A Distinct Smoothened Mutation Causes Severe Cerebellar Developmental Defects and 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 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 . 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-

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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=, and RP, 5=-TTATAGGAACCCGGATGGT nology; 1:100) and loading control ␤-actin (Abcam; 1:2,500). Corre- G-3=. Subsequently, 5 ng of genomic DNA (n ϭ 5 mice per genotype)— sponding horseradish peroxidase-conjugated secondary antibodies were SmoA1 hemizygous, SmoA2 hemizygous, and WT—was amplified using obtained from Santa Cruz Biotechnology and used at 1:5,000 dilution. the same qPCR conditions. The comparative threshold cycle (CT) method Proteins were detected by incubating membranes in chemiluminescent was used to calculate Smo copy numbers in SmoA1 and SmoA2 transgenic substrate (ECL kit; Pierce), followed by exposure to Kodak X-Omat sci- mouse genome relative to normal Smo in the WT reference genome. entific imaging film. Mouse pathology and immunohistochemistry. Mice were anesthe- Transplantation experiments. All experiments involving animals tized using CO2 inhalation, the cerebella were removed, and tissues were were conducted in accordance with the NIH Guide for the Care and Use of snap-frozen for RNA studies and GNP isolation or fixed in 10% parafor- Experimental Animals with approval from the Fred Hutchinson Cancer maldehyde for pathological examination. Tissue blocks were paraffin em- Research Center Institutional Animal Care and Use Committee (IR1457). bedded, cut into 4-␮m sections, and stained with hematoxylin and eosin GNPs were harvested from P5 SmoA2 mice. Orthotopic transplants of (H&E) using standard methods. Immunohistochemical analyses were 2-␮l cell suspensions containing 1 ϫ 106 SmoA2 GNPs were carried out in carried out as follows: (i) for NeuN, a mouse-on-mouse procedure devel- recipient athymic nude mice (Nu/Nu; Jackson Laboratory) as previously oped at the Fred Hutchinson Cancer Research Center (FHCRC) Experi- described (37). mental Histopathology Shared Resource was used with NeuN primary Behavioral tests. The RotaRod test was conducted using a RotaRod antibody (Millipore; 1:100) to create an antibody complex with a rabbit (model 7650; Ugo Basile Comerio, Italy), accelerating from 3 to 30 rpm anti-mouse Fab fragment addition of adding it to the tissue, followed by over a 5-min period. The mice were given a trial run prior to the timed Biocare Mach2 rabbit polymer; (ii) for calbindin, rabbit primary antibody run, where the latency to fall (the time it takes the mouse to fall off the rod, (Millipore; 1:500) followed by biotinylated goat anti-rabbit secondary measured from the start of the test) was recorded. antibody–Vectastain Elite avidin-biotin complex (Vector Laboratories) Footprint assay. The bottom of each foot was coated with nontoxic was used; (iii) for Ki67, rat primary antibody (Dako Tec3; 1:100) followed paint, and the mouse was allowed to walk through a small tunnel on white by biotinylated goat anti-rat antibody–streptavidin-horseradish peroxi- paper. Stride length (the distance between two hind paw prints) and dase (HRP) was used; and (iv) for S100, rabbit primary antibody (Dako; stance width (the distance between opposite hind paw prints perpendic- 1:400) followed by Dako Envision mouse polymer was used. Slides were ular to the walking trajectory) were calculated. Each mouse was given a developed using 3,3=-diaminobenzidine (Dako DAB Plus) reagent fol- trial run before the final run. lowed by Dako Autostainer hematoxylin counterstain. Sections were vi- Modified SHIRPA. SHIRPA stands for SmithKline Beecham Pharma- sualized with a Nikon E800 microscope, and images were captured using ceuticals; Harwell, MRC Mouse Genome Centre and Mammalian Genet- the CoolSnap cf color camera (FHCRC Scientific Imaging Core). ics Unit; Imperial College School of Medicine at St. Mary’s; Royal London qRT-PCR. For quantitative reverse transcription PCR (qRT-PCR) Hospital, St Bartholomew’s, and the Royal London School of Medicine analyses, mRNA was isolated from either snap-frozen tissue or GNPs phenotype assessment. Our laboratory developed an abbreviated behav- from postnatal day 5 (P5) SmoA1, SmoA2, and WT mice harvested using ioral test based on SHIRPA that we called the modified SHIRPA. A single previously described techniques (22), using the RNAeasy kit (Invitrogen) observer evaluated mice for weight, physical phenotype (tremor [1, none/ followed by DNase (Qiagen) treatment and conversion to cDNA using the mild; 2, marked], body position [1, elongated; 2, hunched/rounded], and ABI High Capacity cDNA reverse transcription kit (Life Technologies). tail position [1, horizontally extended; 2, dragging/straub]), and behavior Reactions were set up using TaqMan Master Mix and run on an ABI phenotype (grooming [1, slow/casual; 2, none/excessive], spontaneous

