Wnt part ii 2013

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Wnt part ii 2013

  1. 1. Wnt Signaling: a case study with new research solution Zhong Wu, Ph.D. R&D Scientist Sample & Assay Technologies
  2. 2. Topics to cover Overview of Wnt signaling Function of Wnt signaling Research tools to study Wnt signaling Sample & Assay Technologies
  3. 3. Topics to cover Overview of Wnt signaling Wnt discovery and family members Canonical Noncanonical Function of Wnt signaling Research tools to study Wnt signaling Sample & Assay Technologies
  4. 4. Wnts and receptors Discovery of Wnt Murine oncogene Int-1 Homolog to Drosophila segment polarity gene Wingless Wnt proteins 19 members Highly conserved Secreted glycoproteins Activate cell surface receptors Wnt receptors FZD/LRP receptor complex 10 members of FZD LRP5 and LRP6 coreceptor Sample & Assay Technologies
  5. 5. Different Wnt signaling pathways Sci. Signal. 1, re9 (2008) Sample & Assay Technologies
  6. 6. Canonical Wnt pathway Nat Rev Cancer. 2008;8:387-98 Sample & Assay Technologies
  7. 7. Noncanonical Wnt pathway Wnt/JNK Wnt/calcium CN Cell polarization in epithelial sheet Polarized cell movements Larger overlap with Wnt/PCP Cell adhesion Cardiac differentiation Gene expression through NFAT, HDAC Nat Rev Neurosci. 2010;11(2):77-86 Sample & Assay Technologies
  8. 8. What dictates different Wnt pathways? Different Wnt proteins Wnt1,Wnt3A, Wnt8 - canonical Wnt5A, Wnt11 - noncanonical Receptor mediated Wnt/Frizzled/LRP – β-catenin signaling Frizzled – PCP and Ca2+ signaling Wnt/Ryk – Src signaling Wnt/Ror2 – JNK signaling Sci. Signal. 1, re9 (2008) Sample & Assay Technologies
  9. 9. Topics to cover Overview of Wnt signaling Wnt discovery and family members Canonical Noncanonical Function of Wnt signaling Development Tissue homeostasis Pathological condition Research tools to study Wnt signaling Sample & Assay Technologies
  10. 10. Wnt signaling in embryonic development Cell fate determination Proliferation Migration, motility and polarity specification Tissue patterning Organogenesis and organization of the body plan Almost every development process Axial formation Mesoderm specification Production of presomitic mesoderm Direct patterning of the nervous system Suppress chondrogenic potential of joint interzone cells Sample & Assay Technologies
  11. 11. Wnt signaling in tissue renewal Zebrafish tail fin and heart regeneration Maintain stem-cell like fate Intestinal epithelium Skin Hematopoietic cells Neural stem cells Nature 434, 843-850 (2005) & Development 134, 479-489 (2007) Sample & Assay Technologies
  12. 12. Wnt signaling in developmental malformation Development cell 17,9-26 (2009) Sample & Assay Technologies
  13. 13. Wnt signaling in cancer Eur J Cancer 45,2709-2769 (2009), Nature 434, 843-850 (2005) & Wnt homepage Sample & Assay Technologies
  14. 14. Wnt signaling in CNS-related diseases Alzheimer’s disease Reduced β-catenin signaling Tau hyperphosphorylation by GSK3β LRP6 association Parkinson’s disease Wnt-β-catenin and Wnt-PCP Dopaminergic development and neurogenesis Schizophrenia Wnt1 overexpression SNPs in FZ3 DISC1 interacts with GSK3β Nat Rev Neurosci. 2010;11(2):77-86 Sample & Assay Technologies
  15. 15. Wnt signaling in other pathological condition High bone mass and Osteoporosis LRP5 activating (LRP5G171V) LRP5 lose of function mutation Type II diabetes Wnt5B TCF7L2 (TCF4) Pulmonary fibrosis Activated β-catenin signaling N Engl J Med 2002;346:1513-1521, Am J Hum Genet, 2004,75:832-43 Sample & Assay Technologies
  16. 16. Topics to cover Overview of Wnt signaling Wnt discovery and family members Canonical Noncanonical Function of Wnt signaling Development Tissue homeostasis Pathological condition Research tools to study Wnt signaling – A real case study about Wnt pathway activity Sample & Assay Technologies
  17. 17. Study Wnt pathway activity Why Wnt pathway activity Involving in many physiological and pathological process Mutations of Wnt components lead to altered pathway activity Current methods to study Wnt pathway activity β-catenin translocation Reporter assay Wnt target genes Western blot of active β-catenin Sample & Assay Technologies
