Gli1 Is Important for Medulloblastoma Formation in Ptc1ю /А Mice

Gli1 is important for medulloblastoma formation in Ptc1 þ /À mice

Hiromichi Kimura1, Daniel Stephen2, Alexandra Joyner2 and Tom Curran*,1

1Department of Developmental Neurobiology, St Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105, USA; 2Howard Hughes Medical Institute and Developmental Genetics Program, Skirball Institute of Biomolecular Medicine, Department of Cell Biology, New York University School of Medicine, New York, NY 10016, USA

Germline mutations in the human homolog of the pathway signaling genes, PTCH1 (10%), smoothened, patched1 (PTCH1) are associated with basal cell nevus SMOH (6%)and suppressor-of-fused, SUFU (9%)have carcinoma syndrome (BCNS or Gorlin syndrome), which been reported to occur in sporadic medulloblastoma is characterized by developmental anomalies, radiation (Raffel et al., 1997; Vorechovsky et al., 1997; Wolter hypersensitivity and a predisposition to medulloblastomas et al., 1997; Xie et al., 1997; Reifenberger et al., 1998; and skin tumors. Patched1 (Ptc1) functions as a receptor Dong et al., 2000; Zurawel et al., 2000a; Taylor et al., for Sonic hedgehog (Shh) in a wide range of biological 2002). Mice heterozygous for Ptc1 display several processes. Binding of Shh to Ptc1 results in activation of features of Gorlin syndrome and they develop both Smoothened (Smo), which in turn stimulates expression of rhabdomyosarcoma and medulloblastoma (Goodrich downstream target genes including Ptc1 and Gli1. Gli1 is et al., 1997). Previously, we reported that Ptc1 þ /À mice a member of a family of DNA-binding zinc-finger on a C57Bl/6 background exhibit a 14% incidence of proteins, including Gli2 and Gli3, that function in medulloblastoma by 10 months of age (Wetmore et al., transcription control. Here, we report that inactivation 2000). All medulloblastomas examined from Ptc1 þ /À of both Gli1 alleles in Ptc1 þ /À mice significantly reduces mice expressed Gli1 and the remaining wild-type Ptc1 spontaneous medulloblastoma formation. Therefore, Gli1 allele (Wetmore et al., 2000; Zurawel et al., 2000b). is not only a marker of pathway activation but also plays a Thus, medulloblastoma formation in Ptc1 þ /À mice functional role in medulloblastoma formation. Interest- occurs as a consequence of haploinsufficiency of Ptc1. ingly, Gli2 levels were elevated in medulloblastoma cells Since Ptc1 functions to repress Smo, these results imply but not in normal granule neuron precursors during that reduced expression of Ptc1 leads to increased cerebellar development in mice lacking Gli1. In cultured pathway signaling and ultimately tumorigenesis. fibroblasts, Gli1 was more potent than Gli2 at inducing Hedgehog (Hh)is a secreted protein that is highly cell transformation. These results demonstrate that Gli1 conserved from flies to mammals (Ingham and McMa- plays a central role in medulloblastoma formation in hon, 2001). The three mammalian Hh family members, Ptc1 þ /À mice and that Gli2 may also contribute to Shh, Indian hedgehog (Ihh)and Desert hedgehog oncogenesis. (Dhh), are involved in cell growth, proliferation and Oncogene (2005) 24, 4026–4036. doi:10.1038/sj.onc.1208567 differentiation (Ingham and McMahon, 2001). Shh- Published online 4 April 2005 deficient mice exhibit embryonic lethality with skeletal abnormalities, defects in neuronal development and Keywords: medulloblastoma; Patched1; Gli1 small body size (Chiang et al., 1996). In the cerebellum, Shh is detectable in Purkinje cells as early as embryonic day (E)17.5 in mice (Lewis et al., 2004). Ptc1-null mice also show lethality at E 9.5 as a consequence of ectopic activation of Smo (Goodrich et al., 1997). Binding of Introduction Shh to Ptc1 results in derepression of Smo and increased downstream signaling, which results in elevated expres- Basal cell nevus syndrome (BCNS or Gorlin syndrome), sion of Gli1 (Ingham and McMahon, 2001). The Gli which is associated with heterozygous germline muta- family of zinc-finger transcription factors is composed of tion of patched1 (PTCH1), is characterized by a large three members, Gli1, Gli2 and Gli3 in mice and humans. body size, skeletal abnormalities, radiation sensitivity, Targeted disruption of the zinc-finger region together soft tissue sarcoma, basal cell carcinoma and medullo- with the adjacent 43 amino acids (Gli1zfd)or disruption blastoma (Gorlin, 1987). Medulloblastoma, the most in the entire Gli1 N-terminus and zinc-finger region by common malignant pediatric brain tumor, is a primitive lacZ insertion (Gli1lZ)in mice shows no obvious neuroectodermal tumor that arises in the developing biological effect (Park et al., 2000; Bai et al., 2002). In cerebellum. Mutations in the sonic hedgehog (SHH) contrast, disruption of Gli2 or Gli3 in mice results in severe skeletal and neural defects and embryonic or *Correspondence: T Curran; E-mail: [email protected] perinatal lethality (Hui and Joyner, 1993; Mo et al., Received 24 November 2004; revised 19 January 2005; accepted 28 1997). Double mutation of Gli1 and Gli2 causes more January 2005; published online 4 April 2005 severe developmental defects than loss of Gli2 alone Gli1 is important for medulloblastoma formation in Ptc1 þ /À mice H Kimura et al 4027 (Park et al., 2000; Bai et al., 2002). These findings Substitution of the transactivation domain in Gli1 suggest that Shh signaling is critical for normal with the VP16 transactivation domain (NF-Gli1VP16) development and that Gli1 and Gli2 have overlapping resulted in higher transcriptional activity than wild- functions. This hypothesis was confirmed directly by type Gli1. Mutants lacking the entire Zn-finger motif substitution of Gli1 into the Gli2 locus (Bai and Joyner, (amino acids 240–394)(Gli1 zfd240–394)did not show any 2001). luciferase activity (NF-Gli1zfd240–394). The expression level Increased activity of the Shh pathway is associated of the Gli protein constructs was confirmed using anti- with oncogenesis in many cell types. For example, FLAG and anti-Gli1 C-terminal antibodies (data not overexpression of either Shh, Gli1, Gli2 or a constitu- shown). tively active mutant of Smo under the control of a keratin promoter in mice causes basal cell carcinoma Gli1 and Gli2, but not Gli3 transform cultured fibroblasts (Oro et al., 1997; Grachtchouk et al., 2000, 2003; Nilsson et al., 2000; Oro and Higgins, 2003). Further- We used a retroviral vector system to examine the more, in primary rat fibroblasts, overexpression of Gli1 transforming activity of Gli family members and Gli1 induces morphological transformation in cooperation mutants in NIH 3T3 cells and 208F rat fibroblasts. The with the oncoprotein E1A (Ruppert et al., 1991), Gli constructs that were active in the Gli-luc reporter suggesting that deregulation of Smo signaling promotes assay, F-Gli1, NF-Gli1 and NF-Gli1VP16 were all tumorigenesis. Recently, it was shown that ectopic capable of inducing expression of the endogenous Ptc1 expression of Shh in granule cells in the developing and Gli1 genes in NIH 3T3 cells (Figure 1B). Gli2 also cerebellum of Gli1lZ mice induced medulloblastoma induced both Gli1 and Ptc1 expression at lower levels formation (Weiner et al., 2002), suggesting that Gli1 is than those induced by Gli1, whereas NF- Gli1zfd240–394 not required for tumorigenesis. However, since Gli and Gli3 did not activate the endogenous targets. These family members exhibit functional redundancy and data confirm the results of the luciferase assay that Gli2 compensation, and mice with targeted deletions in is a weaker transactivator than Gli1 (Figure 1A). We different Gli genes show distinct phenotypes, it is found that Shh induces Gli transcriptional activity in important to investigate the specific role of each family 208F cells as determined by the Gli-luc reporter assay member in a mouse model of spontaneous tumor (data not shown). We next examined the morphological formation. Here we investigate the contribution of transformation potential of Gli family members and Gli1 to oncogenesis in mice heterozygous for Ptc1.We Gli1 mutants in 208F cells (Miao and Curran, 1994) found that medulloblastoma formation in Ptc1 þ /À mice (Figure 1C). Only the constructs capable of causing was dramatically reduced but not completely eliminated transactivation (F-Gli1, NF-Gli1, NF-GliVP16 and F- in the absence of Gli1. Thus, Gli1 is a crucial Gli2)were able to induce morphological transformation contributor to medulloblastoma formation in Ptc1 þ /À (Figure 1C, b, c, f and g). These results imply that mice, but it is likely that Gli2 shares this oncogenic activation of target genes by Gli1 or Gli2 is required for function. morphological transformation. Gli3 did not induce transformation (Figure 1C, h), and F-Gli2 exhibited less transforming activity than F-Gli1 (Figure 1C, g). Furthermore, NIH3T3 cells expressing F-Gli1 or NF- Results Gli1VP16 were able to form colonies in soft agar (Figure Transcription properties of Gli proteins 1D, b and c). Cells expressing F-Gli2 grew in agar, but they formed smaller colonies than those expressing F- As a first step in investigating the oncogenic potential of Gli1 or NF-Gli1VP16 and they arose after a longer Gli proteins, we compared their transcriptional activity period of culture (Figure 1D, d). Cells expressing F-Gli1 using a Gli luciferase (Gli-luc)reporter construct that or F-Gli2 grew more readily in soft agar in the presence provides a convenient and sensitive marker for tran- of Shh than in the absence of Shh, whereas F-Gli3- scription activity (Sasaki et al., 1999). Comparison of expressing cells did not form colonies in soft agar even the transcriptional activities of Gli family members when cultured in the presence of Shh (data not shown). revealed that Gli1 exhibited the strongest transcriptional Taken together, these results confirm that Gli1 is the activity, Gli2 possessed approximately half the activity strongest activator among Gli family members and that of Gli1, whereas Gli3 was not active in this assay. Since Gli1 induces cell transformation through its transcrip- Gli1 is localized in the cytoplasm in 293 T and NIH 3T3 tion activation function. Although NF-Gli1VP16 is a cells (Koyabu et al., 2001), we added a nuclear stronger activator and it induced enhanced cell growth localization sequence (NLS), derived from SV40 Large in agar, it is not as efficient as wild-type Gli1 at inducing T antigen, into the N-terminus of Gli1 and Gli1 morphological transformation. This suggests that the mutants. We expected that the NLS fused mutants activation domain of Gli1 may confer specificity for would have higher transcriptional activity; however, this target genes involved in transformation. Although Gli2 was not the case (Figure 1A). The addition of an NLS induces morphological transformation, it is less potent was not sufficient to enhance nuclear translocation of than Gli1. Since Gli2 induces expression of endogenous Gli1. While the majority of NLS-FLAG (NF)-Gli1 was Gli1, transformation may be accomplished through the localized in the cytoplasm (data not shown), it was just activation of common target genes either directly or as active as wild-type Gli1 in the Gli-luc assay. indirectly.

