Alex Gregorieff

Publications of Alex Gregorieff

• In situ hybridization to identify gut stem cells.

  Authors: Alex Gregorieff, Hans Clevers

  Current protocols in stem cell biology. 01/2010; Chapter 2:Unit 2F.1.

  In recent years, considerable effort has been directed towards identifying the repertoire of genes specifically expressed in adult stem cells. In this unit, we describe an in situ hybridizationIn recent years, considerable effort has been directed towards identifying the repertoire of genes specifically expressed in adult stem cells. In this unit, we describe an in situ hybridization protocol adapted for the analysis of gene expression in the intestinal mucosa. This methodology allows researchers to quickly visualize the expression profile of putative stem cell markers with a high degree of sensitivity and resolution.

• The Ets-domain transcription factor Spdef promotes maturation of Goblet and Paneth cells in the intestinal epithelium.

  Authors: Alex Gregorieff, Daniel E Stange, Pekka Kujala, Harry Begthel, Maaike van den Born, Jeroen Korving, Peter J Peters, Hans Clevers

  Gastroenterology. 06/2009;

  BACKGROUND & AIMS:: Stems cells within the intestinal epithelium generate daughter cells that undergo lineage commitment and maturation through the combined action of the Wnt and Notch signalingBACKGROUND & AIMS:: Stems cells within the intestinal epithelium generate daughter cells that undergo lineage commitment and maturation through the combined action of the Wnt and Notch signaling cascades. Both pathways, in turn, regulate transcription factor networks that further define differentiation toward either enterocytes or 1 of 3 secretory cell lineages (Paneth, goblet or enteroendocrine cells). In this manuscript, we investigated the role of the Wnt-responsive, Ets-domain transcription factor Spdef in the differentiation of goblet and Paneth cells. METHODS:: The in vivo function of Spdef was examined by disrupting the Spdef gene in mice ( Spdef-/- mice) and analyzing the intestinal phenotype using a range of histological techniques and DNA microarray profiling. RESULTS:: In accordance with expression data, we found that loss of Spdef severely impaired the maturation of goblet and Paneth cells and conversely led to an accumulation of immature secretory progenitors. Spdef appears to positively and negatively regulate a specific subset of goblet and Paneth cell genes, including Cryptdins, Mmp7, Ang4, Kallikreins, and Muc2. CONCLUSION:: Spdef acts downstream of Math1 to promote terminal differentiation of a secretory progenitor pool into Paneth and goblet cells.

• Giving APCmin tumours a SPARC.

  Authors: Alex Gregorieff, Hans Clevers

  Gut. 11/2007; 56(10):1341-3.

• Wnt signaling mediates regional specification in the vertebrate face.

  Authors: Samantha A Brugmann, L Henry Goodnough, Alex Gregorieff, Philipp Leucht, Derk ten Berge, Christophe Fuerer, Hans Clevers, Roel Nusse, Jill A Helms

  Development (Cambridge, England). 10/2007; 134(18):3283-95.

  At early stages of development, the faces of vertebrate embryos look remarkably similar, yet within a very short timeframe they adopt species-specific facial characteristics. What are the mechanismsAt early stages of development, the faces of vertebrate embryos look remarkably similar, yet within a very short timeframe they adopt species-specific facial characteristics. What are the mechanisms underlying this regional specification of the vertebrate face? Using transgenic Wnt reporter embryos we found a highly conserved pattern of Wnt responsiveness in the developing mouse face that later corresponded to derivatives of the frontonasal and maxillary prominences. We explored the consequences of disrupting Wnt signaling, first using a genetic approach. Mice carrying compound null mutations in the nuclear mediators Lef1 and Tcf4 exhibited radically altered facial features that culminated in a hyperteloric appearance and a foreshortened midface. We also used a biochemical approach to perturb Wnt signaling and found that in utero delivery of a Wnt antagonist, Dkk1, produced similar midfacial malformations. We tested the hypothesis that Wnt signaling is an evolutionarily conserved mechanism controlling facial morphogenesis by determining the pattern of Wnt responsiveness in avian faces, and then by evaluating the consequences of Wnt inhibition in the chick face. Collectively, these data elucidate a new role for Wnt signaling in regional specification of the vertebrate face, and suggest possible mechanisms whereby species-specific facial features are generated.

