Opening the primary mouth with Wnt antagonists

CAMBRIDGE, Mass. –Whitehead researchers have identified a novel mechanism that operates during formation of the “primary mouth”, the first opening between the outside of the embryo and the intestine.

According to findings published recently in Development, inhibitors of the Wnt signaling pathway direct breakdown of the basement membrane during primary mouth formation, in embryos of the frog Xenopus. The Wnt pathway is important in a wide array of developmental processes and in cancer. The basement membrane is a thin sheet of proteins and glycoproteins (proteins with linked sugars) that regulates the cohesiveness of neighboring sheets of cells, and harbors signaling molecules that control cell fate and cell death.

“This is the first demonstration that basement membrane integrity is maintained by the Wnt pathway,” says Whitehead Member Hazel Sive,  “and is an important step in understanding the metabolism of the basement membrane.”

In frogs, as in other vertebrate embryos, the basement membrane separates two layers of cells at the site of the initial mouth opening, called the primary mouth.  Previous work from Amanda Dickinson, a postdoctoral researcher in the Sive laboratory, showed that the earliest known step in primary mouth formation is dissolution of the basement membrane, just in the region where the primary mouth will form. 

In order to investigate what prompts the basement membrane to dissolve, Dickinson identified genes that are more active in the developing primary mouth than in the surrounding tissues. At the top of her list were the frzb-1 and crescent genes, which are highly expressed in the region that will form the primary mouth.  These genes encode the secreted Frizzled Related Proteins (sFRPs), Frzb-1 and Crescent, which are known to inhibit the Wnt pathway, by binding Wnt protein, preventing it from reaching receptors on the cell surface. After ablation of Frzb-1 and Crescent function, Dickinson showed that the primary mouth basement membrane did not break down, and the primary mouth did not form.

However, because Frzb-1 and Crescent are known to be important in a variety of regions of the early embryo, Dickinson wanted to make sure that these genes were acting specifically in the primary mouth. She therefore developed “face transplants” – replacing the primary mouth of a normal frog embryo with the primary mouth from a frog without frzb-1 and crescent gene function. This technique was highly successful, showing that Frzb-1 and Crescent are acting locally in the developing primary mouth. Consistently, when Dickinson increased Wnt protein levels, using face transplants, no basement membrane breakdown occurred and no primary mouth formed, again indicating that Wnt protein inhibits primary mouth formation. Dickinson next gathered evidence that the Wnt pathway acts by promoting expression of laminin and fibronectin, two proteins essential for basement membrane formation and function. Thus, putting too much Wnt protein into the primary mouth increased levels of fibronectin and laminin RNA, while decreasing Wnt function led to a sharp decrease in levels of these RNAs.  

“These results are very exciting,” notes Sive, “because we have had almost no handle on the signals that control basement membrane metabolism during development.”

Is Wnt signaling the whole story? “Almost certainly not”, says Sive, “there are other proteins that will be involved, and we need to identify these, but we think that Wnts may be pivotal regulators of the basement membrane in normal and pathological processes.”

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Hazel Sive’s primary affiliation is with Whitehead Institute for Biomedical Research, where her laboratory is located and all her research is conducted. Sive is also a professor of Biology at the Massachusetts Institute of Technology.

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Dickinson, A. J., & Sive, H. L. (2009). The Wnt antagonists Frzb-1 and Crescent locally regulate basement membrane dissolution in the developing primary mouthDevelopment136(7), 1071-1081.



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