Oncogene Gli1 is important for medulloblastoma formation in Ptc1 þ /À mice H Kimura et al 4028

Figure 1 Oncogenic potential of Gli family members and Gli1 mutants. (A)NIH 3T3 cells were transfected with FLAG-tagged (F),or SV40 large T nuclear localization sequence (NLS)-FLAG tagged (NF), deletion mutants of Gli1, F-Gli2 and F-Gli3 together with Gli- luc and a Renilla luciferase construct. Luciferase activity was measured 48 h after transfection. Luciferase activity from the empty vector (Vector)was designated as 1. ( B)Expression of endogenous Gli1 target genes in NIH 3T3 cells after Gli1, mutant Gli1, Gli2 and Gli3 virus infection. Western analysis showing expression of endogenous and exogenous Gli1, endogenous Ptc1 and F-Gli family was performed at 48 h postinfection. The control lane contains whole-cell lysate from parental NIH 3T3 cells. Numbers on the left indicate molecular mass (kDa). (C)Morphological transformation by Gli family members. 208F cells were infected with MSCV, or pBabe-puro viruses at a MOI of 0.001 (viruses encoding Gli1 and Gli1 mutants)and 10 (pBabe F-Gli2, F-Gli3).Photomicrographs were taken 2 weeks after infection, except for the Gli2 image, which was taken 5 weeks after infection. (a)MSCV, (b)MSCV F-Gli1, (c)MSCV NF- Gli1, (d)MSCV NF- DAD, (e)MSCV NF-Gli1 zfd240À394, (f)MSCV NF-Gli1VP16, (g)pBabe-puro F-Gli2, (h)pBabe-puro F-Gli3. ( D) Growth of Gli1 transduced cells in soft agar. NIH 3T3 cells were infected with virus and suspended in IMDM containing 11.25% FBS with 0.3% agar and plated on top of 0.6% IMDM bottom agar supplemented with 15% FBS. Cells were cultured for 30 days (F-Gli1 and NF-Gli1VP16)or 40 days (F-Gli2).(a)MSCV, (b)F-Gli1, (c)NF-Gli1VP16, (d)F-Gli2

