Wnt signaling affects cell adhesion and neuronal differentiation

Abstract

The mammalian central nervous system (CNS) consists of the brain and the spinal cord. The entire CNS is derived from the walls of a fluid-filled neural tube that is formed at an early stage of embryonic development. The tube itself becomes the adult ventricular system. Thus, by examining how this tube changes during the course of fetal development, we can understand how the brain is organized and how the different parts fit together.

The Wnts (Wingless-Int) seem to have important, but as yet not clearly understood roles as determinants of cell proliferation, migration and differentiation during both early and late phases of CNS development. This thesis investigates how Wnt signalling affects the cytoskeleton, cell adhesion and neuronal differentiation in the developing brain.

We begun by studying transgenic mouse embryos that overexpress Wnt7a in the neural stem cells. To our surprise the embryos displayed a disturbed neurulation. The neural tube shape and rigidity was affected. We demonstrated that this was due to reduced levels, and impaired distribution, of actin microfilaments, β-catenin, and N-cadherin at the neural tube adherens junctions. Formation of the neural tube is mainly accomplished by changes in cell shape and cell-cell interactions of committed ectodermal cells. We also showed that expression of the Wnt PCP signalling gene Vangl2 (Van gogh-like 2) was increased. Wnt7a´s influence on neuronal differentiation was studied by investigation of β-tubulin III expression, a marker for early differentiated neurons. The pattern of β-tubulin III positive cells suggested a delay in neuronal development in Wnt7a overexpressing embryos.

Finally we investigated Vangl2 s effect on the cytoskeleton. For this purpose we studied Vangl2 transfected HEK293T, MDCK and C17.2 cell lines, transgenic mouse embryos that overexpressed Vangl2, and loop-tail Vangl2 loss-of-function embryos. We were able to show that the components of the adherens junctions i.e. actin, β-catenin and N-cadherin, were affected in a similar way as in the Wnt7a overexpressing embryos. The cell polarity and its effect on the actin cytoskeleton was determined by investigating the monomeric GTPases that are members of the Rho protein family: Rac1 and RhoA. Our results show that a balance between Rac1 and Vangl2 is essential for cell adhesion and mobility. Furthermore we could see that both a loss of function and a gain of function of Rac1/Vangl2 leads to changes in the actin cytoskeleton and disturbs adherens junctions.

Since Vangl2 and Wnt7a overexpressing mouse embryos displayed similar changes in the distribution of actin, N-cadherin, Scrb1 and β-catenin, we suggest in this thesis that Wnt7a can affect the cytoskeleton, cell adhesion and adherens junctions via Vangl2, Rac1 and P-JNK.