1. Wnt Signaling: a case study with new research solution
Zhong Wu, Ph.D.
R&D Scientist
Sample & Assay Technologies

2. Topics to cover
Overview of Wnt signaling
Function of Wnt signaling
Research tools to study Wnt signaling
Sample & Assay Technologies

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. 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. Different Wnt signaling pathways
Sci. Signal. 1, re9 (2008)
Sample & Assay Technologies

6. Canonical Wnt pathway
Nat Rev Cancer. 2008;8:387-98
Sample & Assay Technologies

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. 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. 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. 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. 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. Wnt signaling in developmental malformation
Development cell 17,9-26 (2009)
Sample & Assay Technologies

13. Wnt signaling in cancer
Eur J Cancer 45,2709-2769 (2009), Nature 434, 843-850 (2005) & Wnt homepage
Sample & Assay Technologies

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. 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. 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. 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. Cignal reporter from SABiosciences
Two modality
Dual luciferase
GFP
Two systems
Plasmid
Lenti
Over 40 pathways
Wnt and more
Sample & Assay Technologies

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. 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. 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. 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. 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. β-catenin targeted siRNA from QIAGEN
Sample & Assay Technologies

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. 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. Gene content on Wnt signaling target PCR
Array
Sample & Assay Technologies

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. 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. 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. Wnt pathway PCR Array
Signaling
Function
Sample & Assay Technologies

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. 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. 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. 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. 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. 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. Probability of pathway activation
Sixteen genes were identified as Wnt
signature genes
Sample & Assay Technologies

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. 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. 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