses of known Shh pathway targets in 10T1/2 cells, were used to maximize the chance of identifying novel target genes upon activation by Shh transfection. Stimulation with conditioned media was used to complement the transfection studies. Validation of microarray leads by Northern blotting allowed us to establish 11 targets of Shh stimulation in 10T1/2 cells, seven of which were induced (Thrombomodulin (Thbd), Glucocorticoid induced leucine zipper (GILZ), Brain factor 2 (BF-2), Nuclear receptor subfamily 4, group A, member 1 (Nr4a1), Insulin-like growth factor 2 (IGF2), Peripheral myelin protein 22 (PMP22), Lim and SH3 Protein 1 (LASP1)), and four of which were repressed (Secreted frizzled related proteins 1 and 2 (SFRP-1 and SFRP-2), Macrophage inflammatory protein-1 gamma (MIP1-γ), and Anti-mullerian hormone (Amh)). The majority of these represent novel downstream genes not previously reported as targets of Shh.

In this work cDNA microarrays have proved an extremely powerful tool for the discovery of Shh target genes, however it proved important to address limitations of this approach. The spotted UniGene clone set was found to have evidence of cross-contamination, and 16% of clones did not match their expected identity. Similar observations with arrayed cDNA clone sets have been reported by other researchers (Halgren et al., 2001). Thus it was crucial to individually sequence and validate pure plasmid species from each clone of interest to determine true identities. Only genes showing consistent and repeatable induction or repression, and confirmed independently by Northern blotting, are presented as Shh target genes in this work.

In preliminary experiments it became apparent that there was an unexpected lag after Shh transfection before downstream transcriptional responses were observed. This was further supported by the microarray data, where targets were not detected prior to the 48 h timepoint. It was shown that this was due to a requirement for 10T1/2 cells to reach high density before a full response to Shh was initiated. A similar effect has previously been described in the NIH3T3 cell line (Taipale et al., 2000), and it will be interesting to see if density-dependent cofactors of Shh activation are identified in future studies.

Several target genes discovered by our microarray approach have previously been described in other systems. IGF2 is implicated as a Hh target due to its elevation in rhabdomyosarcoma and normal tissue from Patched knockout mice (Hahn et al., 2000). The corroboration of such findings further validates our model system. SFRP-1 and 2 have previously been reported as targets in presomitic mesoderm where both genes are induced by Shh (Lee et al., 2000). In contrast Shh inhibited the expression of both genes in 10T1/2 cells, showing that target genes may respond differently in different tissue contexts, presumably under the control of additional factors.

A number of the verified downstream targets of Shh show altered expression in human cancer tissue, or are known to be involved in tumourigenesis. For example LASP1 is amplified frequently in breast carcinomas, where it is thought to cause cytoskeletal changes during neoplastic transformation (Bieche et al., 1996). Thrombomodulin also shows expression changes in a variety of cancers, and has been hypothesized to play a role in tumour invasion and metastasis (Wilhelm et al., 1998).

Putative roles in apoptosis control are features of a number of the identified Shh target genes, including Nr4a1, GILZ, SFRP-1 and SFRP-2 (D'Adamio et al., 1997; Melkonyan et al., 1997; Li et al., 2000). The aberrant expression of such genes when Hh pathway is perturbed may be a factor in tumour formation. SFRPs bind Wnt proteins and are thought to regulate Wnt function (reviewed by Polakis, 2000). The Wnt pathway, and the genes controlled by it, are strongly implicated in cancer formation, particularly in colorectal cancer, where mutations in the tumour suppressor APC activate a Wnt response through the stabilization of β-catenin (reviewed by Taipale and Beachy, 2001). The Hh pathway may provide a mechanism to modulate Wnt signalling in vertebrates through the regulation of SFRP-1 and SFRP-2, and predispose cells to neoplasia when this control is disrupted.

