Cellular actions and activities within and between cells occur through a complex signalling system. These signalling pathways are made up of proteins, and control gene expression in an organism by passing signals from outside into the cell through receptors on the cell’s surface. Wnt signalling is one such pathway and is involved in embryonic development in both invertebrates and vertebrates. An evolutionarily conserved process, it is similar across species from fruit flies to humans. It plays a key role in determining the body axis, a crucial step in establishing the overall body plan of an organism. It is also involved in differentiation of cells — a process where epithelial cells are modified into specialized cells — and in cell migration. Disruption of the Wnt pathway in humans is known to be associated with limb deformities and also cancer. Wnt pathways are of two types — canonical pathways which involve the protein β-catenin, and non-canonical pathways that function without it.
Recent experiments using zebrafish have helped a team of scientists from the Tata Institute of Fundamental Research (TIFR), Mumbai and the Indian Institute for Science Education and Research (IISER), Pune to uncover the mechanism through which canonical Wnt signalling influences the formation of appendages. Zebrafish are ideal model organisms to study canonical Wnt signaling as they lay large clutches of eggs and have a short developmental cycle. In addition, they are well-studied organisms whose entire genome has already been sequenced.
“Our research shows that Wnt signaling controls the cell shape by regulating expression of laminins, which are extra-cellular matrix components essential for cell-to-matrix adhesion” says Mahendra Sonawane, Professor at TIFR Mumbai. The presence of a correlation between Wnt signalling and cell patterning was confirmed through a series of experiments. Under normal conditions, the epithelial cells extracted from the median fin of zebrafish embryos were elongated at the distal end (the side away from the body) and polygonal towards the proximal end (the side of the fin that is attached to the body). Active Wnt signalling was indicated by the presence of β-catenin in the nucleus of these cells. By manipulating levels of Wnt signalling, it was discovered that the intensity of β-catenin in cells increased along a gradient from the distal end towards the proximal end. The role of the Extra Cellular Matrix (ECM) in cell patterning was also determined. ECM provides a base for the cells to adhere and is known to regulate cell shapes. Using dyes to stain the epithelial cells of the median fin, the scientists observed a gradual decrease in deposition of laminins, major components of ECM, from the distal end towards the proximal end-opposite to the direction of Wnt signalling. Further experiments using embryos treated with morpholino, a laminin inhibitor, showed that the shape of the cells at the distal end had transformed from elongated to polygonal shape, thus indicating high deposition of laminins to beresponsible for elongation of cells at the distal end.
Once a correlation between canonical Wnt signalling, cell patterning and laminin deposition was established, scientists set about to test the link between canonical Wnt signalling and pattern of laminin deposits within the tissue. Gene transcripts of embryos treated to enhance and inhibit the function of β-catenins revealed an increase in the canonical Wnt signalling with a corresponding increase in laminin lama α5
levels (known to be associated with development of appendages). This decreased when Wnt signalling was reduced, thus confirming that canonical Wnt signalling controlled the patterns of formation of epithelial cells by regulating the levels of laminins. Wnt signalling is not restricted to appendages but is also observed to occur in various tissues in developing embryos — such as midbrain-hindbrain boundary, retina, somites, notochord- where active morphogenesis takes place. According to Sonawane, the results of this study not only provide a better understanding of the canonical Wnt signalling pathway, but also warrant further investigation to check if Wnt signaling controls laminin expression during cancer metastasis and whether this regulation can be subjected to therapeutic intervention.