(287e) Spatiotemporal Analysis Reveals Divergent Effects of Activating Mutations on Developmental Signaling

According to the Centers for Disease Control (CDC), approximately one in every six children in the U.S. is born with a developmental disability. A large class of such abnormalities, believed to affect ~1/1000 human births, results from activating mutations in components of the highly conserved RAS/ERK signaling pathway. Known as RASopathies, these disorders are characterized by a broad spectrum of phenotypes, including craniofacial malformations, heart defects, and neurocognitive delay. While many such defects have been successfully mimicked by introducing disease-causing mutations into model organisms, much less is understood about effects of these mutations on developmental dynamics and the emergence of the observed phenotypes. To explore these effects, we used a combination of imaging and genetic experiments with multiple activating mutations introduced into the early Drosophila embryo, a powerful model for studies of the RAS/ERK signaling with high spatiotemporal resolution. We focused on a panel of activating mutations in MEK, an ERK kinase, and analyzed their effects on the first wave of ERK activation in the blastoderm embryo. Our results revealed that the effects of activating mutations are highly dependent on cell context: they cause ectopic ERK activation in cells that do not receive endogenous signals and reduce ERK activation in stimulated cells. Importantly, the observed reduction of ERK activation leads to a pronounced effect on downstream gene expression and causes loss-of-function morphological phenotypes. Our results can be compactly summarized using a testable mathematical model, whereby the reduced ERK activation is a result of a negative feedback loop induced by premature pathway activation. In addition to describing the data, the model makes several predictions, all of which are supported by the results of additional genetic, imaging, and biochemical experiments. Furthermore, the divergent effects on activating mutations on ERK signaling are conserved in zebrafish, suggesting the generality of the proposed model, in which mutations that are activating at the level of the molecule can cause loss-of-function phenotypes at the systems level. Our results provide a first step in the quantitative understanding of large class of developmental abnormalities and present rational guidelines for thinking about their origins and potential treatment.