Wnt signaling in early brain development and disease

Sammanfattning: Wnt/Planar Cell Polarity (PCP) signaling is involved in many cellular processes throughout the development of the embryo. These include control of proliferation, differentiation, migration and cell fate decisions. Deregulation of Wnt/PCP signaling can lead to many developmental abnormalities and cancer. This thesis investigates the effects of altered Wnt/ PCP signaling during neurulation in mouse embryos, its importance in cell-cell adhesion and its role in pediatric cancer. The developing central nervous system (CNS) comprises different patterning centers consisting of groups of cells with organizer-like properties. These produce signals that influence the fate, histogenic organisation and growth of adjacent tissues, resulting in spatial patterning. Members of the Wnt gene family are among few secreted proteins expressed in patterning centers of the developing CNS, such as the pallial-subpallial boundary (PSB) and the cortical hem. This suggests that they might have a role in demarcating and specifying regions in the developing brain. We decided to investigate the influence of Wnt/PCP signaling on forebrain development and regionalization. The method we chose was transgenic overexpression of Wnt7a, Wnt7b and later the PCP signaling component Vangl2, in neural progenitor cells. To our surprise we found drastic changes in early embryonic stages with a decreased body size, smaller forebrain structures and even neurulation defects starting from the midbrain region and further rostral. Analyses of Wnt7a transgenic embryos (paper I) showed that the neural tube adherens junctions, i.e. specialized tight junctions, were affected. This was manifested by decreased expression and impaired distribution of actin microfilaments, N-Cadherin and beta-catenin – the latter both a component of the cytoskeleton and a mediator of canonical Wnt signaling. Since PCP signaling acts on actin and the cytoskeleton, we investigated the downstream signaling components of this pathway and found an increased expression of Vangl2 and a misdistribution of Scribble1. In our following study (paper IV) we confirmed and further described the neurulation defects, the deregulation of adherens junctions and the altered distribution of cytoskeletal components, by analyzing transgenic mouse embryos overexpressing Vangl2 and Vangl2 mutant (loop-tail) mouse embryos. We could demonstrate that Vangl2 targets the Rho family small G-protein Rac1 to sites of actin polymerization at the cell’s adherens junctions. It is known that Wnt signaling can affect the proliferation and differentiation of neural progenitor cells, and that beta-catenin signaling affects target genes that control these cellular processes. Analyses of the neuronal population of transgenic Wnt7a and Wnt7b embryos showed decreased numbers of newly born neurons (beta-tubulin III+ cells) and an aberrant positioning along their migrational axis (paper II), which we concluded to be due to a delay in neuronal differentiation. This mechanism was corroborated and further investigated in Wnt7b transgenic embryos, which also displayed a decreased proportion of proneural transcription factors Tbr1 and Tbr2. However, the proportion of pax6 expression was similar to the wild type, suggesting an unaffected neural progenitor cell pool (paper III). Finally, we set out to investigate the influence of Wnt/PCP signaling on pediatric tumor cell characteristics and viability (paper V). For long, the PCP signaling pathway has been considered to counteract parts of the canonical Wnt signaling pathway, acting in part as a tumor suppressor. In our study we characterized several medulloblastoma and neuroblastoma cell lines for their native expression of Wnt/PCP components. We found that an increase in PCP protein expression correlated with decreased levels of phosphorylated beta-catenin. In neuroblastoma cells, PCP gene knockdown increased, while PCP gene over-expression decreased tumor cell viability in an in vitro MTT assay. These results correlated well with clinical data and survival estimates from open access databases, including gene array data from several neuroblastoma patients. In conclusion, the results presented in this thesis increase our knowledge on Wnt/PCP signaling and how it can affect the correct closure of the neural tube. Also, we discuss its influence on neural stem/progenitor cell behavior and differentiation, where imbalances on Wnt/PCP components have great implications on the characteristics and viability of pediatric tumors.