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Summary
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.
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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.
Key words: Wnt signalling, Intestine, Transcription, T cell factor (Tcf), Stem cell
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