October 2012 Volume 32 Number 20 mcb.asm.org 4105 Dey et al. activity [1, moderate, mouse covers all quadrants of cage; 2, slow, 1 to 3 ternal granular layer (EGL) consisting of aberrantly migrating quadrants; 3, none/darting/circling], and locomotor activity [the number GNPs (Fig. 1D). There is a lack of normal foliation and ectopic of times the mouse places at least one of its paws on the side of the cage progenitor-like cells in the adjacent parenchyma and along the over a 2-min period]). pial surface (Fig. 1D). At P14, the SmoA2 cerebellum continues to RNA isolation and gene expression analysis. Total RNA was isolated manifest extensive dysplasia with massive hypercellularity (Fig. from TRIzol lysates of each cerebellum from P5 WT, Smo/Smo, and 1E). The dysplastic regions consist of normal progenitor cells and SmoA2 cerebella (n ϭ 3 animals per genotype) using the Promega SV-96 RNA isolation kit. Microarray analysis was performed using custom-de- atypical cuboidal to spindle-shaped cells with indistinct cell bor- signed Affymetrix arrays. Extracted RNA was quantified with RiboGreen ders and a scant amount of eosinophilic fibrillar cytoplasm with RNA quantitation reagent (Invitrogen), and its quality was assessed by irregularly round to fusiform nuclei, reminiscent of medulloblas- use of the Agilent RNA 6000 Pico kit (Agilent, Santa Clara, CA) in an toma. These features strongly suggest a primitive phenotype and Agilent 2100 Bioanalyzer (Agilent). Samples were amplified and labeled possible neoplastic transformation. However, the atypical cells in using the Ovation WB protocol (NuGen Technologies, San Carlos, CA), the SmoA2 P14 cerebellum also have morphological similarities to according to the manufacturer’s instructions. The resulting amplified the normal GNPs remaining in the outer EGL of the WT P14 cDNAs were hybridized to Affymetrix gene expression chips (Mouse Ro- cerebellum that are still undergoing migration. setta Custom Affymetrix 1.0; Affymetrix, Santa Clara, CA). Images were By P28, the WT cerebellum attains its mature size and shape analyzed with Affymetrix GeneChip Operating Software (GCOS) and (Fig. 1C). Although 100% of the SmoA2 cerebella remain dysplas- processed further to derive sequence-based intensities by use of the robust tic in adult mice, the cytoarchitecture is more mature than at the multiarray average (RMA) algorithm. The Rosetta Resolver system (Ro- setta Biosoftware, Seattle, WA) was used to calculate fold changes and hypercellular P14 stage (Fig. 1F). The cells in the dysplasia are ratio P values for the differential expression of genes in each of three morphologically identical to the mature granule neurons in the replicates of SmoA1 or SmoA2 mice versus a virtual pool of age-matched WT cerebellum. At this stage, atypical cells, if any, are exclusively WT controls (n ϭ 3). P values were calculated using the Rosetta intensity- positioned along the pial surface, clearly separate from the adja- based Affymetrix error model. Genes that were present in at least two of cent dysplastic regions. the three replicates for SmoA1 and SmoA2 with a P value of Ͻ 0.001 and an Phenotypic differences between SmoA1 and SmoA2 mice do absolute average fold change of Ն2 were considered significantly differ- not result merely from differences in Smoothened expression. ently expressed genes. For clustering analysis, we first filtered the data to To determine whether the cerebellar developmental disorganiza- remove data from probe sets whose expression levels did not significantly tion phenotype observed in SmoA2 mice in comparison to SmoA1 vary across the samples (defined as the top 50% least variable). The filtered mice stems from differences in transgene copy numbers and the data were then clustered using traditional hierarchical clustering. The Gene Ontology (GO) enrichment analysis was carried out using a hy- resulting expression of the SmoA1 and SmoA2 transgenes, we as- pergeometric test for each unique category. We selected significantly en- sessed differences at the genomic level, as well as total Smo mRNA riched categories, defined as any with a false discovery rate (FDR) of and protein. Ͻ10%. We approximated transgene copy numbers in genomic Statistical tests. A Student t test was used to determine if differences DNA samples from SmoA1 (n ϭ 5) and SmoA2 (n ϭ 5) lines by observed between measurements obtained from two groups were statisti- comparing copies of Smo to WT mouse genomic DNA, using cally significant. previously described qPCR-based methodologies (19, 24). Microarray data accession number. Microarray data have been de- qPCR analysis showed that SmoA1 and SmoA2 mice contain posited in the NCBI Gene Expression Omnibus (GEO) and are accessible 39.1 Ϯ 7.7 (standard error of the mean [SEM]) and 6.9 Ϯ 1.5 through GEO series accession number GSE34593. times more copies of Smo than the WT reference, respectively (Fig. 2A and B). RESULTS We investigated Smo expression by measuring total Smo SmoA2 and SmoA1 mutations in the Smoothened receptor have mRNA and protein, that is, expression from the respective trans- vastly different effects on cerebellar development. SmoA2 genes, as well as endogenous Smo. By qRT-PCR, we measured (S537N) and SmoA1 (W539L) are activating point mutations that levels of total Smo and Smo-dependent canonical targets of the lead to constitutive Shh signaling and were originally identified in Shh pathway (Gli1, Gli2, Ptch1, and Ptch2)inSmoA1 and SmoA2 human cancer cases of medulloblastoma and basal cell carcinoma, cerebella relative to