  18. 18. Cignal reporter from SABiosciences Two modality Dual luciferase GFP Two systems Plasmid Lenti Over 40 pathways Wnt and more Sample & Assay Technologies
  19. 19. Reporter assay vs Wnt target genes Reporter assay Relatively simple High throughput Rely on TRE Possible disconnection from endogenous gene expression Wnt target genes? Endogenous genes Readout based on multiple genes No quantification as a whole set Quantitative Wnt target genes? – Develop Wnt gene expression signature with companion scoring Sample & Assay Technologies
  20. 20. Proposed work flow for Wnt gene expression signature development Microarray 1 or 2 cell lines stimulation Initial list of Response genes inhibition Expanded dataset Mathematic model classification Real-time PCR Initial gene list ~ 20 cell lines signature gene sensitivity Compare to other pathway activity readout Small subset of signature genes Sample & Assay Technologies
  21. 21. Experiment setup for whole genome gene expression profiling Whole genome gene expression profiling after altering Wnt/β-catenin pathway activity Illumina bead chip by SABio Service Core Treatment Stimulation – Wnt3a Inhibition – siRNA for β-catenin (Qiagen FlexiTube siRNA) Wnt Pathway activity readout Wnt target genes (Wnt signaling target PCR array) Active β-catenin protein levels Cell lines 293H FHC Sample & Assay Technologies
  22. 22. SABiosciences service core Complete service for genomic and expression analysis that delivers robust and reproducible results. Whole genome Illumina expression profiling Illumina genotyping Pathway/focused panel Mutation profiling Methylation PCR array miRNA PCR array Individual gene/Locus Mutation Methylation qRT-PCR SYBR Green Sample prep Cell, tissue or biofluids Fixed tissue Sample & Assay Technologies
  23. 23. Complete solution for gene silencing from QIAGEN Predesigned siRNA FlexiTube siRNA or siRNA Premix FlexiPlate siRNA QIAGEN AllStars RNAi controls Custom siRNA synthesis HP Custom siRNA – 20 nmol Plasmid based RNAi SureSilencing shRNA plasmids Transfection reagents HiPerFect transfection reagent HiPerFect HTS reagent FlexiTube siRNA Premix siRNA/DNA cotransfection Attractene Transfection Reagent Sample & Assay Technologies
  24. 24. β-catenin targeted siRNA from QIAGEN Sample & Assay Technologies
  25. 25. Experiment setup for whole genome gene expression profiling Whole genome gene expression profiling after altering wnt/β-catenin pathway activity Illumina bead chip service by SABio Service Core Treatment Stimulation – Wnt3a Inhibition – siRNA (Qiagen flexible tube siRNA) Wnt Pathway activity readout Wnt target genes (Wnt signaling target PCR array) Active β-catenin protein levels Cell lines 293H FHC Sample & Assay Technologies
  26. 26. What is Wnt signaling target PCR Array? • 84 genes responsive to Wnt pathway transduction • 5 Housekeeping Genes • Genomic DNA Contamination Control • Reverse Transcription Controls (RTC) n=3 • Positive PCR Controls (PPC) n=3 Supported PCR Array Formats • • • • 96-Well Plate 384-Well Plate 100-Well Disc 96x96 Chip Over 100 different pathways available! Sample & Assay Technologies
  27. 27. Gene content on Wnt signaling target PCR Array Sample & Assay Technologies
  28. 28. RT2 PCR Arrays Work Flow control experimental •cDNA Synthesis (C-03 kit) – Genomic DNA Removal Step (5 min.) – Reverse Transcription Step (20 min.) •Load Plates – 1 Sample per PCR Array – 2 minutes with multi-channel pipet •Run 40 cycle qPCR Program – Standard cycling conditions for all Real Time PCR Instruments – 2 hours •Upload and Analyze Data (FREE) – 15 minutes from Raw Ct to Fold Change Data Sample & Assay Technologies
  29. 29. Check pathway activity with active β-catenin and Wnt target gene PCR array sinon Serum free wnt3a 293H 8 7 RNA 24 36 48h Serum free wnt3a siRNA FHC fold change siRNA 57 293H CTNNB1 c-myc 6 cyclin D1 5 fra-1 4 axin2 sox9 3 2 RNA 24 siCTNNB1 1 69 72h 0 0 400 0 400 wnt3a (ng/ml) total beta-catenin 7 SF+wnt CTNNB1 SF 10% FBS SF+wnt 293H 6 fold change active beta-catenin SF 10% FBS FHC 5 4 3 FHC c-myc cyclin D1 fra-1 axin2 CCND2 2 1 0 GAPDH 0 200 0 200 wnt3a (ng/ml) Sample & Assay Technologies