• The Paneth cell alpha-defensin deficiency of ileal Crohn's disease is linked to Wnt/Tcf-4.

  Authors: Jan Wehkamp, Guoxing Wang, Irmgard Kübler, Sabine Nuding, Alex Gregorieff, Anke Schnabel, Robert J Kays, Klaus Fellermann, Oliver Burk, Matthias Schwab, Hans Clevers, Charles L Bevins, Eduard F Stange

  Journal of immunology (Baltimore, Md. : 1950). 10/2007; 179(5):3109-18.

  Ileal Crohn's disease (CD), a chronic mucosal inflammation, is characterized by two pertinent features: a specific decrease of Paneth cell-produced antimicrobial alpha-defensins and the presence ofIleal Crohn's disease (CD), a chronic mucosal inflammation, is characterized by two pertinent features: a specific decrease of Paneth cell-produced antimicrobial alpha-defensins and the presence of mucosal-adherent bacteria. A mutation in NOD2, the muramyl dipeptide recognition receptor, is found in some patients, which leads to an even more pronounced alpha-defensin decrease. However, the underlying mechanism remains unclear for the majority of patients. In this study, we report a reduced expression in ileal CD of the Wnt-signaling pathway transcription factor Tcf-4, a known regulator of Paneth cell differentiation and alpha-defensin expression. Within specimens, the levels of Tcf-4 mRNA showed a high degree of correlation with both HD5 and HD6 mRNA. The levels of Tcf-4 mRNA were decreased in patients with ileal disease irrespective of degree of inflammation, but were not decreased in colonic CD or ulcerative colitis. As a functional indicator of Tcf-4 protein, quantitative binding analysis with nuclear extracts from small intestine biopsies to a Tcf-4 high-affinity binding site in the HD-5 and HD-6 promoters showed significantly reduced activity in ileal CD. Furthermore, a causal link was shown in a murine Tcf-4 knockout model, where the comparably reduced expression of Tcf-4 in heterozygous (+/-) mice was sufficient to cause a significant decrease of both Paneth cell alpha-defensin levels and bacterial killing activity. Finally, the association between Paneth cell alpha-defensins and Tcf-4 was found to be independent of the NOD2 genotype. This new link established between a human inflammatory bowel disease and the Wnt pathway/Tcf-4 provides a novel mechanism for pathogenesis in patients with ileal CD.

• Rapid loss of intestinal crypts upon conditional deletion of the Wnt/Tcf-4 target gene c-Myc.

  Authors: Vanesa Muncan, Owen J Sansom, Leon Tertoolen, Toby J Phesse, Harry Begthel, Elena Sancho, Alicia M Cole, Alex Gregorieff, Ignacio Moreno de Alboran, Hans Clevers, Alan R Clarke

  Molecular and cellular biology. 12/2006; 26(22):8418-26.

  Inhibition of the mutationally activated Wnt cascade in colorectal cancer cell lines induces a rapid G1 arrest and subsequent differentiation. This arrest can be overcome by maintaining expression ofInhibition of the mutationally activated Wnt cascade in colorectal cancer cell lines induces a rapid G1 arrest and subsequent differentiation. This arrest can be overcome by maintaining expression of a single Tcf4 target gene, the proto-oncogene c-Myc. Since colorectal cancer cells share many molecular characteristics with proliferative crypt progenitors, we have assessed the physiological role of c-Myc in adult crypts by conditional gene deletion. c-Myc-deficient crypts are lost within weeks and replaced by c-Myc-proficient crypts through a fission process of crypts that have escaped gene deletion. Although c-Myc(-/-) crypt cells remain in the cell cycle, they are on average much smaller than wild-type cells, cycle slower, and divide at a smaller cell size. c-Myc appears essential for crypt progenitor cells to provide the necessary biosynthetic capacity to successfully progress through the cell cycle.

• Expression pattern of Wnt signaling components in the adult intestine.

  Authors: Alex Gregorieff, Daniel Pinto, Harry Begthel, Olivier Destrée, Menno Kielman, Hans Clevers

  Gastroenterology. 08/2005; 129(2):626-38.