age-matched WT controls. We accounted for respectively (31, 40, 45). In our previous studies, we described the the differences in cellular composition in SmoA1 and SmoA2 cer- SmoA1 transgenic mouse medulloblastoma model, which ex- ebella by using purified GNP lysates from all genotypes. We chose presses the SmoA1 transgene driven by the GNP-specific fragment this specific developmental stage because (i) the phenotypes of of the NeuroD2 (ND2) gene promoter, causing constitutive Shh SmoA2 and SmoA1 are distinct at P5 and (ii) GNP isolation at P5 signaling exclusively in the cerebellum (16, 18). allows accurate assessment of Smo and Shh target levels in a ho- In this study, we have characterized the SmoA2 transgenic mogeneous cell population. Our results show that (i) although at mouse model with a similarly designed transgene expressing the an mRNA level, SmoA2 mice have ϳ3.8-fold higher Smo mRNA SmoA2 mutation. Comparative histopathological analyses show levels than SmoA1 mice, there is no meaningful difference (less striking differences in phenotypes—while SmoA1 mice have than 2-fold) in the levels of activation of the Shh pathway, as is largely normal development of the cerebellum (Fig. 1G to I) sim- evident from similar levels of target mRNA in SmoA1 and SmoA2 ilar to that of WT mice (Fig. 1A to C), SmoA2 mice have severe mice (Fig. 2C). More importantly, at the protein level, there is no cerebellar malformations (Fig. 1D to F). To ensure that the phe- difference in total Smo protein expression between SmoA1 and notypes observed were not simply due to position effects at the SmoA2 mice (Fig. 2D). sites of transgene insertion, we generated and evaluated multiple The above-mentioned results demonstrate that the phenotypic transgenic lines, which manifested similar phenotypes. differences between SmoA1 and SmoA2 mice, with the latter being At P5, the SmoA2 cerebellum has an extended, undefined ex- more penetrant, are not merely due to differences in transgene

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FIG 1 The SmoA2 and SmoA1 mutations have vastly different effects on the development of the mouse cerebellum. H&E staining of representative sagittal sections shows development of the mouse cerebellum at P5, P14, and P28 in WT (A to C), SmoA2 (D to F), and SmoA1 (G to I) mice. (A to C) The asterisks indicate fissure formation during foliation at P5, and the dotted arrow denotes the anteroposterior (A-P) axis. At P14, formation of the molecular layer, Purkinje cell layer, and IGL is near completion, with remnants of the EGL remaining (arrow). At P28, the mature cerebellum is fully formed. (D to F) The SmoA2 cerebellum has an ill-defined expanded EGL with loss of foliation. The arrows point to ectopic clusters of cells at P5 and P14. In the P28 adult SmoA2 cerebellum, atypical cells with neoplastic morphology, if any, are concentrated along the superficial surface separated from the dysplastic region (dotted line). (G to I) The SmoA1 cerebellum closely parallels the development of the WT cerebellum, except for a slight thickening of the EGL at P14 (H, arrows). In the P28 adult SmoA1 cerebellum, neoplastic cells, if any, localize to the superficial surface of the cerebellum (I, arrows). The insets in panels A, D, and G show high-magnification views of the EGL and PC layer at P5, the inset in panel E shows a mix of atypical and normal cells at P14, and the inset in panel F shows the concentration of atypical cells along the superficial surface. Scale bars, 500 ␮m (A to I) and 25 ␮m (insets). expression but stem from the biological differences between the SmoA2 mice develop cerebellar tumors in a dysplastic cerebel- two activating mutations. lar milieu (Fig. 3A) compared to the SmoA1 mouse tumors, which Medulloblastomas in the SmoA2 model develop in a dysplas- form in an otherwise normally developed cerebellum (Fig. 3D) tic milieu. GNPs in the proliferative EGL are known to be the and are preceded by an expansion of the EGL at P14 (18)(Fig. 1H). source of medulloblastomas caused by hyperactive Shh signaling The frank solid tumors are densely cellular, with oval to spindle- (15, 18, 30, 35, 46). The SmoA2 mice, as shown earlier, lack an shaped cells (Fig. 3B and F) arranged in undefined sheets, short organized EGL, and the entire laminar cytoarchitecture of the cer- streams, and bundles supported on a scant fibrovascular stroma. ebellum remains dysplastic throughout development (Fig. 1D to Multifocally neoplastic cells along vessels and form nu- F). To understand the nature of tumor formation in these mice, merous pseudorosettes. Normal cellular morphology (Fig. 3C and we carried out comparative histopathological analyses on SmoA2 E) is distinct from that of adjacent tumor cells. Although during and SmoA1 tumors. The histological criteria we used for tumor development the entire SmoA2 cerebellum appears hypercellular definition were the same as those established in our previous study with atypical cells and other features of neoplasia (Fig. 1D to F), (18). In addition to our initial report of hyperplasia or lack of even in the absence of a defined EGL, the solid tumors are local- tumors in the SmoA2 line (16), our subsequent experiments, as ized in anatomic regions along the pial surface (Fig. 3A). described in this study, have revealed tumorigenesis. A natural history study to determine the clinical incidence of