Medulloblastoma incidence in Ptc1 þ /À mice is severely C57Bl/6 and 129Sv background (Wetmore et al., 2000) reduced in the absence of Gli1 with Gli1zfd mice (Park et al., 2000). Surprisingly, of the 10 Ptc1 þ /À Gli1 þ /zfd mice obtained in the ﬁrst generation, To examine whether Gli1 contributes to medulloblasto- six Ptc1 þ /À Gli1 þ /zfd mice developed medulloblastoma by ma formation, we crossed Ptc1 þ /À mice on a mixed 31 weeks of age (Figure 2a). The remaining mice showed

Oncogene Gli1 is important for medulloblastoma formation in Ptc1 þ /À mice H Kimura et al 4029 a

100 Ptc1+/+Gli1+/zfd 80

40 Ptc1+/-Gli1+/zfd 20 Medulloblastoma-free (%)

0 0 10 20 30 40 50 60 Weeks

b Ptc1+/-xGli1zfdmice 6

Number of mice with medulloblastoma 0 Figure 3 Expression of Gli1zfd, Gli2, Ptc1 and N-myc in the <10 10-14 15-18 19-22 23-26 27-30 31-34 35-38 39-42 43< developing cerebellum. (a)Western blot analysis showing Gli1 weeks protein levels in the developing cerebellum at postnatal days five Figure 2 Medulloblastoma development in Ptc1 þ /À mice with (P5 CB). þ / þ and zfd/zfd indicate extracts from the normal targeted mutation of Gli1.(a)Medulloblastoma-free curve in the cerebellum and the homozygous zinc-finger deletion mutant (zfd) cerebellum, respectively. Extracts (100 mg/lane)from the cerebella first generation of Ptc1 þ /À Gli1 þ /zfd (diamonds, E)(n ¼ 10)and or whole cell extracts (20 mg/lane)prepared from 208F cells infected Ptc1 þ / þ Gli1 þ /zfd (squares, ’)(n ¼ 5)mice. ( b)Distribution of medulloblastoma formation by age in Ptc1 þ /À mice crossed with with either MSCV control virus (Control)or MSCV F-Gli1 virus (F-Gli1)were subjected to Western blot analysis. Numbers indicate Gli1zfd mutant mice on a mixed C57Bl/6 background (n ¼ 259). The Gli1 numbers of medulloblastoma in Ptc1 þ /À Gli1 þ / þ mice (white molecular mass (kDa). (b)Northern analysis showing RNA levels in P5 CB. RNA from each sample was subjected to Northern column, n ¼ 13), Ptc1 þ /À Gli1 þ /zfd mice (black column, n ¼ 19)and analysis using gene-specific antisense probes for Gli1, Gli2, Ptc1 Ptc1 þ /À Gli1zfd/zfd mice (gray column, n ¼ 2)are shown at each age and N-myc as indicated in the panels. Bracket and asterisks indicate distinct Gli1 transcripts. lZ/lZ indicates extracts from the lacZ knock-in mutant (lZ)cerebellum. Arrowheads indicate the loca- tions of Ethidium Bromide (EtBr)stained 28S and 18S ribosomal RNA (rRNA)used as loading controls no signs of medulloblastoma by 53 weeks of age. This increase in tumor incidence was surprising as Gli1zfd240–394 does not exhibit any DNA-binding activity or transcrip- We expanded the number of mutant mice in the tion activity and it fails to induce cell transformation in cohort by crossing Ptc1 þ /À Gli1 þ /zfd mice from the first culture (Figure 1A and C). Furthermore, mice expres- generation to mice from the first, second and third sing Gli1zfd272–423 under the control of the Wnt1 enhancer generations, and we observed all the mice for 12 months do not show any histological abnormalities or activation (Table 1). Approximately 24.5% of Ptc1 þ /À Gli1 þ / þ of Shh target genes in the neural epithelium (Park et al., mice developed medulloblastoma (n ¼ 13/53). Similarly, 2000). Although Gli1zfd transcripts are expressed in the 21.8% of Ptc1 þ /À Gli1 þ /zfd mice developed medulloblas- mutant mice (Park et al., 2000), we did not detect any toma (n ¼ 19/87). However, only 3.4% of Ptc1 þ /À Gli1zfd/zfd protein product in extracts from the developing cere- mice developed tumors (n ¼ 2/58)and no tumors were bellum (Figure 3a). Therefore, Gli1zfd is likely a null identified in Ptc1 þ / þ Gli1zfd/zfd mice (n ¼ 0/61). Thus, allele as opposed to a gain-of function mutant. The high Ptc1 þ /À Gli1 þ /zfd mice have a similar incidence of incidence of medulloblastoma was reduced to the level medulloblastoma as Ptc1 þ /À Gli1 þ / þ mice (Table 1). reported previously (Goodrich et al., 1997; Wetmore There is no significant difference in the medulloblastoma et al., 2000)when we crossed mice from the next three incidence of Ptc1 þ /À Gli1 þ / þ mice and Ptc1 þ /À Gli1 þ /zfd generations. Thus, the initial increase in medulloblasto- mice (P ¼ 0.8359). These data indicate that the high ma incidence likely reflects the segregation of modifier incidence of medulloblastoma in the first generation genes present in both C57Bl/6 and 129Sv mice. Ptc1 þ /À Gli1 þ /zfd mice likely results from a genetic

Oncogene Gli1 is important for medulloblastoma formation in Ptc1 þ /À mice H Kimura et al 4030 Table 1 Targeted disruption of Gli1 reduces medulloblastoma incidence in Ptc1+/À mice Ptc1+/À Gli1+/+ Ptc1+/À Gli1+/zfd Ptc1+/À Gli1zfd/zfd Ptc1+/+ Gli1zfd/zfd

Medulloblastoma 13/53 (24.5 %)19/87 (21.8 %) 2/58 (3.4 %) a 0/61 (0 %) Rhabdomyosarocoma 1/53 (1.88 %)6/87 (6.9 %) 0/58 (0 %) 0/61 (0 %)

aReduction in medulloblastoma incidence in Ptc1+/À Gli1zfd/zfd mice was highly signiﬁcant compared to Ptc1+/À Gli1+/+ mice (P ¼ 0.0015)and Ptc1+/ À Gli1+/zfd mice (P ¼ 0.0016). Po0.01 is considered signiﬁcant