  BACKGROUND & AIMS: In the intestine, the canonical Wnt signaling cascade plays a crucial role in driving the proliferation of epithelial cells. Furthermore, aberrant activation of Wnt signaling isBACKGROUND & AIMS: In the intestine, the canonical Wnt signaling cascade plays a crucial role in driving the proliferation of epithelial cells. Furthermore, aberrant activation of Wnt signaling is strongly associated with the development of colorectal cancer. Despite this evidence, little is known about the precise identity and localization of Wnts and their downstream effectors in the adult intestine. To address this issue, we examined the expression pattern of all Wnts, Frizzleds (Fzs), low-density lipoprotein receptor-related proteins, Wnt antagonists, and T-cell factors in the murine small intestine and colon and adenomas. METHODS: Embryonic, postnatal, and adult intestinal samples were subjected to in situ hybridization by using specific RNA probes for the various genes tested. RESULTS: Our analysis showed high expression of several signaling components (including Wnt-3, Wnt-6, Wnt-9b, Frizzled 4, Frizzled 6, Frizzled 7, low-density lipoprotein receptor-related protein 5, and secreted Frizzled-related protein 5) in crypt epithelial cells. We also detected Wnt-2b, Wnt-4, Wnt-5a, Wnt-5b, Frizzled 4, and Frizzled 6 in differentiated epithelial and mesenchymal cells of the small intestine and colon. Finally, several factors (Frizzled 4, T-cell factor 1, lymphoid enhancer factor, Dickkopf 2, Dickkopf 3, and Wnt-interacting factor) displayed differential expression in normal vs neoplastic tissue. CONCLUSIONS: Our study predicts a much broader role for Wnt signaling in gut development and homeostasis than was previously anticipated from available genetic studies and identifies novel factors likely involved in promoting canonical and noncanonical Wnt signals in the intestine.

• Wnt signalling induces maturation of Paneth cells in intestinal crypts.

  Authors: Johan H van Es, Philippe Jay, Alex Gregorieff, Marielle E van Gijn, Suzanne Jonkheer, Pantelis Hatzis, Andrea Thiele, Maaike van den Born, Harry Begthel, Thomas Brabletz, Makoto M Taketo, Hans Clevers

  Nature cell biology. 05/2005; 7(4):381-6.

  Wnt signalling, which is transduced through beta-catenin/TCF4, maintains the undifferentiated state of intestinal crypt progenitor cells. Mutational activation of the pathway initiates theWnt signalling, which is transduced through beta-catenin/TCF4, maintains the undifferentiated state of intestinal crypt progenitor cells. Mutational activation of the pathway initiates the adenomacarcinoma sequence. Whereas all other differentiated epithelial cells migrate from the crypt onto the villus, Paneth cells home towards the source of Wnt signals--that is, the crypt bottom. Here, we show that expression of a Paneth gene programme is critically dependent on TCF4 in embryonic intestine. Moreover, conditional deletion of the Wnt receptor Frizzled-5 abrogates expression of these genes in Paneth cells in the adult intestine. Conversely, adenomas in Apc-mutant mice and colorectal cancers in humans inappropriately express these Paneth-cell genes. These observations imply that Wnt signals in the crypt can separately drive a stem-cell/progenitor gene programme and a Paneth-cell maturation programme. In intestinal cancer, both gene programmes are activated simultaneously.

• Wnt signaling in the intestinal epithelium: from endoderm to cancer.

  Authors: Alex Gregorieff, Hans Clevers

  Genes & development. 05/2005; 19(8):877-90.

  The Wnt pathway controls cell fate during embryonic development. It also persists as a key regulator of homeostasis in adult self-renewing tissues. In these tissues, mutational deregulation of theThe Wnt pathway controls cell fate during embryonic development. It also persists as a key regulator of homeostasis in adult self-renewing tissues. In these tissues, mutational deregulation of the Wnt cascade is closely associated with malignant transformation. The intestinal epithelium represents the best-understood example for the closely linked roles of Wnt signaling in homeostatic self-renewal and malignant transformation. In this review, we outline current understanding of the physiological role of Wnt signaling in intestinal biology. From this perspective, we then describe how mutational subversion of the Wnt cascade leads to colorectal cancer.