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FIG 2 Determination of Smo transgene copy number and expression in SmoA1 and SmoA2 transgenic mice. (A) Standard curves obtained by qPCR amplifi- cation of Smo-Exon 6, Exon 10, and control loci RNaseP and Gapdh from 10-fold serial dilutions of WT mouse genomic DNA (190 to 0.019 ng). The data are plotted as cycle threshold versus log picogram DNA. The transgene copy number was confirmed by two sets of Smo and control locus primers. (B) Transgene copy number in SmoA1 and SmoA2 hemizygous mice estimated relative to endogenous Smo in the reference WT genomic DNA (n ϭ 5 per genotype). (C) mRNA expression of canonical Shh target genes Gli1, Gli2, Ptch1, and Ptch2, as well as Smo (endogenous plus transgene), as determined by qRT-PCR analysis using mRNA from WT, SmoA1,orSmoA2 purified P5 GNP lysates (3 independent pools per genotype). The bars represent the average fold change of each target in SmoA1 and SmoA2 relative to WT controls. ␤2m was used as the endogenous control for data normalization. Each assay was run in triplicate. The asterisks denote statistically significant differences in mRNA expression between SmoA1 and SmoA2 (P Ͻ 0.01; determined by Student t test). (D) Representative Western blot image and densitometric analyses of total Smo protein from WT, SmoA1,orSmoA2 purified P5 GNP protein lysates (3 independent pools per genotype) show no difference in total Smo expression between the genotypes. The bars represent the average fold change of Smo protein relative to the WT control. ␤-Actin was used as the loading control. The error bars represent SEM. tumor formation in SmoA2 mice (n ϭ 235) showed 2% of the plastic properties and lead to aggressive tumorigenesis. The allo- SmoA2 mice manifested clinical symptoms of tumor formation at graft tumors, similar to the spontaneously arising tumors in the 2 months of age, which increased to 46% by 4 months and 76% by transgenic SmoA2 model, extend from the outer surface multifo- 6 months (Fig. 3G), similar to the SmoA1 model (18). The main cally, compressing the cerebellum, and are a highly cellular, unen- clinical symptoms, as previously described (18), were weight loss, capsulated mass composed of two populations of cells: round cells protruded head as a result of tumor formation beneath the skull, with hyperchromatic nuclei and spindle-shaped cells with oval to hunched posture or head tilt resulting from hydrocephalus or elongated nuclei (Fig. 3I). nerve impingement, and lethargy. Histological analyses using The simultaneous manifestation of cerebellar developmental asymptomatic SmoA2 mice showed 80% of SmoA2 mice (n ϭ 15) defects, as well as neoplastic changes, as early as P5 in the SmoA2 have subclinical neoplastic lesions by 2 months, which increases to mice makes this a powerful model to study the temporal progres- 100% by 4 months of age (n ϭ 16). sion of an embryonal tumor like medulloblastoma. The presence of neoplastic features as early as P5 indicated that The SmoA2 mutation independently increases neuronal pro- the SmoA2 mutation confers transformative potential on precur- liferation and affects neuronal migration in the cerebellum. The sor cells early in development, much before the onset of clinical Shh signaling pathway plays a crucial role in proliferation of GNPs disease. To investigate the oncogenic potential of SmoA2 precur- by acting as a mitogen during postnatal cerebellar development sors, we transplanted P5 SmoA2 GNPs orthotopically into immu- (44). We therefore characterized the proliferative status of the nocompromised recipient mice. All five recipients succumbed to SmoA2 cerebellum at different developmental stages. The first de- aggressive tumors between 20 and 30 days after transplantation velopmental stage we investigated was embryonic day 15.5 (Fig. 3H), confirming that the SmoA2-expressing GNPs have neo- (E15.5), since the EGL, consisting of proliferating GNPs, is

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FIG 3 Comparative histopathology of SmoA2 and SmoA1 medulloblastomas. (A) Representative images from horizontal cerebellar sections show formation of SmoA2 tumors (arrow) in an aberrantly developed cerebellum on the superficial surface. m, meninges. (B and C) Pleomorphic neoplastic cells with multifocal pseudorosettes (B) are distinct from uniformly round granule neurons (GN) and Purkinje cells (PC) (C) in adjacent dysplasia. (D) SmoA1 tumors (arrow) form adjacent to a normally developed cerebellum also along the superficial surface. (E and F) Normal cellular morphology (E) is distinct from that of adjacent tumor cells (F). (G) Cumulative tumor incidence in SmoA2 and SmoA1 (18) mice at 2, 4, and 6 months of age, based on clinical symptoms. (H) Representative allograft tumor in athymic recipient mice (n ϭ 5) transplanted orthotopically with P5 SmoA2 GNPs. (I) High-magnification image showing a highly cellular mass with two populations of cells, round cells with hyperchromatic nuclei (white arrows) and spindle-shaped cells with oval to elongated nuclei (black arrows). T, allograft tumor; Cbl, normal cerebellum of recipient. Scale bars, 100 ␮m.

