MAPK pathway: a role in development, disease and behaviour

Abstract

Mutations in the RAS-RAF-MEK-ERK (MAPK) pathway give rise to a range of developmental disorders collectively referred to as the RASopathies. De novo germline mutations in patients suffering from these syndromes promote similar phenotypes, which include heart abnormalities, characteristic facial features, cutaneous malformations, gastrointestinal malfunctions, failure to thrive and a spectrum of mental retardation. Although many RASopathies patients show a propensity to develop early-onset benign and malignant tumours, Cardio-faciocutaneous (CFC) syndrome patients do not seem to share this predisposition, with the exception of an increased number of naevi. CFC syndrome is caused by mutations in BRAF, MEK1 or MEK2, with the majority of patients harbouring BRAF mutations. Intriguingly, both kinase-activating and kinase-impaired mutations have been identified in CFC patients. Here, I use the zebrafish system to address the activity of the CFC syndrome alleles and the MAPK pathway in a developmental context and test the potential of small molecule inhibitors to restore normal development. I established an assay for the activity of CFC, melanoma and engineered BRAF and MEK human mutated alleles in vivo. Using zebrafish as an animal model organism, a panel of 31 mutant and wild-type BRAF, MEK1 and MEK2 alleles were expressed in early zebrafish embryos to assess their role in development. Irrespective of the predicted kinase activity, all embryos expressing BRAF and MEK mutant alleles reproducibly manifested the cell movement phenotype during gastrulation. Consistent with aberrant fibroblast growth factor (FGF) signalling and defective gastrulation, in situ hybridisation against convergence-extension markers showed misregulated convergence-extension movement patterns in CFC zebrafish embryos. Finally, I performed whole embryo RNA expression microarrays to identify genes regulated downstream of the CFC mutations, and I discuss the potential for a possible link to some of the phenotypes associated with a CFC zebrafish model. I established that the CFC, BRAF and MEK mutant embryos are sensitive to inhibition of MEK signalling by small molecules. Importantly, a time-window of treatment was identified which was sufficient to restore normal gastrulation movements and to prevent the developmental side effects promoted by the inhibitors at later stages of development. In order to begin considering the therapeutic potential of small molecules in developmental disorders (at least in our model system), the effect of low concentrations of the inhibitors in the normal formation of diverse tissues was thoroughly examined during zebrafish development. From these studies, I identified a concentration of MEK inhibitor that could be administered in a continuous fashion to prevent CFC-associated cell movement defects during gastrulation, without additional later developmental defects. Finally, I addressed the role of MEK-ERK signalling in a specific behavioural phenotype in zebrafish. Many RASopathies patients suffer from mental retardation and experience learning and attention difficulties. Research in our laboratory has identified a novel zebrafish behaviour induced by enhanced cAMP signalling, where the zebrafish seek shaded areas in their environment and exhibit frequent defensive shoaling behaviour. I used western blotting to establish that enhanced cAMP signalling activates the MAPK signalling pathway and, in collaboration with members our laboratory, that this phenotype can be suppressed by administration of the PD325901 MEK inhibitor. While we do not yet know the effect of CFC syndrome mutations on this behaviour, we suggest that altered MEK-ERK signalling may underlie important features of vertebrate behaviour.