Wnt Signaling Inhibition Deprives Small Intestinal Stem Cells of Clonogenic Capacity

Abstract

SummaryThe Wnt pathway plays a crucial role in self‐renewal and differentiation of cells in the adult gut. In the present study, we revealed the functional consequences of inhibition of canonical Wnt signaling in the intestinal epithelium. The study was based on generation of a novel transgenic mouse strain enabling inducible expression of an N‐terminally truncated variant of nuclear Wnt effector T cell factor 4 (TCF4). The TCF4 variant acting as a dominant negative (dn) version of wild‐type (wt) TCF4 protein decreased transcription of β‐catenin‐TCF4‐responsive genes. Interestingly, suppression of Wnt/β‐catenin signaling affected asymmetric division of intestinal stem cells (ISCs) rather than proliferation. ISCs expressing the transgene underwent several rounds of division but lost their clonogenic potential and migrated out of the crypt. Expression profiling of crypt cells revealed that besides ISC‐specific markers, the dnTCF4 production downregulated expression levels of epithelial genes produced in other crypt cells including markers of Paneth cells. Additionally, in Apc conditional knockout mice, dnTCF activation efficiently suppressed growth of Apc‐deficient tumors. In summary, the generated mouse strain represents a convenient tool to study cell‐autonomous inhibition of β‐catenin‐Tcf‐mediated transcription. genesis 54:101–114, 2016. © 2016 The Authors genesis Published by Wiley Periodicals, Inc.

Wnt Effector TCF4 Is Dispensable for Wnt Signaling in Human Cancer Cells

“…Intestines were fixed in 4% formaldehyde ( v / v ; Sigma-Aldrich) in PBS overnight, and embedded in paraffin using an automatic tissue processor (Leica, Wetzlar, Germany); 5-µm sections were stained according to the protocol published previously [ 34 ]. Briefly, specimens were deparaffinized in xylene, and antigen retrieval was performed in a steam bath by immersing the slides into 10 mM citrate buffer pH 6.0.…”

“…Sections were counterstained with hematoxylin (PENTA). β-galactosidase was visualized using the 5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside (X-gal; Sigma-Aldrich) substrate, followed by counterstaining using nuclear fast red stain (DiaPath) [ 34 ]. Periodic acid Shiff (PAS) staining was performed using the P.A.S.…”

Glucose and amino acid in enterocyte absorption metabolism and maturation

“…As a Wnt target gene, the transcription factor Ascl2 acts as a master regulator of intestinal stem cells, cooperating with b-catenin/Tcf to activate fundamental genes. In particular, suppression of Wnt/β-catenin signaling, rather than clonogenic capacity, affects the asymmetric division of those cells (148,149). The greater contents of eukaryotic protein translational pathway initiation factors 4E (eIF4E) protein and it's binding protein 1 γ-isoform to phosphorylated (Pi) eukaryotic elongation factor 2 (eEF2) protein in crypt than upper villi suggest that the crypt cells have higher translational efficiency than upper villus cells (6).…”

Tcf7l1 protects the anterior neural fold from adopting the neural crest fate

“…In order to verify this hypothesis, we performed rescue experiments using two mouse strains conditionally expressing either dominant Tcf repressor or activator. As a dominant repressor we used the mouse strain Rosa26 dnTCF4fl/+ , which contains the N-terminally truncated form of human TCF7L2 (dnTCF4) targeted into the ROSA26 locus and inserted 3′ to the lox-STOP TdTomato (TdT) cassette (originally referred to as Rosa26 +/tdTomato ) (Janeckova et al, 2016). This form cannot bind β-catenin and thus acts as a dominant repressor of Wnt/β-catenin targets.…”

“…(Harada et al, 1999). Generation of Rosa26 dnTCF4fl/+ was described by Janeckova et al (2016). Briefly, human TCF7L2 coding sequence lacking the N-terminal domain (amino acids 1-31) was cloned into the ROSA26 targeting vector, which contains arms for homologous recombination into the ROSA26 locus, in the same way as when generating Rosa26 ctβcat-Tcf7l1fl/+…”