formed by this stage (3) and the ND2 promoter driving the SmoA2 pared to WT controls (Fig. 4A and B). This feature is maintained transgene is activated at ϳE11 (23). Immunohistochemical (IHC) at P5 with disorganized proliferating cells, which persist at P14 in staining for the proliferation marker Ki67 shows an increased SmoA2 mice (Fig. 4F and G) as opposed to WT mice, where pro- number of proliferating progenitors in the expanded EGL com- liferation is high at P5 but nearly complete by P14 (Fig. 4C and D).

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FIG 4 The SmoA2 mutation has independent effects on neuronal proliferation and neuronal and glial organization. (A and B) Ki67 IHC identifying proliferating GNPs marks the EGL (arrow), which is expanded in SmoA2 mice compared to WT mice at E15.5. IHC analysis for Ki67 in WT cerebella at P5, P14, and P28 (C to E) compared to that in SmoA2 cerebella (F to H) shows disorganized regions of proliferating GNPs (arrows) in SmoA2 at P5 compared to the defined EGL in the WT. At P14, while a few proliferating cells remain in the outer EGL (arrow) in the WT, there is an abundance of proliferating GNPs in SmoA2. At P28, proliferation is complete in the WT, whereas Ki67-positive cells are present along the superficial surface of the SmoA2 cerebellum. (I and J) SmoA2 tumors have an abundance of Ki67-positive cells (I), which are undetectable in the dysplastic regions of the SmoA2 cerebella (J). (K to M) H&E staining of representative sagittal cerebellar sections showing that while the EGL is expanded in both the PtchF/F-Math1-Cre and SmoA2 models, the mechanisms underlying foliation and neuronal migration to form the laminar cytoarchitecture are preserved in the former as opposed to the SmoA2 cerebellum. n ϭ 3 per group. Scale bars, 100 ␮m.

In the P28 SmoA2 cerebellum, the Ki67-positive cells remain on SmoA2 tumors have extensive Ki67 staining compared to the dys- the outer surface of the cerebellum in the same region (Fig. 4H) plastic regions, where Ki67 is undetectable (Fig. 4I and J). where the atypical cells are localized, as shown in Fig. 1F. There are Next, we investigated whether the abnormal foliation and mi- no Ki67-positive cells in the P28 WT cerebellum (Fig. 4E). The gration observed in the SmoA2 cerebellum is a consequence of

4110 mcb.asm.org Molecular and Cellular Biology SmoA2 Causes Cerebellar Developmental Defects excess GNPs generated from Shh hypersignaling that could poten- laminar cytoarchitecture (11). The granule neurons carry sensory tially overwhelm and deregulate cell migration processes. To ad- inputs to the cerebellum, while the Purkinje cells are the primary dress this, we compared the P5 cerebella of the SmoA2 mice to an motor output from the cerebellum, relaying motor information to additional Shh-driven medulloblastoma model, the Patched con- higher brain centers. To assess potential abnormalities in motor ditional knockout mice (PtchF/F Math1-Cre) (46)(Fig. 4K, L, and coordination resulting from neuroanatomic defects of the SmoA2 M). At P5, the PtchF/F Math1-Cre mice have a vastly increased cerebellum, we used a modified neurobehavioral assessment tool number of GNPs, which may give rise to nodular structures in based on SHIRPA (33) (see Materials and Methods for details). some regions (46). However, neuronal migration to form the lam- We investigated the physical phenotype (weight, tremor, body inar architecture and mechanisms underlying foliation appear to position, and tail position) and the behavioral phenotype (groom- be preserved (Fig. 4L). This shows that the neuronal disorganiza- ing, locomotor activity, and spontaneous motor activity) (see tion in the SmoA2 cerebellum is not solely a consequence of un- Movie S1 in the supplemental material). Additionally, we con- controlled GNP proliferation. ducted the accelerated RotaRod assay to test fore and hind limb SmoA2 mutation causes aberrations in neuronal and glial or- coordination and balance in both adult SmoA2 and age-matched ganization. To investigate the cellular characteristics of the cere- WT control mice. No differences in physical phenotype were ob- bellar dysplasias observed in the SmoA2 mice, we identified gran- served, and except for a decrease in locomotor activity (P Ͻ 0.01), ule neurons and Purkinje cells, as well as Bergmann glia, that are there were no significant differences in behavior phenotype mea- critical in GNP migration using antibodies for NeuN, Calbindin, sures between WT and SmoA2 mice (Table 1). Importantly, there and S100, respectively. By immunohistochemical techniques, we was no difference in RotaRod performance between SmoA2 and analyzed three distinct stages of cerebellar development, namely, WT mice, demonstrating that these mice were not deficient in P5, P14, and P28, as well as SmoA2 tumors and nontumor cere- motor coordination and balance. We also conducted a footprint bellar dysplasia in adult mice. All three markers showed massive analysis using stride length and stance width measures (9) in both disruptions in the organization of granule neurons, Purkinje cells, SmoA2 and WT mice, which demonstrated no evidence of ataxic and radial glia in SmoA2 mice (Fig. 5D to F, J to L, and P to R) gait, a common consequence of cerebellar dysfunction (Table 1). compared to the WT littermate controls (Fig. 5A to C, G to I, and Together, these data reveal no overt abnormalities in