Wnt signaling in gut development and homeostasis

The Wnt pathway controls diverse biological processes during embryonic development. In the adult, Wnts maintain the balance between cell division and cell specialisation in tissues such as the hemapoetic system, skin, and the intestine. Genetic modifications which activate the Wnt pathway are also closely linked to unrestricted cell growth and malignancy. In this thesis we focused on the specific role of the Wnt pathway during normal intestinal development and homeostasis. To this end, we utilized so-called knockout mice which harbour mutations in the essential componenents of the pathway and studied the molecular consequences of these mutations by in situ hybridization, a technique which allows one to visualize the expression of gene products in tissues. In Chapter 2 we studied the consequences of deleting two Wnt effector proteins Tcf4 and Tcf1 compound null embryos. Mouse lacking the gene products of both Tcf1 and Tcf4 showed severe caudal truncations of the body, as well as duplications of the neural tube. Unlike other mutations affecting Wnt signaling, paraxial mesoderm formation was not impaired and early caudal markers, such as T, were unaffected. Analysis of endodermal markers uncovered early and specific defects in hindgut expansion and later an anterior transformation of the gastro-intestinal tract. Our results reveal a novel role for Wnt signalling in early gut morphogenesis and suggest that specific Wnt-driven patterning events are determined by the unique tissue distribution of Tcf/Lef family members. In Chapter 3 we performed a large scale in situ hybridisation screen to examine the expression pattern of all Wnts, Fzs, LRPs, Wnt antagonists and TCFs in the murine small intestine, colon and adenomas. Our analysis revealed high expression of several signaling components (including Wnt-3, Fz-7,) in crypt epithelial cells. We also detected gene products such as Wnt-2 and Fz-6 in differentiated epithelial and/or mesenchymal cells of the small intestine and colon. Finally, several factors (TCF-1, Dkk-3) displayed differential expression in normal versus neoplastic tissue. This study predicted a much more complex role for Wnt signaling in gut development and homeostasis than was previously anticipated. In Chapter 4 we found that inactivation of Tcf4 in the embryonic intestine and conditional deletion of the Wnt receptor Frizzled-5 in the adult intestine abrogated a specific Paneth cell genetic program. Paneth cells secrete microbicidal peptides, such as cryptdins, important in fighting bacterial infections. Moreover these Paneth cells reside at the very bottom of the intestinal crypts. In Tcf4 and Fz-5 mutant mice we observed a defect in the production of Paneth cell gene products and a scattering of Paneth cells throughout the crypt-villus axis. Conversely, adenomas in APC mutant mice and colorectal cancers in man inappropriately expressed these Paneth cell genes, providing additional support that the expression of these genes is driven by active Wnt signals. Furthermore, these observations implied that Wnt signals in the crypt can separately drive a stem-cell/progenitor gene program and a Paneth cell maturation program. Finally in Chapter 5 we studied in more detail the defects associated with loss of Tcf4 in the intestine. Deletion of Tcf4 results in neonatal death and a complete loss of proliferative stem cells in the intestine. By monitoring the gene products of specialized cell-types we found that absorptive cell markers (ie. Fabp1, Creb3l3, Nr1h4, etc.) were upregulated in Tcf4 knockout embryos. Concomitantly, loss of Tcf4 resulted in specific downregulation of secretory lineage markers (ie. Tff3, Chromogranin B, and Spink4, etc.) and crypt progenitor markers (ie. c-Myc, c-Myb, TcfAP4, etc). Further analysis suggested that Tcf4 promotes early commitment of secretory lineages through activation of the basic helix-loop-helix transcription factor, Math1. Moreover we found that Tcf4-mediated effects on cell fate were independent of any changes in the expression of Hes family members. Finally our results imply a model whereby Tcf4 coordinates renewal of progenitor cells, repression of enterocyte differentiation and commitment towards secretory lineages via Math

Proteomic Profiling of Enteroid Cultures Skewed Towards Development of Specific Epithelial Lineages

