The Investigation of the Effects of RASopathy MEK1 Mutations on Zebrafish Embryonic Morphology and Heart Development

Abstract

The RASopathies are a set of developmental disorders known to be caused by mutations in components of the Ras/mitogen-activated protein kinase (MAPK) pathway, which plays a critical role in development and growth. Cardio-facio-cutaneous (CFC) syndrome is a RASopathy that is caused by activating mutations in the kinase MEK1 and has been associated with congenital heart defects. Zebrafish are an ideal model organism to further understand the effects of CFC on the human heart due to their transparency during cardiogenesis. Since mutants of MEK1 have been reported to demonstrate an oblong phenotype due to increased convergence and extension, the readily-measurable ratio of the major to minor axis was used as a semi-quantitative screen for activity. To do this, we injected mutant MEK1 mRNA into embryos and quantified the ratio at 12 hours post fertilization. It was concluded that the RASopathy variants of MEK1 had intermediate ratios when compared with the negative control of wild type MEK1 and the positive control of constitutively active MEK1. This overexpression technique was also used to assess the effects of these mutations on left/right (L/R) patterning with respect to heart jogging. While previous literature has reported that increased activity of RASopathy mutations has a correlation to the disruption of the directionality of normal heart jogging that indicates a malformation with the L/R patterning component Kupffer’s Vesicle (KV), we have gathered data that suggests that the malformations of KV that affect the L/R patterning of heart jogging are possibly due to the overexpression of the Ras/MAPK proteins, rather than over activation of the pathway. However, in order to verify this further, it is necessary to generate a transgenic line of zebrafish with a CFC mutation in its mek1 locus. To this end, we attempt to utilize homologous recombination induced Abstract 7 with CRISPR/Cas9 genome editing to even better understand the developmental effects of these mutations on heart morphogenesis. Heart development and formation will be able to be further characterized in these mutant zebrafish. Gaining a greater understanding of how the heart is affected in zebrafish can provide insight into how RASopathy mutations are affecting patients with congenital heart defects. This can potentially aid development of treatments for individuals with a RASopathy.