cerebellar M to O). function in SmoA2 mice despite massive disorganization in the At P5, the SmoA2 cerebellum has an ill-defined outer region cytoarchitecture. negative for NeuN and an inner region of NeuN-positive mature SmoA2 and SmoA1 mutations lead to unique transcriptional neurons similar to the forming internal granular layer (IGL) of the profiles in the mouse cerebellum. Since the SmoA2 and SmoA1 WT cerebellum (Fig. 5A and D). At P14 in the SmoA2 cerebellum, cerebella have different cell compositions, we first determined if there are clusters of NeuN-positive neurons marking islands of the cell-autonomous effects of GNP-specific SmoA2 and SmoA1 differentiated cells amid a vast expanse of NeuN-negative cells are represented in the whole cerebella. We compared, by qRT- (Fig. 5E), in stark contrast to the WT cerebellum, where differen- PCR, activation of the Shh pathway through its canonical targets tiated NeuN-positive cells are located in the nearly complete IGL in whole cerebellar lysates versus in purified GNPs, as determined at this stage (Fig. 5B). At P28 in the SmoA2 cerebellum, a majority earlier (Fig. 2C and D). Our results show similar results in whole of the neurons are NeuN positive, representing a differentiated cerebellar lysates, as well, thereby demonstrating that despite dif- state with clusters of NeuN-negative cells toward the outer surface ferences in cell composition, cell-autonomous effects of the mu- of the cerebellum (Fig. 5F), the same region where clusters of tations in GNPs were represented in whole-cerebellar analysis Ki67-positive proliferating cells are observed (Fig. 4H). (Fig. 6A). Calbindin immunostaining shows that the Purkinje cells have Next, to assess transcriptional changes downstream of SmoA2 severely disorganized dendritic arbors and axonal processes in the and SmoA1, we evaluated global gene expression profiles of P5 SmoA2 cerebellum, and the cell bodies fail to align in the charac- SmoA2, SmoA1, and WT age-matched whole cerebella. We chose teristic monolayer array at all the developmental stages analyzed this specific developmental stage because (i) the phenotypes of (Fig. 5J to L) in comparison to WT controls (Fig. 5G to I). Com- SmoA2 and SmoA1 are robust and distinct at P5 and (ii) at P5, pared to the WT controls (Fig. 5M to O), S100 staining of the GNPs undergo proliferation, migration, and differentiation, and radial glia showed atypical glial tangles with entrapped granule therefore, expression profiling could capture key differences in neurons in ectopic locations in the SmoA2 mice (Fig. 5P to R). multiple processes. Hierarchical cluster analysis showed that while The SmoA2 tumors show a heterogeneous pattern of NeuN the gene expression signature is unique for each group, at P5, staining (Fig. 5S), absence of Calbindin-positive Purkinje cells transcriptionally, the WT and SmoA1 cerebella resemble each (Fig. 5T), and sparse to absent S100-positive glia (Fig. 5U). The other more closely than either resembles SmoA2 cerebella (Fig. SmoA2 adult dysplasias, on the other hand, consist of NeuN-pos- 6B), consistent with their phenotypes (Fig. 1A, D, and G). itive neuronal cells (Fig. 5V). The dysplastic regions also consist of Compared to WT controls, we identified 106 transcripts in disorganized Purkinje cells (Fig. 5W) and disorganized glial fibrils SmoA2 and 67 in SmoA1 that differed uniquely by an absolute (Fig. 5X). These results demonstrate that the GNP-specific expres- change of Ն2-fold (P Ͻ 0.001) (Fig. 6C; see Table S2 in the sup- sion of SmoA2 leads to widespread disruptions in the migration plemental material). This highlights the complex transcriptional and organization of other neuronal and glial cell types in the cer- changes induced by the SmoA2 mutation that potentially underlie ebellum. the intriguing phenotype. We confirmed by qRT-PCR, using pu- Severe defects in cerebellar morphology do not cause overt rified GNP mRNA lysates, a subset of genes that were uniquely anomalies in motor coordination in SmoA2 mice. The classic upregulated in the SmoA2 cerebella or in both SmoA1 and SmoA2 functions of the cerebellum, which are regulation of balance and cerebella, according to the gene expression data (Fig. 6D). Bcl11b, motor coordination, are thought to be dependent on its organized MyoD, Pou4f2, and Cbln4 were uniquely upregulated in SmoA2

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FIG 5 SmoA2-induced aberrations in neuronal and glial organization in the developing cerebellum. (A to F) NeuN IHC analysis at P5, P14, and P28 develop- mental stages comparing WT cerebella (A to C) to SmoA2 cerebella (D to F) showing disorganized clusters of differentiated granule neurons (GN) amid abundant NeuN-negative cells at P5 and P14 in SmoA2 cerebella. (G to L) Calbindin IHC for Purkinje cells showing monolayer organization in WT (G to I) and ectopic clusters in SmoA2 (J to L) with disorganized dendritic arbors and axonal collaterals at P5, P14, and P28. (M to R) S100 IHC analysis for radial glia comparing WT cerebella (M to O) to SmoA2 cerebella (P to R) showing disorganized, entangled glial processes in the SmoA2 cerebellum at P5, P14, and P28. (S to X) SmoA2 tumors show heterogeneous NeuN staining, absence of PC, and heterogeneous S100 staining (S to U), whereas adjacent dysplasias are composed of mature but disorganized GN, PC, and radial glia (V to X). n ϭ 3 per group. Scale bars, 100 ␮m.

