A Sprouty4 reporter to monitor FGF/ERK signaling activity in ESCs and mice

Developmental biology

Vidur Garg^1,3¹  Claire S. Simon^1²  Dhruv Raina^4,1³  Sophie M. Morgani^1,2⁴  Nestor Saiz^1⁵ 

[1] Biochemistry, Cell and Molecular Biology Program, Weill Cornell Graduate School of Medical Sciences, New York, NY 10065, USA^3;Department of Genetics, University of Cambridge, Cambridge CB2 3EH, UK^5;Department of Systemic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany^4;Developmental Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA^1;Wellcome Trust-Medical Research Council Centre for Stem Cell Research, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK^2

关键词: Sprouty4;    FGF/ERK;    FGF signaling;    ESCs;    Mouse embryo;    Fluorescent reporter;    Live imaging;    Quantitative image analysis;   

DOI  :  10.1016/j.ydbio.2018.06.017

学科分类：生物科学（综合）

来源: Academic Press

【 摘 要 】

The FGF/ERK signaling pathway is highly conserved throughout evolution and plays fundamental roles during embryonic development and in adult organisms. While a plethora of expression data exists for ligands, receptors and pathway regulators, we know little about the spatial organization or dynamics of signaling in individual cells within populations. To this end we developed a transcriptional readout of FGF/ERK activity by targeting a histone H2B-linked Venus fluorophore to the endogenous locus of Spry4, an early pathway target, and generated Spry4H2B-Venus embryonic stem cells (ESCs) and a derivative mouse line. The Spry4H2B-Venus reporter was heterogeneously expressed within ESC cultures and responded to FGF/ERK signaling manipulation. In vivo, the Spry4H2B-Venus reporter recapitulated the expression pattern of Spry4 and localized to sites of known FGF/ERK activity including the inner cell mass of the pre-implantation embryo and the limb buds, somites and isthmus of the post-implantation embryo. Additionally, we observed highly localized reporter expression within adult organs. Genetic and chemical disruption of FGF/ERK signaling, in vivo in pre- and post-implantation embryos, abrogated Venus expression establishing the reporter as an accurate signaling readout. This tool will provide new insights into the dynamics of the FGF/ERK signaling pathway during mammalian development.

【 授权许可】

CC BY