Other newly identified Shh target genes have putative roles in growth regulation. PMP22 encodes a protein component of the myelin sheath of peripheral nerves which is also implicated as a growth factor (Jetten and Suter, 2000). Though this gene has not previously been implicated in Hh signalling, Dhh is involved in the development of the peripheral nerves and may regulate PMP22 during this process. Another induced gene, Thrombomodulin (Thbd), encodes a receptor that forms a complex with thrombin to reduce the rate of blood clotting. It is unclear what role this gene might play in embryonic mesodermal cells, however Thbd knockout mice display embryonic lethality before the cardiovascular system develops, suggesting a function for Thbd in development independent of its anticoagulant activities (Healy et al., 1995; Weiler-Guettler et al., 1996). Further studies have proposed mechanisms by which Thbd and Thrombin may be involved in controlling cell proliferation (reviewed by Freedman, 2001), though a link to the Hh pathway has not previously been described.

Expression of Gli1 in 10T1/2 cells caused an expression change for some Shh target genes but not others, suggesting Gli1 is not an exclusive mediator of Shh signalling in embryonic mesodermal cells. Strong evidence already exists for bifurcation of the pathway, with Shh regulating at least one target gene, COUP-TFII, through a Gli independent mechanism (Krishnan et al., 1997). Expression changes in IGF2, BF-2, Amh and SFRP-2 in response to Gli1 transfection provide a further level of evidence for these genes as bona fide downstream targets of Shh signalling. DNA binding studies have indicated that human Gli1 binds the nine base pair consensus sequence GACCACCCA (Kinzler and Vogelstein, 1990), with functional assays confirming a number of closely related sequences are also bound by Gli proteins in a range of species (Alexandre et al., 1996; Sasaki et al., 1997; Gustafsson et al., 2002; Yoon et al., 2002). Analysis of available upstream promoter sequence obtained from the Celera mouse database showed a number of putative and known Gli binding motifs to be present in each of the four Shh target genes shown to be regulated by Gli1. This suggests that the regulation of these genes by Gli1 is via a direct mechanism, though this is speculative and awaits detailed functional studies.

Two recent studies have also used microarrays to investigate aspects of the Hh signalling pathway. Kato et al. (2001) transfected a neuroepithelial cell line with a Shh expression construct and identified two target genes, whilst Yoon et al. (2002) used RNA derived from transformed foci generated by stable Gli1 expression, revealing 30 Gli1 target genes in rat epithelial kidney cells. The genes identified in these studies are distinct from those reported in this work. This may reflect tissue specific pathway responses, differences in potency and timing of stimulation methods, or a lack of overlap between cDNA sets used in studies to date.

An investigation of one gene family of interest showed that although the transcriptional regulator GILZ is a strongly regulated target of Shh in 10T1/2 cells, the related gene TSC-22 does not show evidence of regulation in this cell type, even though it has previously been identified as a down-regulated target of Gli1 in kidney cells (Yoon et al., 2002). This highlights the fact that Hh proteins can induce quite different responses in different cell types and tissue environments. Presumably Hh signalling is influenced considerably by a variety of fine tuning mechanisms and regulating factors, which vary spatially and temporally, allowing Hh to display such a diverse range of patterning functions in the developing embryo.

Our finding of both previously known and novel target genes in the 10T1/2 system shows the power of this approach for Shh target gene discovery in pluripotent mesoderm. The diverse functions of Shh target genes identified in 10T1/2 cells are a reflection of the plasticity of this cell type. Microarrays used in this work contained a relatively small percentage of genes from the mouse genome, and future studies will be possible with substantially larger cDNA sets. Expression studies with the murine target genes and their human homologues in normal tissues and NBCCS related tumours will further elucidate their importance in development and disease. It is hoped that the discovery of such genes controlled by hedgehog signalling will provide vital clues to the aetiology of various human neoplasms, and that understanding of their roles may provide greater opportunities in the future design of anti-tumour therapies.

