2. Academic Validation
  3. Assessment of the FRET-based Teen sensor to monitor ERK activation changes preceding morphological defects in a RASopathy zebrafish model and phenotypic rescue by MEK inhibitor

Assessment of the FRET-based Teen sensor to monitor ERK activation changes preceding morphological defects in a RASopathy zebrafish model and phenotypic rescue by MEK inhibitor

  • Mol Med. 2024 Apr 9;30(1):47. doi: 10.1186/s10020-024-00807-w.
Giulia Fasano 1,  Stefania Petrini 2,  Valeria Bonavolontà 1,  Graziamaria Paradisi 1 3,  Catia Pedalino 1,  Marco Tartaglia 4,  Antonella Lauri 5
Affiliations

Affiliations

  • 1 Molecular Genetics and Functional Genomics, Ospedale Pediatrico Bambino Gesù, IRCCS, Rome, 00146, Italy.
  • 2 Microscopy facility, Research laboratories, Ospedale Pediatrico Bambino Gesù, IRCCS, Rome, 00146, Italy.
  • 3 Department for Innovation in Biological Agro-food and Forest systems (DIBAF), University of Tuscia, Viterbo, 01100, Italy.
  • 4 Molecular Genetics and Functional Genomics, Ospedale Pediatrico Bambino Gesù, IRCCS, Rome, 00146, Italy. marco.tartaglia@opbg.net.
  • 5 Molecular Genetics and Functional Genomics, Ospedale Pediatrico Bambino Gesù, IRCCS, Rome, 00146, Italy. antonella.lauri@opbg.net.
PMID: 38594640 DOI: 10.1186/s10020-024-00807-w
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 occurs as somatic events in childhood leukemias.

Method: Here, we evaluated the application of a FRET-based zebrafish ERK reporter, Teen, and used 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 the modulation of ERK phosphorylation obtained by low-dose MEK Inhibitor treatment to early development, 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 monitor and quantify pharmacologically- and genetically-induced ERK activity modulations in early embryos, representing a useful tool in pre-clinical applications targeting RAS-MAPK signaling.

Keywords

ERK; FRET; RASopathies; Zebrafish embryos.

Figures
Products