  30. 30. Experiment problem and approach Q: Why the effect of Wnt stimulation on FHC cells was weaker than that on 293H cells? Hypothesis: Defect in Wnt signaling pathway transduction in FHC cell? Approach: Expression profiling of 293H and FHC cells on Wnt pathway genes with Wnt Signaling Pathway PCR array. Sample & Assay Technologies
  31. 31. Wnt pathway PCR Array Signaling Function Sample & Assay Technologies
  32. 32. Differential expression of receptors and ligands in 293H vs FHC cell 293H vs FHC FZD5 293H FZD3 Wnt5A Wnt3 FHC Sample & Assay Technologies
  33. 33. From microarray to PCR - Custom PCR array Wnt3a 400 ng/ml siCTNNB1 sinon siCTNNB1 8 fold change 7 CTNNB1 293H c-myc 6 cyclin D1 5 fra-1 4 axin2 sox9 3 2 1 0 0 400 0 wnt3a (ng/ml) 400 – + – – + + + – Custom PCR array TMEM2 MYC FGFR3 PODXL SMAD6 CALM1 TMED1 ASB13 LEF1 FEZ2 SKP2 PGM1 MRPL54 DIMT1L SMAD3 MKX SCD5 MTSS1 CA2 CCND2 LAPTM4B NAV2 HSPA12A SORBS2 C3orf31 MT1A C5orf13 AKT1 MKKS MTP18 DPM3 ZAK ILK W DR54 DPYSL2 CNTNAP2 CHSY1 ICK NEFL RPRML PIGY MRFAP1 FAM125B NUAK1 CYP4V2 HES5 FTSJ1 TMEM98 GARNL4 CCND1 LOC653506 HCFC1R1 FAM44B CXADR IGFBP5 TARBP2 EYA2 CERK PRMT6 SH3D19 PHB THOC7 REEP5 FAM134B 12 Genes 8 Samples 24 Genes 4 Samples 32 Genes 3 Samples 48 Genes 2 Samples 96 Genes 1 Sample Sample & Assay Technologies
  34. 34. Identify Wnt signature genes with Custom Wnt response gene PCR array CCD1079SK Wnt3a 400 ng/ml LiCl 20 uM – – + – – – + – BJ U105MG Lncap – + – – Custom Wnt response gene PCR array – + – – + – active CTNNB1 GAPDH U373MG siCTNNB1 MCF-7 HT29 LnCap HepG2 – + – + – + – + – + CTNNB1 GAPDH Sample & Assay Technologies
  35. 35. Interesting question during experiment wnt3a X Y LEF1 Q: What’s the mechanism involved in differential Wnt target gene expression between HepG2 and IMR90 cells? Hypothesis: Epigenetic silencing of Wnt pathway transducers in HepG2 cell? Approach: Examine methylation status of Wnt pathway transduction genes with Wnt signaling EpiTech Methyl qPCR array. Sample & Assay Technologies
  36. 36. EpiTech Methyl qPCR Array Protocol Overview Innovative technology enabling fast and accurate detection of CpG island DNA methylation of individual genes, and disease or pathway focused gene panels (0.5 – 4 ug of Genomic DNA) Raw Ct Values Sample & Assay Technologies
  37. 37. Differentially methylated genes identified by EpiTech Methyl qPCR Wnt signaling array 100 90 Unmethylated 80 HepG2 % Total input DNA Methylated 70 60 50 40 30 20 10 0 FZD1 FZD2 FZD9 SOX17 WIF1 100 90 Unmethylated IMR90 % Total input DNA 80 Methylated 70 60 50 40 30 20 10 0 FZD1 FZD2 FZD9 SOX17 WIF1 Sample & Assay Technologies
  38. 38. Probability of pathway activation Sixteen genes were identified as Wnt signature genes Sample & Assay Technologies
  39. 39. Sensitivity of Wnt signature genes HCT116 (CCT036477, 6h) 0 1.25 2.5 5 10 293H (CHIR99021, 24h) 20 µM 0 1.25 2.5 5 10 20 µM Active-β-catenin β GAPDH Pathway activity based on Wnt gene signature Sample & Assay Technologies
  40. 40. Summary Overview of Wnt signaling Wnt discovery and family members Canonical - β-catenin Nonncanonical – Wnt/JNK & Wnt/Ca2+ Function of Wnt signaling Development – many aspects Tissue homeostasis – maintain stem cell like feature Pathological condition – cancer, bone, nervous Wnt gene signature development with various tools – A gene expression signature to measure Wnt pathway activity PCR array – study gene expression changes related to Wnt signaling siRNA – gene silencing for functional Wnt pathway study EpiTech Methyl array – explore mechanism of gene expression changes Cignal Reporter assay – assess pathway activity of Wnt signaling Sample & Assay Technologies
  41. 41. Additional Resources Questions? Contact Technical Support 9 AM – 6 PM Eastern M – F Contact: 1-888-503-3187 OR support@SABiosciences.com E-Learning Center: Recorded Seminars Sample & Assay Technologies

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