Wnt signaling in adult intestinal
stem cells and cancer - an insight
from transgenic mice
Vladimir Korinek
Department of Cell and Developmental Biology
Institute of Molecular Genetics, v.v.i., Prague, CZ
Hierarchical model of adult tissue - all tissues contain three basic cell compartments
stem cells (101-104 divisions/life)
selfrenewal via asymetric cell division
progenitors
(Transit Amplifying cells; TA cells)
differentiated cells
Two fundamental questions of stem cell biology
How to find stem cells? How to prove that you have them?
Find unique marker(s) and perform functionality tests:
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transplantation to recipient = reconstitution of given tissue in vivo
reconstitution of given tissue in vitro
lineage tracing experiment track stem cell and its progeny in mouse
The gastrointestinal epithelia:
the most rapidly renewing tissue of the human body
Stem cells reside in the crypts
Small intestine
villi: non-dividing cells
life span – 5 days
Colon
colon lumen: non-dividing cells
life span – 5 days
crypts: dividing cells
crypts: dividing cells
stem cells
stem cells
Ki67 staining in the small intestine
villi: non-dividing cells
The crypts contain both dividing and non-dividing cells
crypts: dividing cells
Stem cell in the epithelium differentiates in four main cell
types
© Crosnier et al., 2006; Cheng & Leblond, 1974
Assymetric division of stem cell:
one stem cell and its progeny is shown in blue
Signaling pathways governing the architecture of the intestinal epithelium
© Krausova & Korinek, 2012
The Wnt signaling pathway (a simplified schema)
The Wnt gene family
mammals: 19 members (i.e. Wnts)
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all metazoans contain
elements of Wnt signaling
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genes activated by Wnt signaling:
c-Myc, Cyclin D1, Twist, Axin2
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Wnt signaling regulates various
cellular processes during
development and in adult tissues
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aberrant activation can
lead to cancer
For details visit the Wnt signaling homepage:
http://www.stanford.edu/group/nusselab/cgibin/wnt/
adopted from Reya and Clevers, 2006
b-catenin, a protein with a dual function: adhesion and signaling
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b-catenin binds cadherins
the protein is highly conserved
during evolution
Adherens junctions:
strong mechanical attachments between adjacent cells
anti-b-catenin staining
P P
P P
protein stability
cadherin
transactivation domain
Nuclear b-catenin can be detected in the crypts
of the large (and small) intestine
© van de Wetering et al., 2002
In the mouse the Wnt-signaling deficient gut displays dramatically
reduced numbers of epithelial cells
Existence of gene families complicates
gene knockout studies
(problems with redundancy)
wild type
19 Wnt genes
Tcf4 KO
4 Lef/Tcf genes
© Korinek et al. 1998
The APC gene, the main tumor suppressor gene in the intestine
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polyp, a small clump of cells that forms on the lining of the intestine
1-10% of polyps progress to malignancy (carcinoma)
the majority of colorectal cancer is sporadic (no familial history)
10-15% of colorectal cancers occur in dominantly inherited patterns (Lynch syndrome, FAP syndrome)
Polyp observed during
colonoscopy
Resected carcinoma
© www.wikipedia.org
• inactivation of APC is the rate-limiting step in tumor initiation
• APC mutations are found in >40% sporadic colorectal tumors
Rizk & Barker, 2012
Aberrant Wnt signaling in colorectal tumors
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APC binds b-catenin and mediates b-catenin degradation
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APC-deficient colorectal tumors display constitutively active Wnt signaling
Tumor histology
(b-catenin + hematoxyline stainings)
Situation in tumor cell
(no Wnt added but the APC gene is mutated)
anti-b-catenin staining in tumor; detail
1900-2007: search for intestinal stem cells
= search for markers of these cells
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Cheng and Leblond (1974): description of CBC cells = Crypt Base Columnar cells
→ prediction that CBC cells are the intestinal stem cells
van de Wetering et al. (2002): high throughput screen for Wnt signaling target genes in the intestinal cells
Barker et al. (2007): Lgr5 is the marker of CBC stem cells
Lgr5=leucine-rich-repeat-containing G-protein coupled receptor 5
Transmission Electron Microscopy (bottom of the crypt)
© Porter et al. 1997
Paneth cell
CBC cells
Lgr5 mRNA in situ hybridization
in the small intestine
© Barker et al. 2007
Labeling of Lgr5-positive cells using knock-in technique
© Barker et al., 2007
Lgr5-EGFP-IRES-CreERT knock-in mouse
Lgr5+ cells
(green fluorescence)
in the small intestine
CBC stem cells (Lgr5)
Paneth cells (lysosyme)
© Snipert et al. 2010
Growing organoid derived from one Lgr5+ cell
Epithelial organoids (or „miniguts“) –
an in vitro system to study the gut epithelium
freshly isolated crypt
2 days
Organoids culturing conditions:
EGF
noggin (BMP signaling inhibitor)
R-spondin (Wnt signaling agonist)
7 days
9 days
Organoid transplantation assay