mice, and Isl1 was upregulated in both SmoA1 and SmoA2 mice, pared to SmoA1 mice include cell and neuronal fate specification showing consistency between the microarray data using whole and commitment, regulation of neuronal differentiation, neural cerebella and the cell-autonomous effects of the transgenes in pu- crest cell migration, regulation of cell proliferation, neuron migra- rified GNPs. tion, neuronal projection, membrane anchorage, cell matrix ad- To determine key biological processes that are different be- hesion, axonal and dendritic molecules, localization molecules, tween SmoA2 and SmoA1 mice, we used mRNAs over- and under- ligand gated ion channel activity, and thyroid hormone metabo- represented in SmoA2 compared to SmoA1 mice to conduct GO lism, as well as various metabolic processes. In the cell fate com- gene set enrichment analyses (1). The ontological categories rep- mitment GO category, among other transcription factors, the resented by the gene sets up- or downregulated in SmoA2 com- expression of MyoD, encoding a transcription factor that orches-

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TABLE 1 Measures from neurobehavioral assessment WT SmoA2 Test Measure nb Mean SEM n Mean SEM RotaRod Latency to fall (s) 8 222.4 1.04 12 189.8 0.59 Footprint analysis Stride length (cm) 3 63.2 3.84 4 60.4 2.13 Stance width (cm) 3 22.5 0.64 4 23.3 1.03 Locomotor activitya No. of times mouse places at least 1 paw on side of cage during a 2-min assessment 8 12.1 0.07 12 6.5 0.02 a P value Ͻ 0.01. b n, number of mice in each group. trates muscle differentiation, was particularly intriguing, since tern. We are currently exploring the significance of this unex- MyoD is not known to be expressed or to have a function in the pected finding. In summary, the biological processes reflected by mammalian brain. qRT-PCR (Fig. 6D), as well as Western blot the gene expression profiles are complex, and ascertaining specific analysis (data not shown), confirmed this ectopic expression pat- pathways remains a direction for the future. Our data demon- strate the fundamental differences between SmoA2 and SmoA1 at a molecular level that affect distinct biological processes.

DISCUSSION In this study, we have established a mouse model of medulloblas- toma that manifests severe defects in critical pathways of cerebel- lar development, including neuronal proliferation, differentia- tion, and migration, stemming from an activating mutation, SmoA2. Our comparative analysis reveals vastly different pheno- typic effects of the SmoA2 and SmoA1 mutations and thereby demonstrates the complexity of the downstream molecular path- ways regulated by a single molecule. The lack of a significant dif- ference in the levels of activation of the Shh pathway at P5 suggests that the phenotypic differences are not exclusively due to the ex- tent of pathway activation. The similarity in global transcriptional profiles between the WT and SmoA1 mice in early development is in accordance with the phenotype of SmoA1 being indistinguish- able from that of the WT at that stage, whereas the SmoA2 pheno- type and transcriptional profile are unique. We have demon- strated how two activating mutations in identical domains of a single protein can cause unique changes. Identifying molecular pathways uniquely employed by SmoA2 and SmoA1 variants with cues from the GO classification remains an important future di- rection that may provide further insights into the mechanics of the Shh pathway in both normal development and tumorigenesis. The cell of origin of the Shh-driven subtype of medulloblasto- mas has been shown to belong to the granule neuron lineage (12, 15, 16, 18, 30, 35, 46). However, why the niche of the superficial surface of the cerebellum is conducive to tumorigenesis, as seen in several mouse models (18, 30, 46), including SmoA2, has yet to be FIG 6 Transcriptional profiles of SmoA2 and SmoA1 P5 cerebella. (A) mRNA understood. In the Patched heterozygous model (30), ectopic rests expression of canonical Shh target genes Gli1, Gli2, Ptch1, Ptch2, and Smo (endog- (discrete clusters) of preneoplastic cells that have failed to undergo enous plus transgene) as determined by qRT-PCR analysis using mRNA from WT, SmoA2,orSmoA1 P5 cerebella (n ϭ 3 per genotype). The bars represent the proper migration reside in the niche of the EGL, whereas the vast average fold change of each target in SmoA2 and SmoA1 relative to WT controls. majority of GNPs mature and organize correctly. Intriguingly, in Gapdh was used as the endogenous control. Each assay was run in triplicate. (B) the SmoA2 developing cerebellum, the vast majority of GNPs un- Hierarchical cluster analysis showing transcriptional similarity of WT and SmoA1 dergo abnormal migration and manifest neoplastic features early ϭ P5 cerebella in comparison to SmoA2 cerebella (n 3 per genotype). (C) Venn in development. However, the frank tumors remain confined to diagram showing unique and overlapping changes in transcript expression (Ն2- fold absolute