Recently, three‐dimensional small intestinal organoids (enteroids) have been developed from cultures of intestinal stem cells which differentiate in vitro to generate all the differentiated epithelial cell types associated with the intestine and mimic the structural properties of the intestine observed in vivo. Small‐molecule drug treatment can skew organoid epithelial cell differentiation towards particular lineages, and these skewed enteroids may provide useful tools to study specific epithelial cell populations, such as goblet and Paneth cells. However, the extent to which differentiated epithelial cell populations in these skewed enteroids represent their in vivo counterparts is not fully understood. In this study, we have performed label‐free quantitative proteomics to determine whether skewing murine enteroid cultures towards the goblet or Paneth cell lineages results in changes in abundance of proteins associated with these cell lineages in vivo. Our data confirm that skewed enteroids recapitulate important features of the in vivo gut environment, confirming that they can serve as useful models for the investigation of normal and disease processes in the intestine. Furthermore, by comparison of our mass spectrometry data with histology data contained within the Human Protein Atlas, we identify putative novel markers for goblet and Paneth cells

Fate specification and tissue-specific cell cycle control of the <i>Caenorhabditis elegans</i> intestine

Coordination between cell fate specification and cell cycle control in multicellular organisms is essential to regulate cell numbers in tissues and organs during development, and its failure may lead to oncogenesis. In mammalian cells, as part of a general cell cycle checkpoint mechanism, the F-box protein β-transducin repeat-containing protein (β-TrCP) and the Skp1/Cul1/F-box complex control the periodic cell cycle fluctuations in abundance of the CDC25A and B phosphatases. Here, we find that the Caenorhabditis elegans β-TrCP orthologue LIN-23 regulates a progressive decline of CDC-25.1 abundance over several embryonic cell cycles and specifies cell number of one tissue, the embryonic intestine. The negative regulation of CDC-25.1 abundance by LIN-23 may be developmentally controlled because CDC-25.1 accumulates over time within the developing germline, where LIN-23 is also present. Concurrent with the destabilization of CDC-25.1, LIN-23 displays a spatially dynamic behavior in the embryo, periodically entering a nuclear compartment where CDC-25.1 is abundant

Acute WNT signalling activation perturbs differentiation within the adult stomach and rapidly leads to tumour formation

A role for WNT signalling in gastric carcinogenesis has been suggested due to two major observations. First, patients with germline mutations in adenomatous polyposis coli (APC) are susceptible to stomach polyps and second, in gastric cancer, WNT activation confers a poor prognosis. However, the functional significance of deregulated WNT signalling in gastric homoeostasis and cancer is still unclear. In this study we have addressed this by investigating the immediate effects of WNT signalling activation within the stomach epithelium. We have specifically activated the WNT signalling pathway within the mouse adult gastric epithelium via deletion of either glycogen synthase kinase 3 (GSK3) or APC or via expression of a constitutively active β-catenin protein. WNT pathway deregulation dramatically affects stomach homoeostasis at very short latencies. In the corpus, there is rapid loss of parietal cells with fundic gland polyp (FGP) formation and adenomatous change, which are similar to those observed in familial adenomatous polyposis. In the antrum, adenomas occur from 4 days post-WNT activation. Taken together, these data show a pivotal role for WNT signalling in gastric homoeostasis, FGP formation and adenomagenesis. Loss of the parietal cell population and corresponding FGP formation, an early event in gastric carcinogenesis, as well as antral adenoma formation are immediate effects of nuclear β-catenin translocation and WNT target gene expression. Furthermore, our inducible murine model will permit a better understanding of the molecular changes required to drive tumourigenesis in the stomach

