Characterization of Overactive Mutations in the Ras/MAPK Pathway During Drosophila Development

Abstract

RASopathies are a large class of developmental disorders caused by gain-of-function germline mutations in the Ras/MAPK pathway. In this study, we examine the effects of RASopathy mutations in MAPK/ERK kinase (MEK) on Torso signaling in Drosophila melanogaster. Using a quantitative immunofluorescence assay, we found that embryos with overactivating MEK mutations exhibit context-dependent, divergent effects on signaling. In the center of the embryo, the pathway showed ectopic activation, consistent with in vitro analysis of the mutation. However, at the anterior and posterior poles, where the pathway is endogenously activated in the wild-type embryo, the MEK mutants exhibited an attenuated response to upstream signal. These opposing effects were reflected in the downstream phenotype, as MEK mutant embryos exhibited both gain-of-function phenotypes such as anterior-posterior patterning defects and loss-of-function phenotypes such as loss of anterior structures. Furthermore, results from a RNA sequencing screen on the mutant embryos showed that transcription of a negative regulator of the Ras/MAPK pathway, sprouty, was upregulated ~100-fold in the MEK mutant background. This implies that the upregulation of sprouty could be a part of the pathway’s negative feedback response to the RASopathy mutation, and is thus a major candidate for the source of the divergent effects we see on Ras/MAPK signaling in such mutant backgrounds. Thus, we demonstrate that the disease phenotypes of RASopathies are not simply caused by hyperactivation of the pathway, as previously thought, but also by attenuation of endogenous Ras/MAPK signal.