A Distinct Smoothened Mutation Causes Severe Cerebellar Developmental Defects and Medulloblastoma in a Novel Transgenic Mouse Model Joyoti Dey,a,b Sally Ditzler,b Sue E. Knoblaugh,c Beryl A. Hatton,b Janell M. Schelter,d Michele A. Cleary,d Brig Mecham,e Lucy B. Rorke-Adams,f and James M. Olsona,b,g Molecular and Cellular Biology Program, University of Washington, Seattle, Washington, USAa; Clinical Research Divisionb and Comparative Medicine Shared Resource,c Fred Hutchinson Cancer Research Center, Seattle, Washington, USA; Merck Research Laboratories, Merck & Co., Incorporated, Boston, Massachusetts, USAd; Sage Bionetworks, Seattle, Washington, USAe; Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USAf; and Seattle Children’s Hospital, Seattle, Washington, USAg Deregulated developmental processes in the cerebellum cause medulloblastoma, the most common pediatric brain malignancy. About 25 to 30% of cases are caused by mutations increasing the activity of the Sonic hedgehog (Shh) pathway, a critical mitogen in cerebellar development. The proto-oncogene Smoothened (Smo) is a key transducer of the Shh pathway. Activating mutations in Smo that lead to constitutive activity of the Shh pathway have been identified in human medulloblastoma. To understand the developmental and oncogenic effects of two closely positioned point mutations in Smo, we characterized NeuroD2-SmoA2 mice and compared them to NeuroD2-SmoA1 mice. While both SmoA1 and SmoA2 transgenes cause medulloblastoma with similar frequencies and timing, SmoA2 mice have severe aberrations in cerebellar development, whereas SmoA1 mice are largely normal during development. Intriguingly, neurologic function, as measured by specific tests, is normal in the SmoA2 mice despite exten- sive cerebellar dysplasia. We demonstrate how two nearly contiguous point mutations in the same domain of the encoded Smo protein can produce striking phenotypic differences in cerebellar development and organization in mice. he protracted phase of extensive proliferation during cerebel- essary to investigate the unique behavior of driving mutations, Tlar development makes it vulnerable to neoplastic transforma- since the downstream effects may be distinct. Since medulloblas- tion (42). Medulloblastoma, a developmental cancer of the cere- toma results from developmental aberrations (25), investigation bellum, continues to be the most common pediatric brain cancer. of critical milestones in cerebellar development will provide valu- Standard treatments result in neurocognitive impairment and ad- able insights in this area. Toward this goal, we developed the verse quality of life (12, 34). SmoA2 mouse model of medulloblastoma and carried out a Medulloblastomas are categorized based on histological char- comparative analysis with the existing SmoA1 model. SmoA1 acteristics and molecular signatures (12, 41). Genetic aberrations (W539L) and SmoA2 (S537N) mutations, originally identified in leading to hyperactive Sonic hedgehog (Shh) signaling in granule human cancer patients (31, 45), lie in the same transmembrane neuron precursors (GNPs) cause 25 to 30% of medulloblastoma domain of Smo and cause constitutive activation of the Shh path- cases (17). The Shh pathway plays a pivotal role in cerebellar de- way (40). While the SmoA1 mutation has been widely studied, velopment by regulating proliferation of GNPs and foliation (7, very little is known about SmoA2. 44). The Shh subgroup has been widely studied with numerous Through characterization of the SmoA2 model, we show strik- mouse models recapitulating the human disease (26). The overall ing differences between the SmoA1 and SmoA2 mutations at the prognosis in patients with Shh-driven medulloblastomas, how- molecular and cellular levels. While both mutations lead to ever, remains intermediate (41). medulloblastomas, the SmoA2 mutation uniquely causes severe Within the Shh subgroup of human medulloblastoma there exists significant biological and clinical heterogeneity, the under- defects in cerebellar development. Early in development, the two lying molecular basis of which remains to be explored (29, 36). mutations lead to distinct transcriptional profiles affecting differ- Leptomeningeal dissemination observed uniquely in the SmoA1 ent biological processes. Despite disruptions in the cytoarchitec- homozygous (Smo/Smo) mouse model and not other Shh-driven ture thought to be critical for cerebellar function, the SmoA2 mice, models (18) demonstrates disparities in pathology. Inhibition of intriguingly, do not display clinical signs of cerebellar malfunc- the Shh pathway by the Smoothened (Smo) antagonist cyclo- tion. pamine varies based on mutations driving hyperactive signaling (4, 5), leading to differences in therapeutic responses. Aberrations