FRET imaging of Teen sensor captures ERK activation changes preceding morphological defects in a RASopathy zebrafish model and phenotypic rescue by MEK inhibitor

Abstract

Background RASopathies are genetic syndromes affecting development and having variable cancer predisposition. These disorders are clinically related, and are caused by germline mutations affecting key players and regulators of the RAS-MAPK signaling pathway generally leading to an upregulated ERK activity. Gain-of-function (GOF) mutations in PTPN11, encoding SHP2, a cytosolic protein tyrosine phosphatase positively controlling RAS function, underlie approximately 50% of Noonan syndromes (NS), the most common RASopathy. A different class of these activating mutations occur as somatic events in childhood leukemias. Methods Here, we evaluated the application of a FRET-based zebrafish ERK reporter, Teen, and use of fast and quantitative FRET protocols to monitor non-physiological RASopathy-associated changes in ERK activation. In a multi-level experimental workflow, we tested the suitability of the Teen reporter to detect pan-embryo ERK activity correlates of morphometric alterations driven by the NS-causing Shp2D61G allele. Results Spectral unmixing- and acceptor photobleaching (AB)-FRET analyses captured pathological ERK activity preceding the manifestation of quantifiable body axes defects, a morphological pillar used to test the strength of SHP2 GoF mutations. Last, the work shows that by multi-modal FRET analysis we can quantitatively trace back to early development the modulation of ERK phosphorylation obtained by low-dose MEK inhibitor treatment, occurring before the onset of morphological defects. Conclusion This work proves the usefulness of FRET imaging protocols on both live and fixed Teen ERK reporter fish to readily monitoring and quantifying pharmacologically- and genetically-induced ERK activity modulations in early embryos, representing a useful tool in pre-clinical applications targeting RAS-MAPK signaling.