background effect generated by crossing mouse strains Gli1zfd is a gain-of-function mutant. We performed with different modifier genes. This compound modifier Western blot analysis to examine the levels of Gli1 effect was lost in subsequent generations. Therefore, we expression in P5 CB from Gli1zfd/zfd mice (Figure 3a). A conclude that complete loss of Gli1 function signifi- single band of Gli1 protein of approximately 150 kDa cantly reduces medulloblastoma formation in Ptc1 þ /À was identified in the normal P5 CB. Interestingly, the mice (P-value of medulloblastoma incidence in Ptc1 þ /À Gli1zfd mutant protein was not detectable in P5 CB in Gli1 þ / þ compared to Ptc1 þ /À Gli1zfd/zfd mice or Ptc1 þ /À Gli1zfd/zfd mice, indicating that it is either not translated Gli1 þ /zfd compared to Ptc1 þ /À Gli1zfd/zfd mice is 0.0015 or very unstable in GNPCs. These results support the and 0.0016, respectively). There was no haploinsuffi- contention that Gli1zfd does not act as a dominant- ciency effect of Gli1 on medulloblastoma formation in negative mutation during normal development or in Ptc1 þ /À mice. We also observed one rhabdomyosarcoma tumorigenesis in Ptc1 þ /À mice. Next, we performed in 53 Ptc1 þ /À Gli1 þ / þ mice (1.88%), six rhabdomyo- Northern analysis to examine Gli1zfd RNA levels and to sarcomas in 87 Ptc1 þ /À Gli1 þ /zfd mice (6.9%)and no determine whether there is any difference in the rhabdomyosarcomas in Ptc1 þ /À Gli1zfd/zfd (0%)( n ¼ 58) expression of Gli2 and Ptc1 in P5 CB from the mutant or Ptc1 þ / þ Gli1zfd/zfd mice (0%)( n ¼ 61)(Table 1). mice. As a control, we used Gli1lZ/lZ mice (Bai et al., However, there was no statistically significant difference 2002). These mice display an identical phenotype to among the incidences among the four genotypes. The Gli1zfd/zfd mice (Bai et al., 2002). Gli1 RNA was low incidence of rhabdomyosarcoma in Ptc1 þ /À Gli1 þ / þ detectable in P5 CB from both normal and Gli1zfd/zfd mice on the mixed C57Bl/6, 129Sv and Swiss Webster mice but not from Gli1lZ/lZ mice (Figure 3b). Consistent background makes this result difficult to interpret. with previous studies, several Gli1zfd RNA transcripts of The age of onset of medulloblastoma was very similar different lengths were observed (Figure 3b, asterisks in in Ptc1 þ /À Gli1 þ / þ , Ptc1 þ /À Gli1 þ /zfd and Ptc1 þ /À Gli1zfd/zfd Gli1 panel). The expression level of Gli2 in P5 CB in the mice, occurring between 10 and 41 weeks of age Gli1 mutant mice was similar to that observed in the (Figure 2b). The majority of the brain tumors (84.6% normal P5 CB. In addition, Ptc1 and N-myc RNAs were of the medulloblastoma in Ptc1 þ /À Gli1 þ / þ mice and present at the similar levels in the P5 CB from normal 84.2% of the medulloblastoma in Ptc1 þ /À Gli1 þ /zfd mice) and Gli1 mutant mice. These data suggest that loss of were observed by 30 weeks of age. Two Ptc1 þ /À Gli1zfd/zfd Gli1 does not affect Gli2, Ptc1 and N-myc expression mice exhibited medulloblastomas at 24 and 34 weeks of and that the normal level of Gli2 expression is sufficient age. These findings indicate that mice with heterozygous to compensate for loss of Gli1 function during cerebellar loss of Ptc1 developed medulloblastoma by 41 weeks of development. age and mice lacking functional Gli1 and one copy of Ptc1 did not display a delay in tumor formation. Thus, Gli2 levels are elevated in medulloblastomas from Ptc1 þ /À we conclude that the loss of Gli1 function suppresses Gli1 þ /zfd and Ptc1 þ /À Gli1zfd/zfd mice medulloblastoma formation in Ptc1 þ /À mice. We performed Northern analysis to investigate the expression levels of Shh pathway genes in medulloblas- Loss of Gli1 does not result in upregulation of Ptc1, Gli2 toma arising from Ptc1 þ /À, Ptc1 þ /À Gli1 þ /zfd and Ptc1 þ /À and N-myc during normal cerebellar development Gli1zfd/zfd mice on a mixed C57Bl/6, 129Sv and Swiss In the developing cerebellum, Gli1, Gli2, Ptc1, Ptc2 and Webster background (Figure 4). It is technically difficult N-myc are expressed in granule neuron precursor cells to completely separate the normal cerebellum from (GNPCs)in the external germinal layer (EGL)(Wechs- medulloblastoma tissue. Therefore, medulloblastomas ler-Reya and Scott, 1999; Kenney et al., 2003; Lee et al., were collected together with the entire cerebellum to 2003). These genes are highly expressed in medulloblas- minimize differences in tissue composition in our toma and gene expression profiles obtained from mouse studies. RNA was examined by Northern transfer and medulloblastoma are most similar to the profiles hybridization using specific probes. Gli1 RNA was obtained from the developing cerebellum at postnatal detected in both the normal P5 CB and the medullo- day 5 (P5 CB), a structure mostly composed of blastoma samples but not in the adult cerebellum. proliferating GNPCs (Pomeroy et al., 2002; Lee et al., However, low levels of Gli1 are expressed in Bergmann 2003). Although the incidence of medulloblastoma in glial cells as detected by lacZ staining of a knock-in Ptc1 þ /À Gli1 þ / þ and Ptc1 þ /À Gli1 þ /zfd mice was similar allele (Corrales et al., 2004)and in situ hybridization in (Table 1), we wanted to exclude the possibility that this region (Romer et al., 2004). Only low levels of Gli1

