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Cancer Research Takes Flight:

Wnt Signaling in Development and Disease

What do cancers have in common with fruit fly wings? Wnts. The very name of the Wnt (pronounced /wint/) family of secreted signaling molecules proclaims its dual history in developmental biology and cancer research. The "w" comes from wingless, a gene necessary for the proper development of the fruit fly body plan. The "nt" comes from Int oncogenes, first identified near sites of integration of the mouse mammary tumor virus. There are 19 Wnt genes in the human genome. Their tight regulation orchestrates development both embryonically and into adulthood; their misregulation contributes to multiple cancers. The merging of these two lines of research, which is now more than 25 years in the making, has been a boon for both fields.

Terry Yamaguchi, Ph.D. (right), with Postdoctoral Fellow Bill Dunty, Ph.D. (left), and technician Kirstin Biris (center). (Photo: R. Baer)

Terry Yamaguchi, Ph.D., Head of the Cell Signaling in Vertebrate Development Section in CCR’s Cancer and Developmental Biology Laboratory, came to Wnt signaling from developmental biology. His research has taken him from an interest in the late stages of muscle differentiation steadily backwards to the role of Wnts in the earliest steps of cell specification from embryonic stem cells. Now he hopes to define the key molecular events that govern the fate of stem cells in embryonic development and to apply that knowledge to understanding how stem cells contribute to adult tissues normally as well as how abnormal signaling gives rise to cancers.

Location, Location, Location

The mantra of "location, location, location" is as critical for determining cell fate during development as it is for setting the value of real estate, but location works its magic in development through the much more complicated process of gene regulation. Embryonic stem cells are originally pluripotent— capable of developing into almost any cell type, but their fates are gradually refined as they interact with their local environment. The pluripotent embryonic stem cells soon give rise to three germ layers—ectoderm, mesoderm, and endoderm—which give rise to specific tissues. Gradients of secreted signaling molecules activate distinct gene expression programs in the cells of each germ layer, which in turn regulate the cells’ interaction with the gradients of signaling molecules they encounter.

The trick throughout development is to create signals that are sufficiently restricted in time and space that they balance the production of more stem cells (proliferation) with the production of specific cell types (differentiation) to produce exactly and only as many cells as necessary for a specific tissue, a concept known as stem cell homeostasis.

The trick throughout development is to create signals that are sufficiently restricted in time and space that they balance the production of more stem cells (proliferation) with the production of specific cell types (differentiation).

The 19 Wnt ligands are generally expressed in patterns that are tightly regulated in time and space throughout development. Mutation of these genes usually results in dramatic developmental defects, although there appears to be some redundancy in the system so that a single Wnt mutation may leave an embryo seemingly unimpaired. Without Wnt3a, for example, the entire trunk and tail mesoderm fails to form. To similarly "disappear" the lungs, however, requires the double mutation of Wnt2 and Wnt2b. "The primitive streak," described Yamaguchi, pointing to a dark purple line in a micrograph of an eight-day old embryo, "is a source of many secreted signaling molecules, including Wnt3a, which can pattern the entire anteroposterior axis." Whereas it was once believed that the primitive streak was simply a point through which cells transit as they become the mesoderm of the trunk and tail, it now seems that the primitive streak is also a source of stem cells that give rise to the germ layers. "One of the main hypotheses that we are pursuing is that Wnt3a in the primitive streak is required for the maintenance of mesodermal stem cells."

Through a series of genetic experiments, published in the January 2008 issue of Development, Yamaguchi, Postdoctoral Fellow Bill Dunty, Ph.D., and their colleagues have formally demonstrated that Wnt3a works through the well-studied canonical β-catenin pathway to support mesodermal stem cells. β-catenin is normally maintained at low levels in the cellular milieu by the APC/axin complex, which steadily consigns β-catenin to degradation. Wnt signaling sequesters some of the components of the degradation complex, resulting in increased levels of β-catenin, which can then make its way to the nucleus to activate the transcription of a number of target genes.

Sp5 is an example of a Wnt3a/β-catenin target gene that is expressed in primitive streak (PS) stem cells of the mouse embryo (panels A and B) and in adult intestinal crypt stem cells (panel C, arrows) and adenomas (asterisk). (Image: T. Yamaguchi, CCR)

"The main point of this pathway from our perspective is that its stimulation activates a transcriptional program of gene expression. One of the big goals in the lab is to identify what this pathway is doing in the early embryo and identify the target genes through a transcriptional profiling approach." By looking at the gene expression patterns in different Wnt3a and β-catenin mutant mice, Yamaguchi’s team has identified 62 genes that may be regulated by this pathway. Some, like Sp5 and Axin2, are known targets of the Wnt/ β-catenin system in other contexts, some are known oncogenes like Myc, and others appear to be completely novel. Kristin Biris, a technician in Yamaguchi’s group, is using in situ hybridization to determine where these Wnt3a target genes are expressed in early embryos.

One of the most interesting target genes they have studied so far is Mesogenin 1, which is itself not only directly activated by Wnt3a signaling but also appears to operate in a feedback loop to inhibit Wnt3a signaling. Such an inhibitory feedback mechanism could allow high concentrations of Wnt3a, as found in the primitive streak, to support mesodermal stem cell renewal, whereas the effects of lower concentrations of Wnt3a would be inhibited by Mesogenin 1 feedback, turning a gradient of Wnt3a into a threshold that supports either proliferation or the differentiation of mesodermal stem cells.

Into the Crypt

High concentrations of Wnts are not confined to embryonic development. They reappear, among other places, in the adult intestine, where they regulate the intestinal stem cell niche. "Wnts are so conserved, and their expression is so closely associated with stem cell populations, we believe that what we learn from the early embryo may be generally applicable to other stem cells in the adult," said Yamaguchi. He and his colleagues are now beginning to put that belief to the test.

The adult intestine is coated with a single layer of epithelial cells that are responsible for digestion and absorption, as well as for providing a barrier against pathogens. These epithelial cells, of which there are four major types, are replaced every 4–5 days by a process of cellular renewal. The stem cells that give rise to these new cells are found in pockets of cells called the intestinal crypt. Deep in the crypt, new cells are born and mostly migrate upwards and away from the source of their renewal, up into finger-like protrusions of the intestine called villi. Three days after their cellular identity or fate is sealed, they reach the tip of the villus, self-destruct, and are shed away to be replaced by younger cells.