Materials and methods

Cell culture, transient transfection and conditioned media production

C3H/10T1/2 Clone 8 cells (Reznikoff et al., 1973) were obtained from the American Type Culture Collection (at passage 10), and maintained in Dulbecco's Modified Eagle's Medium supplemented with streptomycin (50 μg/ml), penicillin (50 units/ml) and Serum Supreme (9.1%, BioWhittaker). Stock cells were plated at 2000 cells/cm2, never allowed to reach confluence and discarded at passage 20. For pathway activation a construct encoding the active N-terminal domain (amino acids 1–198) of mouse Sonic Hedgehog in pMT21 (pShh-N-PMT21), or a null mutant version of this construct containing a 64 bp deletion of the start ATG region (pΔ64-Shh-N-PMT21), were transiently transfected into cells. Transfection was performed with lipofectamine plus (Invitrogen) 3.5 days after seeding at 2000 cells/cm2, when the cells approximately 80% confluent. Alternatively, conditioned media (diluted 1 : 1) was used for Shh stimulation, with time zero at same density as transfection initiation.

Conditioned media was produced by collecting 3 day post-transfection growth media from pShh-N-PMT21 and pΔ64-Shh-N-PMT21 treated cells, and centrifuging at 3000 g for 5 min to remove cell debris. Media from a number of production plates was pooled to give a large volume stock to ensure stimulation with an equal concentration of Shh in all experiments. This was tested in 10T1/2 cells and shown to be potent in inducing alkaline phosphatase (AP) activity. The activity of conditioned media produced by this method was found to be quite stable when stored at 4°C, and achieved consistently higher levels of AP induction than 1 μg/ml of commercial recombinant Shh protein (#461-SH, R&D systems Inc.) when small scale 7-day quantitative assays were performed in parallel (data not shown).

Activation with Gli1 was by transient transfection of human Gli1 cDNA in the vector pRK7 (pN-Myc-hGli-PRK7; Murone et al., 1999), using empty pRK7 as a negative control. In all activation experiments with Shh or Gli1, a subset of cells grown on coverslips were removed prior to RNA isolation and histochemically assayed for AP to ensure transfection had been successful in initiating a strong pathway response in a large percentage of cells.

Alkaline phosphatase assays

Alkaline phosphatase (AP) activity was detected using a histochemical procedure, modified from Katagiri et al. (1994). Cells were fixed with 4% PFA for 10 min, washed with PBS and incubated with AP staining reagent (0.09 mg/ml Fast Blue BB salt, 0.5% dimethylformamide, 0.1 mg/ml Naphthol AS-MX phosphate, 2 mM MgCl2, 0.1 M Tris-HCl pH 8.5) for 1 h. Additionally, quantitative measurement was performed using the ALP Procedure 104 kit (Sigma Diagnostics), using 0.9% NaCl with 0.2% Triton X-100 for cell lysis. Sample inputs were normalised to total protein concentration measured with Bradford reagent. Each reaction was read in triplicate at 415 nm against p-Nitrophenol standards (Sigma Diagnostics), with a secondary read after acid addition to correct for background lysate absorbance.

Microarray hybridization and analysis

Microarray chips were manufactured by the IMB microarray facility. These consisted of PCR products spotted onto poly-L-lysine coated glass slides using standard protocols. Chips contained 3936 spots which were duplicated in separate blocks on each slide. Spots represented 1594 cDNA clones from the mouse UniGene set (Research Genetics); 1920 cDNA clones from a normalised mouse embryonic branchial arch library (NMEBA; constructed by B Soares, University of Iowa, IA, USA); and a further 422 spots including control spots and various mouse and human clones.

Total RNA samples (40–65 μg) from treatment and pair-matched control samples were labelled with cy-5 or cy-3 dUTP, using oligo d(T) primer and Superscript II reverse transcriptase (Invitrogen). RNA was removed by alkaline hydrolysis. Labelled cDNA was purified using YM-30 microcons (Millipore). Hybridization (in 0.25 mg/ml CotI DNA, 0.5 mg/ml Poly d(A), 4×SSC, 0.5% SDS, 50% formamide) was performed overnight at 45°C, under coverslips in humidified chambers. Slides were washed for 3 min in 0.2×SSC/0.05×SDS, and 2×3 min in 0.2×SSC, prior to obtaining fluorescence images with a Genetic MicroSystems G418 scanner. RNA from each treatment-timepoint combination was hybridized to two independent microarray chips, giving a total of 12 hybridizations.

