Wnt signaling in gut development and homeostasis

Abstract

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 Math1