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  • br Introduction Fibroblast growth factors FGF are a


    Introduction Fibroblast growth factors (FGF) are a family of 18 secreted ligands that bind to four receptor tyrosine kinase (RTK) FGF receptors (FGFR1–4). The interaction of FGFs with their cognate receptors results in the activation of a number of downstream signaling pathways, including the MAPK cascade which involves ERK (Brewer et al., 2016, Ornitz and Itoh, 2015). FGF signaling through ERK (referred to hereafter as FGF/ERK signaling) is evolutionarily conserved from primitive metazoans to mammals (Itoh and Ornitz, 2011, Oulion et al., 2012) and plays a fundamental role in vital cellular processes encompassing proliferation, metabolism, migration, cell survival and differentiation. The FGF/ERK signaling axis is important throughout embryonic development, at post-natal stages for homeostatic regulation, and has been implicated in the progression of many diseases such as cancer and various neuropathies (Dorey and Amaya, 2010, Nies et al., 2015, Kelleher et al., 2013, Turner and Grose, 2010, Su et al., 2014, Tsai et al., 2013, Turner et al., 2016). During early mouse development, FGF/ERK signaling is required to generate and/or maintain the three lineages of the pre-implantation embryo – the embryonic epiblast (Epi), and the extraembryonic primitive endoderm (PrE) and trophectoderm (TE). FGF/ERK regulates maturation of the pluripotent Epi, PrE specification (Chazaud et al., 2006, Frankenberg et al., 2011, Kang et al., 2013, Kang et al., 2017, Nichols et al., 2009, Yamanaka et al., 2010, Krawchuk et al., 2013, Molotkov et al., 2017) and TE proliferation (Nichols et al., 2009, Nichols et al., 1998, Lu et al., 2008, Orr-Urtreger et al., 1993, Rossant and Cross, 2001). FGF is also essential for the isolation and maintenance of the in vitro counterpart of the post-implantation Epi, epiblast stem MDV 3100 (EpiSCs) (Brons et al., 2007, Tesar et al., 2007) and of the TE, trophoblast stem (TS) cells (Nichols et al., 1998, Tanaka et al., 1998). In both vertebrates and invertebrates (Bertrand et al., 2003, Ciruna and Rossant, 2001, Deng et al., 1994, Isaacs et al., 1994, Popovici et al., 2005, Stathopoulos et al., 2004, Sun et al., 1999, Yamaguchi et al., 1994, Matus et al., 2007), FGF signaling is required for efficient cell migration during gastrulation, when the three embryonic germ layers are specified (Deng et al., 1994, Sun et al., 1999, Yamaguchi et al., 1994, Ciruna et al., 1997). Later in development, FGF/ERK is employed in a variety of contexts including the regulation of somitogenesis, branching organogenesis (for example in the lung and kidney), as well as in limb, brain and tooth development (Ornitz and Itoh, 2015). Despite the critical and widespread roles of the FGF/ERK pathway, there is limited information on the pathway's spatiotemporal signaling dynamics and how they correlate with functional outputs. The static distribution of ERK activity has been analyzed via immunoreactivity against its active di-phosphorylated form (ppERK) (Corson et al., 2003). However, due to difficulties in capturing low-level or transient signaling activity, not all tissues with known FGF/ERK activity exhibit robust staining for ppERK, such as the inner cell mass (ICM) of the blastocyst and the primitive streak (PS) of the mouse gastrula (Corson et al., 2003). Hence, sensitive tools that capture dynamics are required to monitor signaling in fixed and live samples at cellular resolution. We found that, from a panel of known FGF/ERK pathway targets, Sprouty4 (Spry4) demonstrated the most rapid and robust transcriptional response to acute ERK activation in mouse embryonic stem cells (ESCs), the in vitro counterpart of the embryonic Epi. We therefore generated a fluorescent reporter ESC line and mouse line carrying an H2B-Venus fusion (Nowotschin et al., 2013) knocked into the mouse Spry4 locus (Minowada et al., 1999). While we noted that the Spry4 reporter disrupted transcription at the endogenous locus, Spry4 mice were viable and fertile. The Spry4 reporter was expressed in known domains of ERK activity including the ICM of the blastocyst, the nascent mesoderm, somites and limb buds. We also observed Venus signal within the visceral endoderm (VE), a previously uncharacterized site of ERK activity, and noted highly localized expression within distinct cell types of adult organs. We validated the specificity of this reporter by inhibiting FGF/ERK, both genetically and pharmacologically, in ESCs and embryos. Both approaches significantly abrogated expression of Spry4 confirming that the reporter represents a bona fide readout for FGF/ERK activity. This reporter represents the first tool to study the transcriptional output of FGF/ERK signaling at single-cell resolution in live mice, and should yield novel insights into pathway regulation during embryonic development and in tissue homeostasis. As Sprouty family members are general RTK pathway regulators whose expression can be controlled by factors other than FGF (Gross et al., 2001, Katoh and Katoh, 2006, Reich et al., 1999, Taniguchi et al., 2007), the reporter may also be utilized to study RTK signaling induced via a variety of ligands in different contexts.