Spot intensities were quantified using ImaGene software (BioDiscovery). Normalization and statistical analysis of local background adjusted signals was performed with spreadsheets, and also using the GeneSpring package (Silicon Genetics). In the former normalization was performed by applying a constant to all values such that the average intensity of spots on both channels was equivalent, in accordance with Hegde et al. (2000). Data was filtered so that spots were only considered for analysis when one or both channels had a significant signal. GeneSpring analysis was performed with the data normalized using the median of signals on each channel as a synthetic positive control.

Clones corresponding to spots of interest were obtained from the IMB microarray facility. Plasmids from pure cultures were sequenced with M13 primers (Hegde et al., 2000). Identity was established using BLAST and the GenBank database (NCBI). Plasmids were digested to liberate insert without poly(A)+ tail were possible, separated on agarose gel and purified with Geneclean (Biol 101), ready for use as Northern probes.

RT–PCR generation of additional cDNA probes

Probes for mouse Thg-1pit and TSC-22 were generated by RT–PCR using M-MLV reverse transcriptase for cDNA synthesis (Invitrogen). Products were purified by Ultraclean PCR cleanup (MoBio) prior to use as probes on Northern blots. Primers for 1530 bp TSC-22 product 5′-tttgaaccaggctgctggag-3′ (forward) with 5′-gcgcagaacgactatacaggtgag-3′ (reverse); and for Thg-1pit as in Fiorenza et al. (2001).

RNA isolation and Northern blotting

Total RNA was prepared using the RNeasy RNA isolation kit (Qiagen), with poly(A)+ isolated using Message Maker (Life Technologies, GIBCO–BRL) when required. Northern analysis of 10 μg total (or 2 μg poly(A)+) RNA was performed by electrophoresis on 1% agarose/formaldehyde gels with transfer to Magna nylon membrane (Osmonics). Pre-hybridization (4 h) and hybridization (overnight) were performed in 5×SSPE, 5×Denhardts, 0.5% SDS in 50% formamide at 42°C. Probes were labelled with 32P by random priming (Rediprime II, Amersham Pharmacia), and purified on G-50 sephadex. Bands were detected by autoradiography using Kyokko High Plus intensifying screens (Fuji). Autoradiograms were quantified using a GS-700 imaging densitometer (Bio-Rad). A 600 bp fragment of the mouse GAPDH cDNA was used as a loading control probe.


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Microarray chips were a generous gift of Dr L Fowles (IMB). The pN-Myc-hGli-PRK7 construct was kindly provided by Dr F de Sauvage (Genentech Inc, San Francisco, CA, USA), whilst pShh-N-PMT21 was a kind gift of the late Dr T Yamada. Thanks to Dr T Ellis and K McCue for critical reading of the manuscript. This work was supported in part by a grant from the Australian National Health and Medical Research Council (NHMRC). The IMB incorporates the Centre for Functional and Applied Genomics, a Special Research Centre of the Australian Research Council. The Cooperative Research Centre for Discovery of Genes for Common Human Diseases is established and supported by the Australian Government's Cooperative Research Centres program. CA Wicking is an NHMRC Senior Research Fellow. WJ Ingram was supported by a Commonwealth Scholarship and Fellowship Plan (CSFP) award.

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Correspondence to Brandon J Wainwright.

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Ingram, W., Wicking, C., Grimmond, S. et al. Novel genes regulated by Sonic Hedgehog in pluripotent mesenchymal cells. Oncogene 21, 8196–8205 (2002).

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  • Sonic Hedgehog
  • downstream target genes
  • C3H/10T1/2
  • microarray

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