in genes outside the Shh pathway also lead to medulloblastomas Received 27 June 2012 Returned for modification 23 July 2012 with Shh signatures in mice, highlighting the widespread interac- Accepted 31 July 2012 tions of the Shh pathway with other networks (26). In several Published ahead of print 6 August 2012 mouse models, medulloblastoma-prone progenitors exit the cell Address correspondence to James M. Olson, [email protected]. cycle and undergo normal neuronal differentiation, suggesting Supplemental material for this article may be found at http://mcb.asm.org/. that factors in addition to initiating mutations contribute to tu- Copyright © 2012, American Society for Microbiology. All Rights Reserved. morigenesis (2) and possibly tumor heterogeneity. doi:10.1128/MCB.00862-12 While broad molecular classifications are important, it is nec- 4104 mcb.asm.org Molecular and Cellular Biology p. 4104–4115 October 2012 Volume 32 Number 20 SmoA2 Causes Cerebellar Developmental Defects MATERIALS AND METHODS 7900HT Fast Real-time PCR System. TaqMan gene expression assays (Ap- Generation of the SmoA1 and SmoA2 transgenic lines. The SmoA1 and plied Biosystems) were used for mouse Gli1, Gli2, Ptch1, Ptch2, and Smo. SmoA2 transgenic mouse lines were previously described (16, 18). Both Data were analyzed using ABI GeneAmp SDS v2.3 software. Primers for lines were generated and maintained on a C57BL/6 background. SmoA2 qRT-PCR (Platinum SYBR green; Invitrogen) verification of microarray hemizygous and SmoA1 homozygous (Smo/Smo model) (18) mice of ei- data were as follows: Bcl11b FP, 5=-ACGCGTAAAGATGAGGCCTTC- ther sex were used for all experiments, except for the transgene copy num- 3=, and RP, 5=-AAGCCATGTGTGTTCTGTGC-3=; MyoD FP, 5=-CCCG ber analysis, where the SmoA1 hemizygous line was used (16). SmoA1 and CGCTCCAACTGCTCTG-3=, and RP 5=-GGCTCGACACAGCCGCACT SmoA2 mutations were originally identified in human cancer cases (31, C-3= (a kind gift from the Stephen Tapscott laboratory); Pou4f2 FP, 5=-C 45) and correspond to W539L and S537N, respectively, in mouse Smo GGAGAGCTTGTCTTCCAAC-3=, and RP, 5=-GCCAGCAGACTCTCAT (40). All mice were maintained in accordance with the NIH Guide for the CCA-3=; Pou4f1 FP, 5=-GACCTCAAAAAGAACGTGGTG-3=, and RP, Care and Use of Experimental Animals with approval from the Fred 5=-TAAGTGTCTCTGGTCCCCTCAG-3=; Cbln4 FP, 5=-TGAGCAAC Hutchinson Cancer Research Center Institutional Animal Care and Use AAGACTCGCATC-3=, and RP, 5=-GTGCCACAAAGACAGATTCC-3=; Committee (IR1457). Ranbp17 FP, 5=-TTAGAGCGCGCGATAATTG-3=, and RP, 5=-TCTGGG Copy number determination. Transgene copy numbers were approx- CTGTCAATCAGTTC-3=; Isl1 FP, 5=-AGATCAGCCTGCTTTTCAGC- imated using a quantitative-PCR (qPCR) approach (Platinum SYBR 3=, and RP, 5=-ATGCTGTTGGGTGTATCTGG-3=; and Aqp1 FP, 5=-TCC green; Invitrogen) based on existing methodologies (19, 24). Briefly, 10- TCCCTAGTCGACAATTCAC-3=, and RP, 5=-TGCAGAGTGCCAATGA fold serial dilutions of wild-type (WT) mouse genomic DNA (ranging TCTC-3=. All assays were run in triplicate and normalized to endogenous from 190 to 0.019 ng) were used to make standard curves to determine the control mouse ␤-2 microglobulin (␤2m)orGapdh for TaqMan assays, efficiency and specificity of each primer pair used in the copy number and Cyclophilin A (Ppia) FP, 5=-GAGCTGTTTGCAGACAAAGTTC-3=, analysis. Primers with 100% efficiency against the gene of interest, Smo and RP, 5=-CCCTGGCACATGAATCCTGG-3= (a kind gift from the Sunil Exon 10, and control loci, RNAseP, were used for copy number estima- Hingorani laboratory), were used for SYBR green assays. tion. The additional primers Smo Exon 6 and control locus Gapdh were Western blot analysis. GNPs from P5 SmoA1, SmoA2, and WT mice used for confirmation. The Smo primers used recognize both the endog- were harvested by previously described techniques (22) and used for prep- enous Smo gene and the transgenes SmoA1 and SmoA2, which differ by a aration of protein lysates using RIPA buffer (Millipore) with Halt protease point mutation from the endogenous loci. The sequences of the primers inhibitor cocktail (Pierce Biotechnology) and Phosphatase Inhibitor are as follows: Smo Exon 6 FP, 5=-CGTGAGTGGCATCTGTTTTG-3=, Cocktail 2 (Calbiochem) and Cocktail 3 (Sigma). Equal amounts of pro- and RP, 5=-AGTAGCCTCCCACAATAAGCAC-3=; Smo Exon 10 FP, 5=- tein from each sample (25 ␮g) were subjected to SDS-PAGE using a Nu- AGAGCAAGATGATCGCCAAG-3=, and RP, 5=-CCATCATGGGAGAC PAGE Novex 4 to 12% Bis-Tris gel (Invitrogen), transferred to a nitrocel- AGTGTG-3=; RNAseP FP, 5=-CTCCCCAAATGGAAGATGAG-3=, and lulose membrane using the X-Cell SureLock Mini cell system RP, 5=-TATTCTACGTTCCGGTGTGG-3=; and Gapdh FP, 5=-AAATGA (Invitrogen), and probed with Smo antibody (H-300; Santa Cruz Biotech- GAGAGGCCCAGCTAC-3=,