Engineering an FGF/ERK Transcriptional Reporter to Detect Drug-Inducible Ligand Secretion in Gastruloids

Abstract

Embryo morphogenesis and the mechanisms required to pattern an embryo through signaling are not fully understood. Recent 3D organoid systems replicate late-gastrulation mammalian development, permitting an investigation of the signaling requirements of embryogenesis while avoiding the experimental constraints of the post-implantation embryo. These structures, called gastruloids, self-organize from aggregates of mouse embryonic stem cells (mESCs) into 3D structures with well-defined anteroposterior axes, allowing us to investigate the role of distinct signaling pathways in driving patterning and morphogenesis. The canonical Ras/ERK signaling cascade is a prime example of such a developmental patterning system and is important in specifying positional information along the body axis, lying downstream of the FGF family of secreted signaling proteins. As such, having local (i.e., optogenetic) user-defined control over FGF secretion in embryonic organoids will be useful both to better understand the signaling requirements for these developmental processes and for engineering more complex organoid systems. Here, we lay the groundwork for optogenetic control of the FGF/ERK signaling pathway by developing a DUSP6 transcriptional reporter that faithfully reads out ERK activity in the gastruloid. We demonstrate that our DUSP6 reporter is sensitive to pharmacological activation and inhibition of ERK and define the minimum ERK threshold to induce reporter activation. We further demonstrate that the DUSP6 reporter is amenable to liveimaging, revealing the dynamics of ERK patterning at the posterior domain of the elongating gastruloid. Finally, we developed a dox-inducible FGF4 secretion platform, showing that (1) mESCs can be engineered to secrete signaling-active ligand, and (2) that our DUSP6 transcriptional reporter is of sufficient sensitivity to detect ligand secreted 6 from neighboring cells. Taken together, our data provides a foundation for future optogenetic ligand-secretion and biosensing experiments to better understand the signaling requirements for a wide array of developmental processes.