Analyzing pathogenic MEK variants in zebrafish

Abstract

Somatic gain-of-function (GOF) mutations in components of the Ras/extracellular signal-regulated kinase (ERK) signaling pathway have been known for some time to cause cancer. Only recently have germline GOF mutations in the same components of the Ras/ERK pathway been shown to cause a common class of developmental disorders, known as RASopathies. Advances in genome sequencing have led to the identification of hundreds of such pathogenic mutations, but so far, the effects of a small subset of these mutations have been investigated using animal models. In Chapter 1, we review the animal models of RASopathies and find that they recapitulate many of the developmental abnormalities found in humans. However, it is not known whether individual mutations translate into different patient-specific phenotypic severity and causative abnormal Ras/ERK signaling dynamics. In the rest of this thesis, we focus on mutations in MEK1, an ERK kinase. In Chapter 2, we report that the aspect ratio of the yolk shape of early zebrafish embryos can be used as a metric to efficiently rank these mutations. Furthermore, the ranking is conserved in other zebrafish-specific and Drosophila-specific assays and is predictive of drug dose needed to reverse the aspect ratio abnormality. These assays can be used to test the strengths of existing and newly found mutations in MEK1 and other pathway components, providing the first step in the development of rational guidelines for patient-specific diagnostics and treatment of RASopathies. In Chapter 3, using spatially-resolved immunofluorescence experiments on embryos mosaic for the GOF MEK1 variants, we find that the MEK1 variants cause both higher and lower levels of Ras/ERK signaling in different parts of the embryo. We also find that some GOF MEK1 variants only have activity in the presence of upstream Ras signaling in zebrafish. Our results suggest that quantitative assays for certain Ras/ERK-dependent developmental processes in the early zebrafish can yield insights into how pathogenic MEK1 mutations work. In Chapter 4, we discuss conclusions and future directions, including the importance of analyzing how these mutations affect the Michaelis-Menten parameters of MEK1 and to generate zebrafish lines heterozygous for RASopathy mutations to analyze the severity of adult phenotypes.