change; P Ͻ 0.001) between P5 SmoA2 and SmoA1 cerebella. (D) the superficial surface of the cerebellum. By design of the trans- Confirmation of differential expression of transcripts between P5 SmoA2 and gene, each GNP should express the SmoA2 oncogenic mutation, SmoA1 whole cerebella as determined in microarray experiments and by qRT- yet dysplasias adjacent to the tumors consist of normally differen- PCR in purified GNP lysates. The bars represent the average fold change of each tiated granule neurons. This suggests that potential cell-extrinsic mRNA target in SmoA2 and SmoA1 GNP lysates relative to the WT (n ϭ 3 inde- pendent pools). Ppia was used as the endogenous control. The asterisks indicate factors in the pial surface might act on a subset of SmoA2 cells statistically significant differences in mRNA expression between SmoA2 and arrested in migration, leading to tumor initiation. The cells that SmoA1 (*, P Ͻ 0.01; **, P Ͻ 0.05; Student t test). The error bars represent SEM. have migrated away from the EGL differentiate or regress. Alter-

October 2012 Volume 32 Number 20 mcb.asm.org 4113 Dey et al. natively, cell-extrinsic factors could attract neoplastic cells to the movement characteristic of cerebellar malfunctions, yet intrigu- pial surface by providing a favorable environment for tumor ingly, SmoA2 mutants with some phenotypic similarities in terms growth. The leptomeningeal membrane, known to secrete chemo- of disruptions in the laminar architecture, with more severe mor- kines and other trophic factors (21, 39), is one potential source of phological abnormalities in multiple cell types, maintain grossly such extrinsic signals. Future experiments to further characterize normal motor behavior. the environmental niche and the resident precursors will provide In summary, through the characterization of the unique phe- further insights into the development of this subtype of medullo- notype of the SmoA2 model, we address key aspects of cerebellar blastoma. development and function, as well as of medulloblastoma biology, An interesting phenomenon observed in the SmoA2 develop- that have yet to be understood. The SmoA2 model is therefore a ing cerebellum is the apparent regression of the hypercellularity valuable addition to the existing mouse models of medulloblas- observed throughout the developing cerebellum. Progenitor cells toma. Medulloblastoma is known to be a cancer resulting from in the developing cerebellum appear to have neoplastic character- deregulated developmental signals. The developmental pheno- istics morphologically, as well as functionally, as shown by trans- type of the SmoA2 model will therefore allow investigation of cer- plantation experiments. However, in a mature SmoA2 cerebellum, ebellar developmental pathways and aberrations thereof that lead the dysplasias histologically seem to have fewer cells and to consist to medulloblastoma formation. of mature neurons. The early neoplastic lesions are confined to the ACKNOWLEDGMENTS superficial surface of the cerebellum, while the rest of the cerebel- lum remains dysplastic. Spontaneous regression has been docu- This work was supported by NIH grants 5R01CA11456705 and mented in certain cancers, such as the highly malignant stage 5R01CA112350. J.D. was supported by Predoctoral Developmental Biol- 1V-S (10, 27), the molecular basis of which is ogy Training Grant 5T32HD007183 from the National Institute of Child Health and Human Development. poorly understood. There are conflicting data regarding the We thank Kyle Pedro and Andrew Richards for assistance with mouse role of apoptosis in spontaneous regression in neuroblastoma colony management; Lindsey Conrad for assistance with database main- (47). We have been unable to detect any significant apoptotic tenance; Ying-tzang Tien at the University of Washington Histopathology cell death by immunohistochemistry for activated caspase 3 Laboratory, Julie-Randolph Habecker and the FHCRC Experimental His- (data not shown). Whether the processes underlying the appar- topathology Shared Resource, Julio Vazquez and David L. McDonald at ent regression observed in the SmoA2 cerebellum include de- FHCRC Scientific Imaging, and the Rosetta Inpharmatics Gene Expres- layed neuronal differentiation programs and caspase-depen- sion Laboratory for profiling studies; Matthew Tudor for compiling the dent and possibly caspase-independent programmed cell results for GEO submission; Andrew Strand for assistance with microar- death, like autophagic degeneration, as implicated in neuro- ray data analysis; Michael LeBlanc for advice with statistical analysis; and blastoma (20), has yet to be determined. Stacey Hansen, Olga Klezovitch, and Michelle Cook Sangar for critical review of the manuscript. Shh signaling acting through the Smo-Gli axis has been shown to regulate foliation, with the extent of foliation proportional to REFERENCES the level of signaling (7, 8). However, increased cell-autonomous 1. Ashburner M, et al. 2000. 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