Optogenetic Interrogation of Crosstalk in Developmental Signaling

Abstract

Signaling systems coordinate cell fate patterning and morphogenetic movements to shape a developing embryo. Although only a handful of different signaling pathways exist, they induce thousands of different cell fates. Spatial patterns, signal strength and duration, and the combination of these elements enable a diversity of cellular responses. To induce specific responses, these features of signaling need to be very precise. Dysregulation in signaling results in the formation of various developmental disorders and cancers, presumably caused by blunders in establishing these combinations of signals. One such class of diseases, RASopathies, is associated with the ERK pathway. The MEK-ERK signaling system is a ubiquitously present pathway that signals via a conserved cascade of protein interactions. In a developing system, the MEK-ERK signaling pathway must occur at the right time and place. Otherwise, developmental disorders and cancer can occur.

Currently, there exists a lack of quantitative understanding of developmental ERK signaling which would help address questions such as when and where ERK is important. We can study perturbations to the ERK pathway with morphological readouts at the broadest level and transcriptional targets most directly affected by ERK activity. We choose to examine the effects of ERK signaling at an intermediate developmental step, crosstalk with the BMP-Smad signaling system, which may be regulated by downstream targets of ERK that evolve with time. The BMP-Smad signaling pathway is involved in patterning and morphogenesis of the embryo through the induction of ventralizing fates and is responsible for the formation of the dorsal-ventral axis.

Here, we report a systematic approach to making optogenetic perturbations to restrict ERK signaling in time and quantifying BMP signaling gradients in response to these perturbations using immunofluorescence staining. We determined that enabling ectopic MEK-ERK pathway activation with externally applied light resulted in downregulation of the BMP-Smad signaling pathway. In preliminary studies, we lay the foundations for the generation of transgenic lines of the optogenetic tool to perform these quantifications more robustly as well as to extend quantitative studies to other systems.