Minimizing off-target signals in RNA fluorescent in situ hybridization

Fluorescent in situ hybridization (FISH) techniques are becoming extremely sensitive, to the point where individual RNA or DNA molecules can be detected with small probes. At this level of sensitivity, the elimination of ‘off-target’ hybridization is of crucial importance, but typical probes used for RNA and DNA FISH contain sequences repeated elsewhere in the genome. We find that very short (e.g. 20 nt) perfect repeated sequences within much longer probes (e.g. 350–1500 nt) can produce significant off-target signals. The extent of noise is surprising given the long length of the probes and the short length of non-specific regions. When we removed the small regions of repeated sequence from either short or long probes, we find that the signal-to-noise ratio is increased by orders of magnitude, putting us in a regime where fluorescent signals can be considered to be a quantitative measure of target transcript numbers. As the majority of genes in complex organisms contain repeated k-mers, we provide genome-wide annotations of k-mer-uniqueness at http://cbio.mskcc.org/∼aarvey/repeatmap

Genetic Variants of Wnt Transcription Factor TCF-4 (TCF7L2) Putative Promoter Region Are Associated with Small Intestinal Crohn's Disease

Reduced expression of Paneth cell antimicrobial α-defensins, human defensin (HD)-5 and -6, characterizes Crohn's disease (CD) of the ileum. TCF-4 (also named TCF7L2), a Wnt signalling pathway transcription factor, orchestrates Paneth cell differentiation, directly regulates the expression of HD-5 and -6, and was previously associated with the decrease of these antimicrobial peptides in a subset of ileal CD. To investigate a potential genetic association of TCF-4 with ileal CD, we sequenced 2.1 kb of the 5′ flanking region of TCF-4 in a small group of ileal CD patients and controls (n = 10 each). We identified eight single nucleotide polymorphisms (SNPs), of which three (rs3814570, rs10885394, rs10885395) were in linkage disequilibrium and found more frequently in patients; one (rs3814570) was thereby located in a predicted regulatory region. We carried out high-throughput analysis of this SNP in three cohorts of inflammatory bowel disease (IBD) patients and controls. Overall 1399 healthy individuals, 785 ulcerative colitis (UC) patients, 225 CD patients with colonic disease only and 784 CD patients with ileal involvement were used to determine frequency distributions. We found an association of rs3814570 with ileal CD but neither with colonic CD or UC, in a combined analysis (allele positivity: OR 1.27, 95% CI 1.07 to 1.52, p = 0.00737), which was the strongest in ileal CD patients with stricturing behaviour (allele frequency: OR 1.32, 95% CI 1.08 to1.62, p = 0.00686) or an additional involvement of the upper GIT (allele frequency: OR 1.38, 95% CI 1.03 to1.84, p = 0.02882). The newly identified genetic association of TCF-4 with ileal CD provides evidence that the decrease in Paneth cell α-defensins is a primary factor in disease pathogenesis

Wnt-reporter expression pattern in the mouse intestine during homeostasis

<p>Abstract</p> <p>Background</p> <p>The canonical Wnt signaling pathway is a known regulator of cell proliferation during development and maintenance of the intestinal epithelium. Perturbations in this pathway lead to aberrant epithelial proliferation and intestinal cancer. In the mature intestine, proliferation is confined to the relatively quiescent stem cells and the rapidly cycling transient-amplifying cells in the intestinal crypts. Although the Wnt signal is believed to regulate all proliferating intestinal cells, surprisingly, this has not been thoroughly demonstrated. This important determination has implications on intestinal function, especially during epithelial expansion and regeneration, and warrants an extensive characterization of Wnt-activated cells.</p> <p>Methods</p> <p>To identify intestinal epithelial cells that actively receive a Wnt signal, we analyzed intestinal Wnt-reporter expression patterns in two different mouse lines using immunohistochemistry, enzymatic activity, <it>in situ </it>hybridization and qRT-PCR, then corroborated results with reporter-independent analyses. Wnt-receiving cells were further characterized for co-expression of proliferation markers, putative stem cell markers and cellular differentiation markers using an immunohistochemical approach. Finally, to demonstrate that Wnt-reporter mice have utility in detecting perturbations in intestinal Wnt signaling, the reporter response to gamma-irradiation was examined.</p> <p>Results</p> <p>Wnt-activated cells were primarily restricted to the base of the small intestinal and colonic crypts, and were highest in numbers in the proximal small intestine, decreasing in frequency in a gradient toward the large intestine. Interestingly, the majority of the Wnt-reporter-expressing cells did not overlap with the transient-amplifying cell population. Further, while Wnt-activated cells expressed the putative stem cell marker Musashi-1, they did not co-express DCAMKL-1 or cell differentiation markers. Finally, gamma-irradiation stimulated an increase in Wnt-activated intestinal crypt cells.</p> <p>Conclusion</p> <p>We show, for the first time, detailed characterization of the intestine from Wnt-reporter mice. Further, our data show that the majority of Wnt-receiving cells reside in the stem cell niche of the crypt base and do not extend into the proliferative transient-amplifying cell population. We also show that the Wnt-reporter mice can be used to detect changes in intestinal epithelial Wnt signaling upon physiologic injury. Our findings have an important impact on understanding the regulation of the intestinal stem cell hierarchy during homeostasis and in disease states.</p

