An improved Erk biosensor reveals oscillatory Erk dynamics driven by mitotic erasure during early development

Abstract

Erk signaling dynamics elicit distinct cellular responses in a variety of contexts. The early zebrafish embryo is an ideal model to explore the role of Erk signaling dynamics in vivo, as a gradient of activated diphosphorylated Erk (P-Erk) is induced by Fgf signaling at the blastula embryonic margin. Here we describe an improved Erk-specific biosensor which we term modified Erk Kinase Translocation Reporter (modErk-KTR). We demonstrate the utility of this biosensor in vitro and in developing zebrafish and Drosophila embryos. Moreover, we show that Fgf/Erk signaling is dynamic and coupled to tissue growth during both early zebrafish and Drosophila development. Signaling is rapidly extinguished just prior to mitosis, which we refer to as mitotic erasure, inducing periods of inactivity, thus providing a source of heterogeneity in an asynchronously dividing tissue. Our modified reporter and transgenic lines represent an important resource for interrogating the role of Erk signaling dynamics in vivo.

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

“…Of note, the morphological rescue obtained is matched by a moderate reduction in ERK activity registered in late (9-11/12hpf) and very early (5.30/6hpf) stages preceding the morphological rescue as observed respectively via live-and AB-FRET in Teen embryos and confirmed by pERK immunoblots on whole-embroys extracts. Together with additional evidence in other mice and fish RASopathies models, including a RAF and SOS1-dependent NS mice models (Pagani et al, 2009;Chen et al, 2010;Holter et al, 2019), BRAF-dependent CFC and NS fish models (Anastasaki et al, 2009(Anastasaki et al, , 2012Bonetti et al, 2014) as well as a NS model in Drosophila (Oishi et al, 2006), and recent developments in FRET-based in vivo applications (Sari et al, 2018;Patel et al, 2019;Wong et al, 2019;Wilcockson et al, 2022), our data support the usefulness of animal models as well as reporter systems to advance pre-clinical applications in the field of RASopathies.…”

Low-frequency ERK and Akt activity dynamics are predictive of stochastic cell division events

“…It is appreciated that M-phase CDK activity (CDK1/CyclinB) can induce ERK-KTR responses [84][85][86] . We do not expect this non-specific behavior to impact our findings because M-phase is a relatively short time period in the cell cycle.…”

Zbtb16 mediates a switch between Fgf signalling regimes in the developing hindbrain

“…RNAscope ISH ( Wang et al, 2012a ) was carried out using the RNAscope Multiplex Fluorescent v2 system as described previously ( Wilcockson et al, 2022 preprint). The fgf3 probes (850161-C4, ACDBio; gift from C. Hill, The Francis Crick Institute, UK) were detected by incubating the embryos with Multiplex FL V2 HRP-C4 for 15 min at 40°C, followed by incubation with tyramide (Sigma-Aldrich, T2879) coupled to fluorescein-NHS ester (Thermo Fisher Scientific, 46410) for 25 min in the dark at room temperature.…”