Signalling through the canonical WNT pathway

coordination (ataxia), swaying of the head being one of the symptoms. Here, the mutated Wnt-1 protein lacks the C-terminal half as a result of a single base pair mutation that creates a premature stop codon.

From mutation analysis in Drosophila and screening for homologous genes in mice, it emerged that Wnt is a member of a large family of genes (at least 19 in mammals), having different functions and different signal transduction pathways. As with the Ras-MAPkinase pathway (see page 327), the study of mutants in genetically accessible organisms greatly facilitated investigations of the role of Wnt in the regulation of proliferation, differentiation, and cell fate in mammalian cells.

Following its discovery, Wnt remained a gene that evaded proper definition as a protein for about 15 years. Its effects were studied through gene amplification and deletion, through injection of mRNA, or, latterly, through translation arrest by applied siRNA. Proteins of the Wnt family are now recognized as secreted palmitoylated glycoproteins.20 The lipid attachment, essential for signalling, renders them insoluble and upon exocytosis they remain associated with the cell membrane. However, Wnt can diffuse into the tissues to a limited extent, over a distance of up to 30 cells, in this way forming morphogenic gradients.21

Wnt signals through -catenin

Mutational analysis in Drosophila led to the discovery of a signal transduction pathway consisting of the ligand (wingless, Wg), its receptor (frizzled, Fz), and downstream components. By scoring for rescue or aggravation of the

phenotype (so-called epistatic tests), these were found to act in the sequence:

dishevelled (Dsh) → shaggy (Sgg) → armadillo (Arm) → pangolin (Pan)

resulting in the expression of the transcription factor engrailed (en) (Figure 14.2)11,22,23 (for review see Willert and Nusse24). The genes involved are named after the mutant phenotypes they give rise to, having either abnormal hair patterns on their body or wings (as in frizzled, shaggy, or dishevelled) or with abnormal larval cuticle formation (armadillo or pangolin).

Understanding these genes and their interrelationships, and how they are integrated into pathways that determine cell fate or polarity, has required a number of different experimental approaches. Sequence analysis and

immunocytochemical experiments have provided evidence that armadillo is a homologue of the mammalian -catenin and that it resembles plakoglobin, a component of desmosomes.30,31 Pangolin encodes a protein homologous to the transcription factor, LEF-1,32 that interacts with -catenin. This interaction is essential for transmission of the Wnt signal into the nucleus.33,34 Armadillo also enters the cell nucleus to form a binary transcription complex with a

Wnt alleles and disease.

Wnt may be regarded as a proto-oncogene (having the potential to cause cancer), but so far no human tumours have been correlated with a Wnt defect. However, certain components

of the Wnt-signalling transduction pathway do qualify as true oncogenes. Dysfunctional Wnt alleles have been found in other human pathologies such as obesity, type 2 (late onset) diabetes, Müllerian duct regression, and the embryo-lethal phenotype tetra-amelia (lack of extremities).19

Wnt in development.

For an informative review of Wnt effects in Drosophila see Klingensmith and Nusse.25 For a well illustrated description of Wnt’s role in morphogenesis in

Drosophila see Martinez-Arias.26 For developmental disorders in mice Wnt-knockouts, see Logan and Nusse.27 A continuing update

of Wnt and its signal transduction pathways, including figures and gene tables, can be found on the Wnt homepage, maintained by the laboratory of Roel Nusse, http://www.stanford.edu/ ~rnusse/wntwindow.html

Regulation of gene expression. Regulation of gene expression is mediated by several mechanisms such

as DNA methylation, ATP-dependent chromatin remodelling, and post-translational modifications of histones, which include acetylation/deacetylation of lysine residues present

in the tail of core histones. The histone acetyltransferases (HATs) act as transcriptional coactivators, and histone deacetylases (HDACs) form part

of transcriptional corepressor complexes. Wnt uncouples Groucho from TCF and in so doing, it also uncouples the associated de-acetylation complex (containing HDAC2). This opens many gene loci for transcription. A mutation in HDAC2, which makes it insensitive to inhibitory factors, predisposes to colorectal cancer.42

FIG 14.2  Examples of Drosophila phenotypes due to mutations in the Wnt pathway.

In the wild-type fly, actin, which forms a single hair, polymerizes in a defined position with uniform orientation within the plane of the epithelial wing cells. Mutations in Fz and Dsh cause a loss of planar polarity of these cells. Polymerization occurs at random points within the plane of the epithelium giving rise to a frizzled (curly) or dishevelled (disorganized) phenotype. Mutations in Arm give rise to a highly segmented embryo resembling the banded shell of an armadillo.

Image of hairs from Wong and Adler 1993,28 originally published in the Journal of Cell Biology 123, 209–221. Images of Drosophila larvae Wt and Arm are kindly provided by Dr Martinez-Arias, Cambridge, UK

Drosophila homologue of the mammalian TCF-1 gene (dTCF)35 (for a review, see Clevers and van de Wetering36).

The closely related TCF-1 and LEF-1 were initially discovered as nuclear proteins involved in lymphocyte development and in the expression of T- cell-receptor complex components.37,38 With the Wnt signal silenced, TCF-1 and LEF-1, together with Groucho (a nuclear protein that recruits histone deacetylase) repress DNA transcription. The ensuing de-acetylation of histone causes dense packing of the DNA in nucleosomes (chromatin condensation) making it inaccessible for RNA polymerases39–41 (see Figure 14.3 and page 288). Binding of -catenin (armadillo) to TCF-1 (or LEF-1) interrupts the interaction with Groucho and its repressor partners, and allows re-acetylation of the DNA. The TCF-1 (or LEF-1) associated with -catenin then initiates transcription of genes leading to proliferation, differentiation or cell death (depending on cell type and context).

From all this it was understood that -catenin is localized either at the cell membrane or in the nucleus and that its presence in the nucleus profoundly alters the course of gene expression. It remains to be seen how Wnt affects the translocation of -catenin from the membrane to the nucleus.