The lineage tracing experiments:
Cre recombinase is used to mark irreversibly one cell and its progeny
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Cre is type I topoisomerase of phage P1
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catalyzes recombination between loxP sites
(34 bps)
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sequence of the loxP site is not present in mammalian genome
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Cre recombinase fused to the estrogen receptor (=CreERT)
is used
Deletion of „floxed“ sequences
“Reporter” mouse
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Rosa26 gene is active in all cell types
lacZ encodes b-galactosidase enzyme (=reporter gene)
genome of the reporter mouse contains floxed transcription blocker
lacZ is produced only when the blocker is recombined out by Cre
intercrossing
CreERT-expressing mouse
Lineage tracing using CreERT-expressing and ROSA26 „reporter“ mouse
Cre recombinase fused to the estrogen receptor (CreERT) can be regulated by tamoxifen
Situation in genomic DNA of cells expressing
CreERT upon addition of tamoxifen
Progeny of the breeding
+ tamoxifen
The CreERT/lacZ induction in the adult Lgr5-EGFP-IRES-CreERT mice by
tamoxifen
Situation in genomic DNA of Lgr5+ cells
+ tamoxifen
Days after tamoxifen injection
© Barker et al. 2007
Our aim: to find genes important for physiology of healthy and diseased
gut tissue
2007: Chromatin Immunoprecipitation (ChIP) with DNA microarray analysis (ChIP-on-chips) of
chromatin isolated from colorectal cancer cells displaying aberrant Wnt signaling
APC-deficient cells: Colo320, DLD1, SW480
Cells harboring oncogenic mutations in b-catenin: LS174T
Antibodies: anti-TCF; anti-b-catenin
Promoter regions (bound by TCF4) of
18 genes were precipitated in all (four)
tested cell lines
Two genes – Troy and Nkd1 – were selected for further study
tumor (APC-Min mouse)
Troy mRNA in situ hybridization
crypts
Generation of Troy reporter mouse using BAC transgenesis
Cre expression/activity = Troy expression
Situation in genomic DNA of Troy+ cells
+ Tamoxifen
TROY-CreERT x Rosa26 reporter mouse
Troy-CreERT2 transgene expression
phenocopies the expression pattern
of endogenous Troy
Long-term labeling of cells (originally) expressing Troy
LacZ staining in the small intestine of
Troy-CreERT x Rosa26 reporter mice
(indicated days after tamoxifen injection)
1 day
3 days
1 day
60 days
60 days
Conclusions of the study
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COLON
Troy is Wnt signaling target gene
Troy is a marker of CBC stem cells
Troy interacts with Lgr5
Troy inhibits (negatively modulates) Wnt signaling
The additional aim:
Analysis of the Nkd1 gene
All details can be found in the recently published article:
The Crew
Bobeš Fafílek
Nikol Baloghová
Kateřina Gajdůšková
Adéla Hlavatá/Monika Horázná
Dušan Hrčkulák
Jitka Stančíková
Collaborators
Zbynek Kozmik & his lab
different mouse strains and ingredients
Radek Sedlacek, Inken Beck (Transgenic Unit)
transgenic mice
Antonio Pombinho & Petr Bartunek
HTS
David Stanek & his lab
microscopy
Hynek Strnad & Jan Paces & Michal Kolar
(Vlcek Lab)
Chips and Bioinformatics
Michaela Krausová
Vít Kříž
Eva Šloncová
Jiří Švec
Lucie Tůmová
Martina Vojtěchová
To produce conditional knockouts you need two mouse strains
(1) Mouse expressing Cre in a specific tissue
(the mouse is generated by transgenesis)
Conditional (= tissue-specific)
deletion of gene of interest
(2) Mouse harboring „floxed“ alleles of gene of interest
(gene targeting in ES cells; floxed is mostly smaller
but essential part of the gene)
Testing Cre-expressing mice (question: in which tissue is Cre produced?)
“Reporter” mouse
(Rosa26 gene/locus is active in all cell types)
Cre-expressing mouse
X
Progeny of the reporter and Cre mouse
Situation in cells
expressing Cre
Example:
Cre is driven by a promoter active in
tracheal endoderm of the lung
+ X-gal
lacZ = b-galactosidase
Inactivation of the floxed-APC gene in Lgr5(Troy)-positive CBC stem cells
without Tamoxifen
+ Tamoxifen
Non-functional APC gene/protein in Lgr5+ cells
APC is deleted in CBC stem cells
Tumor formation in Lgr5-CreERT mouse harboring a conditional allele of
the APC gene
b-catenin-positive tumors in the large and small intestine
14 days after tamoxifen injection (floxed-APC x Lgr5-CreERT2 mice)
0.5 mg of tamoxifen per animal
2 mg of tamoxifen per animal
small intestine
small intestine
colon
The Yin and Yang of the Wnt pathway in the intestine
Nuclear b-catenin can be detected
in the crypts of the healthy large
(and small) intestine
polyp:
aberrant Wnt signaling
Adherens junctions
© van de Wetering et al., 2002
Reminder:
nucleus of the cell
stimulated with Wnt
the crypt: physiological Wnt signaling
APC-Min mice – a mouse model of the intestinal cancer
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produced using chemical mutagenesis of laboratory mice in the lab (Moser et al., 1990)
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APC-Min mice are APC+/- (Su et al., 1992)
Min=Multiple intestinal neoplasia
Reminder:
wild-type APC (2842 AA)
truncated APC protein
(produced in APC-Min mice)
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Wnt signaling in adult intestinal stem cells and cancer