Oncogene Gli1 is important for medulloblastoma formation in Ptc1 þ /À mice H Kimura et al 4031 neurons, was present at high levels in the adult but not the P5 CB (Figure 4, Gabra6 panel). As expected, Gabra6 was detectable in tumors but the levels were much lower than that in the adult cerebellum, indicating that the majority of RNA in medulloblastoma sample is derived from tumor tissue. Previously, we demonstrated that medulloblastoma from Ptc1 þ /À mice exhibit high levels of Gli1 expression (Wetmore et al., 2000). Consistent with the previous results, medulloblastomas from Ptc1 þ /À and Ptc1 þ /À Gli1 þ /zfd mice showed high levels of Gli1 expression (Figure 4). Gli1 transcripts from the Gli1zfd mutant allele were also detected in medulloblastoma from Ptc1 þ /À Gli1zfd/zfd mice but they were present at lower levels compared with those observed in medulloblastomas from Ptc1 þ /À and Ptc1 þ /À Gli1 þ /zfd mice. Three distinct Gli1zfd transcript variants were detected in tumors derived from the Gli1 þ /zfd and Gli1zfd/zfd mutant mice (Figure 4, Gli1 panel, lanes 6, 7, 8 and 9). Gli1 transcripts migrating just above 28S rRNA in the Gli1zfd/zfd tumor sample were expressed at higher levels than in the Gli1 þ /zfd tumor sample (Figure 4, Gli1 panel, asterisks). The Gli1 transcripts likely reflect splicing variants since the targeted Gli1 locus still contains an intact 50UTR (Park et al., 2000). Since Gli1 is a target gene of Gli2, this suggests that Gli2 function in Gli1zfd/zfd tumors may be elevated. Gli2 was detected in all medulloblastomas examined but not in the adult cerebellum although, based on lacZ staining of a knock-in allele, it can be detected in the internal granule layer (IGL)of the cerebellum (Corrales et al., 2004). Interestingly, the levels of Gli2 were elevated in medulloblastoma from Ptc1 þ /À Gli1 þ /zfd and Ptc1 þ /À Gli1zfd/zfd mice compared to that in medulloblastoma from Ptc1 þ /À mice (Figure 4, Gli2 panel, lanes 6, 7, 8 and 9). These data imply that expression of Gli2 is upregulated in medulloblastoma from both Ptc1 þ /À Gli1 þ /zfd and Ptc1 þ /À Gli1zfd/zfd mice. Thus, Gli2 may contribute to medulloblastoma formation in both Ptc1 þ /À Gli1 þ /zfd and Ptc1 þ /À Gli1zfd/zfd mice. Previously, we reported that Ptc1 is expressed from the remaining normal allele in mouse medulloblastomas (Wetmore et al., 2000, 2001; Romer et al., 2004). Interestingly, the level of Ptc1 detected in medulloblas- Figure 4 Expression of the Shh signaling pathway genes and myc þ /À þ /À þ /zfd þ /À þ /À zfd/zfd tomas from Ptc1 , Ptc1 Gli1 and Ptc1 family genes in Ptc1 Gli1 medulloblastoma. Northern zfd/zfd analysis showing expression of Gli1, Gli2, Ptc1, Ptc2, N-myc and Gli1 mice is lower than that detected in P5 CB and c-myc in the normal cerebellum and the cerebellum with adult cerebellum even though these tumors express high medulloblastoma. P5, the developing cerebellum at postnatal day levels of Gli1 and Gli2. In contrast, Ptc2 was highly 5 (lane 1); Adult, adult cerebellum over 9 weeks of age (lane 2); 347, expressed in all medulloblastomas examined and it was 386, 387, cerebellum with medulloblastoma from Ptc1 þ /À mice (lanes 3,4 and 5); 162, 194, cerebellum with medulloblastoma from detected in P5 CB but not in adult cerebellum. Thus, the Ptc1 þ /À Gli1 þ /zfd mice (lanes 6 and 7); 208, 516, the whole level of Ptc1 does not directly correlate with the levels of cerebellum with medulloblastoma from Ptc1 þ /À Gli1zfd/zfd mice Gli1 and Gli2 in medulloblastoma in Ptc1 þ /À mice. (lanes 8 and 9). Numbers indicate mouse identification number. These data imply that in medulloblastoma, the levels of RNA from each sample was analysed using gene-specific antisense Ptc1 RNA are regulated independently from the levels probes for Gli1, Gli2, Ptc1, Ptc2, N-myc, c-myc and Gabra6 as indicated. Asterisks indicate distinct Gli1 transcripts. CB, cerebel- of Gli1, Gli2 and Ptc2. lum. EtBr staining indicates ribosomal RNAs (rRNA)of 28S and In a subset of human medulloblastoma both Gli1 and 18S as loading controls N-myc are present at high levels (Pomeroy et al., 2002). N-myc expression is upregulated by Shh in GNPCs and expression are observed in granule neurons in the adult it promotes proliferation of GNPCs (Kenney et al., cerebellum (Romer et al., 2004). In contrast, we found 2003; Oliver et al., 2003). We examined the expression that Gabra6, which is present in differentiated granule levels of N-myc and c-myc in medulloblastomas arising

Oncogene Gli1 is important for medulloblastoma formation in Ptc1 þ /À mice H Kimura et al 4032 from Ptc1 þ /À Gli1 þ /zfd and Ptc1 þ /À Gli1zfd/zfd mice compared with tumors from Ptc1 þ /À mice. Northern analysis indicated that all medulloblastomas expressed high levels of N-myc. Indeed, tumors from Ptc1 þ /À Gli1zfd/zfd mice exhibited remarkably elevated levels of N-myc expression although Gli1 was absent. C-myc expression was detected in medulloblastomas from Ptc1 þ /À, Ptc1 þ /À Gli1 þ /zfd and Ptc1 þ /À Gli1zfd/zfd mice; however, it did not correlate with the levels of Gli1, Gli2 and N-myc. Thus, it is possible that in the absence of Gli1, N-myc contributes more to tumorigenesis than c-myc.

