A Novel System for Monitoring in vivo Cell Signaling Pathways Involved in Early Embryonic Patterning
Early developmental events, such as the arrangement of the head-tail axis, are fundamentally driven by cell signalling cascades. Such incidents are regulated in a highly complex manner by promoters and inhibitors at many levels of the cascade. This complexity makes it difficult to understand where and when certain signalling occurs, and what effects additional factors have on the signalling system. Nodal signalling, executed by intracellular Smad2/3 signal propagation, is thought to induce the anterior-posterior and head-tail patterning of the early mouse embryo. Target gene outputs of this signalling are fine-tuned by a vast array of modulators; TGBβ co-receptors, extracellular ligand and receptor inhibitors, DNA binding cofactors, and intracellular enhancers and inhibitors. The endogenous target genes of this system cannot be used as a measure of signalling as they themselves feedback on the original system and others, creating diverse signals. In this body of work, we have distilled the Nodal signalling cascade to a single variable by creating a fluorescent genetic reporter to semi-quantitatively measure Smad signalling during early embryonic development. Reporter constructs contain Smad binding elements, a minimal promoter and fluorescent protein elements. Various sensitivity Smad binding elements were created to respond to different thresholds of signalling. Fluorescent microscopy and flow cytometry were used to verify responsiveness of reporter constructs, tested first in a mouse embryonic fibroblast line and subsequently in transgenic embryos. This study will provide an understanding of how extracellular cues dictate gene expression during early embryonic formation. The knowledge acquired from this work may have implications in dairy cattle and human fertility.