β-Catenin stabilizes Cyclooxygenase-2 mRNA by interacting with AU-rich elements of 3′-UTR

Cyclooxygenase-2 (COX-2) mRNA is induced in the majority of human colorectal carcinomas. Transcriptional regulation plays a key role in COX-2 expression in human colon carcinoma cells, but post-transcriptional regulation of its mRNA is also critical for tumorigenesis. Expression of COX-2 mRNA is regulated by various cytokines, growth factors and other signals. β-Catenin, a key transcription factor in the Wnt signal pathway, activates transcription of COX-2. Here we found that COX-2 mRNA was also substantially stabilized by activating β-catenin in NIH3T3 and 293T cells. We identified the β-catenin-responsive element in the proximal region of the COX-2 3′-untranslated region (3′-UTR) and showed that β-catenin interacted with AU-rich elements (ARE) of 3′-UTR in vitro and in vivo. Interestingly, β-catenin induced the cytoplasmic localization of the RNA stabilizing factor, HuR, which may bind to β-catenin in an RNA-mediated complex and facilitate β-catenin-dependent stabilization of COX-2 mRNA. Taken together, we provided evidences for β-catenin as an RNA-binding factor and a regulator of stabilization of COX-2 mRNA

The Tyrosine Kinase Csk Dimerizes through Its SH3 Domain

The Src family kinases possess two sites of tyrosine phosphorylation that are critical to the regulation of kinase activity. Autophosphorylation on an activation loop tyrosine residue (Tyr 416 in commonly used chicken c-Src numbering) increases catalytic activity, while phosphorylation of a C-terminal tyrosine (Tyr 527 in c-Src) inhibits activity. The latter modification is achieved by the tyrosine kinase Csk (C-terminal Src Kinase), but the complete inactivation of the Src family kinases also requires the dephosphorylation of the activation loop tyrosine. The SH3 domain of Csk recruits the tyrosine phosphatase PEP, allowing for the coordinated inhibition of Src family kinase activity. We have discovered that Csk forms homodimers through interactions mediated by the SH3 domain in a manner that buries the recognition surface for SH3 ligands. The formation of this dimer would therefore block the recruitment of tyrosine phosphatases and may have important implications for the regulation of Src kinase activity

Lentiviral-Mediated Transgene Expression Can Potentiate Intestinal Mesenchymal-Epithelial Signaling

Mesenchymal-epithelial signaling is essential for the development of many organs and is often disrupted in disease. In this study, we demonstrate the use of lentiviral-mediated transgene delivery as an effective approach for ectopic transgene expression and an alternative to generation of transgenic animals. One benefit to this approach is that it can be used independently or in conjunction with established transgenic or knockout animals for studying modulation of mesenchymal-epithelial interactions. To display the power of this approach, we explored ectopic expression of a Wnt ligand in the mouse intestinal mesenchyme and demonstrate its functional influence on the adjacent epithelium. Our findings highlight the efficient use of lentiviral-mediated transgene expression for modulating mesenchymal-epithelial interactions in vivo