Expression of Gli2 in cells transformed by Gli1 Overexpression of Gli1 dramatically induced expression of Gli1 and Ptc1 in NIH 3T3 cells 48 h after retroviral infection (Figure 1B). We performed Northern analysis to examine expression of Gli1, Gli2, Ptc1 and Ptc2 RNAs in NIH 3T3 cells in short-term (48 h after retrovirus infection)and long-term (2 weeks after retrovirus infection)cultures (Figure 5).Since F-Gli1 and NF-Gli1VP16 RNAs were transcribed as the F-Gli1 ires GFP and NF-Gli1VP16 ires GFP RNAs in the viral vector, we were able to distinguish the endogenous and exogenous Gli1 RNAs because of the differences in mRNA size. F-Gli1, NF-Gli1VP16 and F-Gli2 all induced expression of both Gli1 and Ptc1 following short-term expression in NIH 3T3 cells (Figure 5, lanes Figure 5 Differential induction of the endogenous Shh pathway 4, 5 and 7). NF-Gli1VP16 induced higher levels of Gli1 genes in NIH 3T3 cells dependent upon the culture conditions. than F-Gli1 (Figure 5, lanes 4, 5, 9 and 10). As expected, Northern blot analysis illustrating expression of Gli1, Gli2, Ptc1 F-Gli2 was capable of inducing Gli1 and Ptc1, although and Ptc2 in NIH 3T3 cells under the short-term (48 h)and long- the levels of Ptc1 observed were lower than those term (2 weeks)culture conditions after retroviral-mediated gene induced by F-Gli1 or NF-Gli1VP16 (Figure 5, lanes 4, 5 transfer. P7 CB, the developing cerebellum at postnatal day 7 (lane 1); parental, NIH 3T3 cells (lane 2); MSCV (control) (lane 3), and 7). In long-term culture of transformed cells, F-Gli1 MSCV F-Gli1 (lane 4), MSCV NF-Gli1VP16 (lane 5), pBabe and NF-Gli1VP16 induced expression of Gli1, Gli2, Ptc1 (control)(lane 6)and pBabe F-Gli2 (lane 7)indicate samples of and Ptc2. In contrast to tumor cells in vivo, in cultured NIH 3T3 cells harvested 48 h after retrovirus infection in culture fibroblasts, Ptc1 expression correlated directly with medium containing 10% FBS (short-time culture)or NIH 3T3 cells harvested 2 weeks after retrovirus infection in culture medium expression of endogenous Gli1 induced in these cells. containing 5% FBS (long-term culture)as indicated (lanes 8–12). F-Gli2 was equally efficient at inducing expression of EtBr staining of 28S and 18S rRNAs is shown as loading controls Gli1 in both short- and long-term cultures (Figure 5, lanes 7 and 12). Consistent with expression of endogenous Gli1 and Ptc1 proteins in short-term culture (Figure 1B), F-Gli2 induced expression of Gli1 and flies to humans. Deregulation of Hh signaling causes Ptc1 in both short- and long-term cultures (Figure 5, severe developmental abnormalities and can lead to lanes 7 and 12). Interestingly, in short-term cultures of oncogenesis. Heterozygous loss of Ptc1 in mice pro- NIH 3T3 cells, expression of either F-Gli1 or NF- motes medulloblastoma formation as a consequence of Gli1VP16 did not stimulate expression of Gli2 and Ptc2 derepression of Smo (Romer et al., 2004). Here, we (Figure 5, lanes 4 and 5). These data suggest that Gli1 investigated the role of Gli1 in spontaneous medullo- and Ptc1 are immediate target genes of Gli1 and Gli2 in blastoma in the Ptc1 þ /À mouse model. In fibroblast cell vitro and they imply that induction of Gli2 and Ptc2 may lines, enforced expression of Gli1 and Gli2 but not Gli3 be regulated indirectly by additional factors during cell induced morphological transformation. Thus, both Gli1 transformation. Taken together, these findings suggest and Gli2 are capable of inducing cell transformation. that activation of Gli1 is important for tumorigenesis Therefore, we tested whether Gli1 is required for tumor and Gli2 may assist in both cell transformation in vitro formation in vivo. We found that loss of Gli1 in Ptc1 þ /À and medulloblastoma formation in vivo by Gli1. mice resulted in a clearly reduced incidence of medulloblastoma. Thus, Gli1 is not only a potential clinical marker in medulloblastoma that have deregulated Shh Discussion signaling pathways but also plays a crucial role in medulloblastoma formation. Interestingly, in the few The Hh signaling pathway plays critical roles in cellular medulloblastomas arising in Ptc1 þ /À Gli1zfd/zfd mice, the responses during development that are conserved from level of Gli2 expression was elevated. Surprisingly, Gli2

Oncogene Gli1 is important for medulloblastoma formation in Ptc1 þ /À mice H Kimura et al 4033 expression was also elevated in medulloblastomas from suppression of Smo activity using a small molecule Ptc1 þ /À Gli1 þ /zfd mice. These findings suggest that there inhibitor reduces Gli1 expression in tumor cells in vivo is a compensatory mechanism in which reduced Gli1 and eradicates medulloblastoma (Romer et al., 2004). expression leads to an increase in Gli2 expression. Taken Here, we demonstrate a remarkable reduction in together, our data show that Gli1 is very important in medulloblastoma incidence in Ptc1 þ /À mice lacking medulloblastoma formation and that Gli2 shares this Gli1 function (Table 1). We also found that medullo- oncogenic function. blastomas from Ptc1 þ /À mice exhibit high levels of Gli1 but not Gli2 expression compared to the developing Role of Gli1 and Gli2 in proliferation of GNPCs cerebellum. Interestingly, the medulloblastoma incidence in Ptc1 þ /À mice was similar to that observed in Formation of the normal cerebellum requires the Ptc1 þ /À Gli11 þ /zfd mice, indicating that there is no precise temporal and spatial regulation of GNPCs haploinsufficiency effect. However, medulloblastoma proliferation. GNPCs proliferate in the EGL of the from Ptc1 þ /À Gli11 þ /zfd mice expressed elevated levels cerebellum, exit the cell cycle, differentiate and migrate of Gli2 (Figure 4). This suggests that increased inward to form the IGL in the postnatal cerebellum. expression of Gli2 may compensate for the reduced Shh, secreted by cells in the developing Purkinje cell level of Gli1 to regulate target genes responsible for plate, controls the dramatic expansion of the granule medulloblastoma formation in Ptc1 þ /À mice. In addi- cell population during this critical phase of cerebellar tion, expression of the mutated Gli1 transcripts in Ptc1 þ / development (Dahmane and Ruiz-i-Altaba, 1999; À Gli11zfd/zfd tumors was increased. This also suggests Wallace, 1999; Wechsler-Reya and Scott, 1999). Mice that Gli2 function may be increased as Gli1 is a target lacking Shh show severe defects including a reduction gene of Gli2. Thus, there is a threshold of Gli in brain size (Chiang et al., 1996). Furthermore, transcriptional activity required for medulloblastoma conditional ablation of Shh in Purkinje cells results formation that can be met by contributions from both in a dramatic reduction of the EGL and the IGL in Gli1 and Gli2. the developing cerebellum (Lewis et al., 2004). In Gli1 is not absolutely required for medulloblastoma addition, the use of small molecule inhibitors of formation as we observed a few tumors in Ptc1 þ /À mice Smo demonstrates that the proliferative activity of lacking Gli1. This is consistent with a prior report GNPCs depends on downstream signaling by Smo showing that ectopic expression of Shh in cerebellar (Frank-Kamenetsky et al., 2002; Romer et al., 2004). anlage, using a retroviral vector, induced medulloblas- Both Gli1 and Gli2 are expressed in the developing toma in both normal and Gli1 mutant mice (Weiner EGL (Dahmane and Ruiz-i-Altaba, 1999; Wallace, et al., 2002). Ptc1 þ /À tumors from mice lacking Gli1 1999; Corrales et al., 2004). However, the cerebellum express high levels of Gli2, suggesting that Gli2 may be develops normally in the absence of Gli1 (Park et al., involved in medulloblastoma formation by deregulating 2000; Bai et al., 2002). In contrast, loss of Gli2 causes Shh signaling. Medulloblastoma formation can be embryonic lethality that can be rescued by a low level induced by ectopic expression of Shh in GNPCs in of Gli1 expression (Bai and Joyner, 2001). Gli2 mutant both normal and Gli1 mutant mice (Weiner et al., 2002). mice have a reduced EGL and greatly reduced During normal cerebellar development, Shh is made by Gli1 expression at birth (Corrales et al., 2004). Thus, Purkinje cells from approximately E 17.5 (Lewis et al., Gli1 and Gli2 have overlapping functions during normal 2004). Thus, autocrine activation of Shh signaling using cerebellar development. Here, we found that, in the a Shh retrovirus may promote transformation by developing cerebellum, the expression levels of Gli2 deregulating Gli2 expression. In fact, we found that and Ptc1 were comparable to those observed in both the transforming activities of Gli1 and Gli2 correlated Gli1zfd/zfd and Gli1lZ/lZ mice. These data suggest that Gli2 with the levels of transcription activity observed in can maintain expression of Gli1 target genes in the culture, indicating that cell transformation is mediated absence of Gli1. This may explain why Gli1 is not by activation of specific target genes. However, essential for proliferation of GNPCs and normal Gli1VP16, although a stronger transcription activator cerebellar development. than Gli1, was less potent at inducing cell transformation, suggesting that the transcription activation domain confers some specificity for target genes involved in Role of Gli1 in medulloblastoma formation in Ptc1 þ /À mice transformation. Previously, it was shown that Gli1 induces transforma- Deregulated Shh signaling and N-myc expression in tion and promotes tumor formation in allografts of cells medulloblastoma from Ptc1 þ /À mice expressing E1A (Ruppert et al., 1991). Gli1 is expressed in approximately a third of human medulloblastoma Several lines of evidence implicate N-myc regulation of and it is present at high levels in the spontaneous tumors D-type cyclins in the control of GNPC proliferation. arising in mice with mutations in Ptc1, DNA ligase IV, Shh induces up regulation of N-myc and increases cyclin-dependent kinase inhibitors or Parp1 (Lee et al., expression of Cyclin D1 and Cyclin D2 without de novo 2003; Tong et al., 2003). Therefore, Gli1 is likely to play protein synthesis in GNPCs (Kenney et al., 2003, 2004; an important role in medulloblastoma formation. This Oliver et al., 2003). Ectopic expression of N-myc induces was underscored by our recent observation that expression of Cyclin D1 and Cyclin D2 in primary