• Hindgut defects and transformation of the gastro-intestinal tract in Tcf4(-/-)/Tcf1(-/-) embryos.

  Authors: Alex Gregorieff, Rudolf Grosschedl, Hans Clevers

  The EMBO journal. 05/2004; 23(8):1825-33.

  Wnt signalling plays a critical role in both initiating and patterning of the anterior-posterior axis during development. Wnts exert their biological effects, in part, by activating specific targetWnt signalling plays a critical role in both initiating and patterning of the anterior-posterior axis during development. Wnts exert their biological effects, in part, by activating specific target genes through members of the TCF/LEF family of transcription factors. To gain new insight into the role of T-cell factors (or Tcf's) during development, we analysed Tcf4 and Tcf1 compound null embryos. These mutants showed severe caudal truncations, as well as duplications of the neural tube. Unlike other mutations affecting Wnt signalling, 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.

• Canonical Wnt signals are essential for homeostasis of the intestinal epithelium.

  Authors: Daniel Pinto, Alex Gregorieff, Harry Begthel, Hans Clevers

  Genes & development. 08/2003; 17(14):1709-13.

  To assess the critical role of Wnt signals in intestinal crypts, we generated transgenic mice ectopically expressing Dickkopf1 (Dkk1), a secreted Wnt inhibitor. We find that epithelial proliferationTo assess the critical role of Wnt signals in intestinal crypts, we generated transgenic mice ectopically expressing Dickkopf1 (Dkk1), a secreted Wnt inhibitor. We find that epithelial proliferation is greatly reduced coincidentally with the loss of crypts. Although enterocyte differentiation appears unaffected, secretory cell lineages are largely absent. Disrupted intestinal homeostasis is reflected by an absence of nuclear beta-catenin, inhibition of c-myc expression, and subsequent up-regulation of p21CIP1/WAF1. Thus, our data are the first to establish a direct requirement for Wnt ligands in driving proliferation in the intestinal epithelium, and also define an unexpected role for Wnts in controlling secretory cell differentiation.

• Expression Pattern of Wnt Signaling Components in the Adult Intestine

  Authors: Alex Gregorieff, Daniel Pinto, Harry Begthel, Olivier Destrée, Menno Kielman, Hans Clevers

  Gastroenterology.

  Background & Aims: In the intestine, the canonical Wnt signaling cascade plays a crucial role in driving the proliferation of epithelial cells. Furthermore, aberrant activation of Wnt signaling isBackground & Aims: In the intestine, the canonical Wnt signaling cascade plays a crucial role in driving the proliferation of epithelial cells. Furthermore, aberrant activation of Wnt signaling is strongly associated with the development of colorectal cancer. Despite this evidence, little is known about the precise identity and localization of Wnts and their downstream effectors in the adult intestine. To address this issue, we examined the expression pattern of all Wnts, Frizzleds (Fzs), low-density lipoprotein receptor-related proteins, Wnt antagonists, and T-cell factors in the murine small intestine and colon and adenomas. Methods: Embryonic, postnatal, and adult intestinal samples were subjected to in situ hybridization by using specific RNA probes for the various genes tested. Results: Our analysis showed high expression of several signaling components (including Wnt-3, Wnt-6, Wnt-9b, Frizzled 4, Frizzled 6, Frizzled 7, low-density lipoprotein receptor-related protein 5, and secreted Frizzled-related protein 5) in crypt epithelial cells. We also detected Wnt-2b, Wnt-4, Wnt-5a, Wnt-5b, Frizzled 4, and Frizzled 6 in differentiated epithelial and mesenchymal cells of the small intestine and colon. Finally, several factors (Frizzled 4, T-cell factor 1, lymphoid enhancer factor, Dickkopf 2, Dickkopf 3, and Wnt-interacting factor) displayed differential expression in normal vs neoplastic tissue. Conclusions: Our study predicts a much broader role for Wnt signaling in gut development and homeostasis than was previously anticipated from available genetic studies and identifies novel factors likely involved in promoting canonical and noncanonical Wnt signals in the intestine.