Oncogene Gli1 is important for medulloblastoma formation in Ptc1 þ /À mice H Kimura et al 4034 GNPCs culture (Ciemerych et al., 2002; Kenney et al., Genetic background effects on tumorigenesis in Ptc1 þ /À 2004). Conditional disruption of N-myc in neuronal mice precursor cells results in severe defects in the cerebellar Previously, we reported that the incidence of medullo- primordium and the postnatal developing cerebellum Ptc1 þ /À (Knoepfler et al., 2002). Furthermore, mice with loss blastoma in mice on a C57Bl/6 background is similar to that on a 129Sv background (Goodrich et al., of both Cyclin D1 and Cyclin D2 exhibit severe 1997; Wetmore et al., 2000). However, Ptc1 þ /À mice on cerebellar developmental defects despite the fact that N-myc is still expressed in the EGL (Ciemerych et al., a CD1 background develop more rhabdomyosarcomas than medulloblastomas (Calzada-Wack et al., 2002; 2002). Medulloblastomas arising in the various mouse Pazzaglia et al., 2002). Here, the first generation of models described above all show high levels of N-myc Ptc1 þ /À Gli1 þ /zfd mice, generated by crossing Ptc1 þ /À as well as Gli1 (Lee et al., 2003). Thus, N-myc mice on a C57Bl/6 background with Gli1 þ /zfd mice may promote medulloblastoma formation as well as derived from 129Sv ES cells but maintained on a Swiss proliferation of GNPCs. We found that N-myc was Webster background, exhibited a 60% incidence (six out highly expressed in medulloblastomas from Ptc1 þ /À, of 10 mice)of medulloblastoma and a 10% incidence Ptc1 þ /À Gli1 þ /zfd and Ptc1 þ /À Gli1zfd/zfd mice as well as in (one out of 10 mice)of rhabdomyosarcoma by 8 months the developing cerebellum. The N-myc level was not of age. Continued intercrossing of these Ptc1 þ /À Gli1 þ /zfd directly correlated with the level of Gli1, but it was correlated with Gli2 expression in medulloblastomas. N- mice resulted in a reduced incidence (21.8%)of medulloblastoma that was similar to the previously myc levels were higher in tumors from Ptc1 þ /À Gli1zfd/zfd reported incidence (Table 1)(Wetmore et al., 2000). mice. These results support the contention that Gli2 These observations of strain-specific effects on both transcriptional activity is higher in Ptc1 þ /À Gli1zfd/zfd tumor incidence and spectra in Ptc1 þ /À mice indicate tumors than in the other tumors. These data suggest that there may be several modifier genes involved in that N-myc is not a direct target of Gli1 but it may be a the generation of medulloblastoma and rhabdo- direct target of Gli2. Alternatively, it is possible that myosarcoma. N-myc expression is regulated by an as yet unidentified Our findings indicate that Gli1 plays an important effect of Shh, independently of Gli1 and Gli2, in the role in medulloblastoma formation in Ptc1 þ /À mice. We developing cerebellum and in medulloblastoma formation. also observed an unanticipated upregulation of Gli2 in the absence of Gli1 during medulloblastoma formation. The ultimate mechanism(s)responsible for controlling Possible other regulatory signals in the Shh pathway target genes in the Shh-Gli1 network and the identity of the genes responsible for tumorigenesis remain to be Previously, it was suggested that wild type Ptc1 mRNA uncovered. Nevertheless, our study supports the use of is undetectable in Ptc1 þ /À medulloblastoma (Berman Gli1 expression as a molecular target for developing et al., 2002). However, we routinely observe low levels of novel therapies to treat the subset of human medullo- transcription from both the normal Ptc1 allele as well as blastoma associated with increased Shh pathway the mutant allele in medulloblastoma from Ptc1 þ /À and signaling. Ptc1 þ /À p53À/À mice (Wetmore et al., 2000, 2001; Romer et al., 2004). Here we show the presence of Ptc1, but no up regulation, in medulloblastoma despite the fact that both Gli1 and Gli2 readily induce expression of endogenous Ptc1 in cultured medulloblastoma cells Materials and methods (Romer et al., 2004). These data suggest that Ptc1 Plasmids, cell culture, transfections and luciferase assays expression may be regulated in both an Shh pathway- dependent and an Shh pathway-independent manner in For immuno-detection, a FLAG, or an NLS plus FLAG GNPCs and medulloblastoma. One explanation for epitope sequence was inserted into the N-terminus of mouse these results is that Ptc1 may be controlled by an Gli1 and Gli2 (a kind gift from Dr Hiroshi Sasaki from RIKEN, Kobe)in pcDNA3.1. by PCR. A series of Gli1 alternative promoter in the tumor cells. The fact that mutants were constructed by site-directed mutagenesis and Ptc1 is not upregulated in medulloblastoma suggests restriction enzyme digestion according to the manufacturer’s that the negative feedback loop, characteristic of Shh instructions (QuikChange, Stratagene, CA, USA). DNA signaling in Drosophila development and mouse embry- fragments in all constructs were verified by sequencing. ogenesis (Goodrich and Scott, 1998), is not functional. 293T cells, NIH 3T3 cells and 208F cells were maintained in Both mouse and human medulloblastomas express high DMEM with 10% FBS and supplements (1 mM Gluta Max levels of several genes associated with GNPCs prolifera- (Invitrogen, CA, USA), 100 U/ml penicillin, 100 mg/ml strep- tion including Cyclin D2, N-myc, CXCR4 and IGF2 as tomycin). For quantification of the Shh pathway response, a well as Gli1 and Gli2, indicating that the Shh pathway is construct containing 8 Â Gli binding sites fused to firefly active (Klein et al., 2001; Pomeroy et al., 2002; Zhao luciferase reporter (a kind gift from Dr Hiroshi Sasaki)(Sasaki et al., 1999)and a Renilla luciferase reporter (phRL-SV40, et al., 2002; Lee et al., 2003). It is possible that the Promega)were used. Luciferase activity was measured 48 h negative feedback loop that modulates physiological after transfection. All luciferase assays were performed Shh signaling is abrogated by an unknown mechanism according to the manufacturer’s instructions (Promega, Dual in tumors and that this contributes to medulloblastoma luciferase assay kit)and were normalized for transfection formation in Ptc1 þ /À mice. efficiency using Renilla luciferase activity.

Oncogene Gli1 is important for medulloblastoma formation in Ptc1 þ /À mice H Kimura et al 4035 Virus production and retrovirus gene transfer tumors were evident, according to NIH-approved institutional guidelines. In each mouse, the presence of a tumor was 293T cells were transfected with helper retrovirus plasmids, confirmed by gross examination of the brain after 12 months pPAM3 and pVSV-G, encoding gag-pol-env and the G of age. If the mouse was not available for examination or if no glycoprotein from vesicular stomatitis virus, respectively, tumor was detected, the cause of death was indicated as together with the murine stem cell virus (MSCV)-internal unknown. Genotyping of Ptc1, Gli1zfd and Gli1lZ alleles was ribosome entry site (ires)-green fluorescent protein (GFP) performed as previously described (Goodrich et al., 1997; Park vectors encoding FLAG-tagged, NLS-FLAG-tagged wild type et al., 2000; Bai et al., 2002). All the mice were handled in or mutant Gli1 proteins, or pBabe-puro vector encoding accordance with institutional guidelines and housed in an FLAG-tagged Gli2 or -Gli3 (The original F-Gli3 clone was a AAALAC accredited, pathogen free environment on a 12 : 12 h gift from Dr Shunsuke Ishii, RIKEN, Tsukuba (Dai et al., light : dark cycle. 1999)). Viruses were harvested 24–60 h post-transfection, pooled and stored on ice. Cells were infected with virus- containing medium plus Polybrene (10 mg/ml). Infectious units Statistic analysis were measured by flow cytometric analysis of GFP expression Fisher’s exact test was used to make comparisons between any levels and by counting the number of drug selected NIH 3T3 two mice groups. P-value o0.01 considered significant. cell colonies. For focus formation assays, cells were plated at 4 Â 105 on 60-mm dishes one day before virus infection, and then they were infected at a multiplicity of infection (MOI)of Northern analysis 0.001 to 10. Culture medium was replaced with DMEM RNA extraction from tissues and cell culture was performed containing 5% FBS and supplements the next day and every 3 using TriPure reagent according to the manufacturer’s days thereafter. For soft agar assays, after virus infection, instructions (Roche Applied Science, Indianapolis, USA). In 2 Â 105 cells were suspended in 1 ml of 0.3% noble agar (BD total, 10 mg of total RNA were separated by electrophoresis Diagnostic Systems, MD, USA)in IMDM culture medium under formaldehyde denaturing conditions. RNA was trans- (Invitrogen, CA, USA)containing 11.25% FBS and supple- ferred onto a NytranSPC membrane (Schleicher and Schuell ments. The cell suspension was plated onto 0.6% bottom noble BioScience, Inc., NH, USA)and hybridized at 67 1C with agar in IMDM with 15% FBS and supplements. antisense RNA probes for mouse Gli1 (nucleotides corresponding to amino acids 271–837), mouse Gli2 (nucleotides Immunoblotting corresponding to amino acids 256–531), mouse Ptc1 (nucleotides corresponding to amino acids 1398–1434 and the 30 Western blot analysis was carried using whole-cell lysates as untranslated region (741 nucleotides)IMAGE clone 3989319), previously described (Kimura and Shiota, 2003)using en- mouse Ptc2 (nucleotides corresponding to amino acids 652– hanced chemiluminescence detection (ECL plus, Amersham, 1182 and 30 untranslated region (577 nucleotides)IMAGE NJ or Supersignal West Femto Maximum Sensitivity Sub- clone 3972649), mouse N-myc (nucleotides corresponding to strate, Pierce, IL, USA). Proteins were separated by SDS– amino acids 2–462)and mouse c-myc (nucleotides correspond- PAGE and transferred onto nitrocellulose (Invitrogen, CA, ing to amino acids 2–439), mouse Gabra6 (nucleotides USA)or Immobilon-P membrane (Millipore).Blots were corresponding to amino acids 315–433 and 30 untranslated probed with the following antibodies: anti-FLAG (M2 or region (521 nucleotides)). IMAGE clones were obtained from polyclonal, 1 : 5000, Sigma), anti-C-terminal Gli1 (1 : 2000 or Research Genetics, Inc., CA, USA. 5000)(Wetmore et al., 2000), anti-Ptc1 (1 : 1000) (a kind gift from Dr Allen Bale, Yale University, CT, USA). Acknowledgements We thank Drs Hiroshi Sasaki for Gli1, Gli2 clones and Gli-luc Animals reporter plasmids, Shunsuke Ishii for F-Gli3 plasmid, Suzanne Ptc1 þ /À mice obtained from Dr Mathew Scott, (Stanford Baker for the VP16 clone, Allen Bale for anti-Ptc1 antiserum, university, CA, USA)were generated on a mixed C57Bl/6 and and Peter McKinnon for N-myc and c-myc clones. We also 129Sv strain background (Goodrich et al., 1997)and were thank Curran lab members for critical reading of the manu- maintained on a C57Bl/6 background (Wetmore et al., 2000). script and helpful comments. We are grateful to Ms Michele Gli1zfd mice were maintained on a Swiss Webster background Connelly for technical assistance. We thank Mehmet Kocak, (Park et al., 2000). Gli1lZ mice were generated from inbred Dana Wallace and Dr James Boyett for statistical analyses. 129SvEv ES cells, backcrossed to Black Swiss strain mice and This work was supported by a grant CA096832 from the maintained on a Swiss Webster background. All mice were National Institutes of Health and the American Lebanese euthanized when they were moribund, or when they showed Syrian Associated Charities (ALSAC)(TC).AJ is an signs of increased intracranial pressure, or when extracranial investigator of the Howard Hughes Medical Institute.

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