br Materials and methods br
Materials and methods
Discussion A reporter of FGF signaling activity has long been sought to facilitate the quantitative real-time analysis of pathway activity at single-cell resolution. Here we report the generation of a Spry4 reporter allele in ESCs and mice that recapitulates known patterns of FGF/ERK signaling during development. The emerging roles of Sprouty genes in cancer and ESC biology (Felfly and Klein, 2013, Lee et al., 2016, Masoumi-Moghaddam et al., 2014) also make this a valuable tool to study the role of signal regulation by Spry4 in these processes. In vitro, the Spry4 signal was elevated in response to FGF ligands and was abrogated by small molecule inhibitors of MEK or in the absence of FGF stimulation (Fig. 2), validating the reporter as a read out FGF/ERK signaling activity. The rapid maturation time of the Venus fluorescent protein (Nagai et al., 2002) renders this a useful tool to study the onset of signaling. The half-life of Venus in ESCs was ~ 9 h (Fig. S1B,C) hence, in its current form, the reporter might be utilized as a short-term trace of signaling history. To visualize more rapid signaling dynamics, additional modifications may be introduced into the Venus construct, for example a destabilizing PEST sequence (Li et al., 1998, Vilas-Boas et al., 2011), or alternatively sensors directly reporting ERK activity could be utilized (Komatsu et al., 2011). The Spry4 reporter exhibited heterogeneity of expression within ESC cultures (Fig. 1D,E). This indicated that individual I-BET-762 may exhibit distinct temporal responses, have different sensitivities to FGF ligands or can activate distinct downstream targets. Alternatively, this could represent a spatially heterogeneous distribution of FGF ligands across the culture. On a population level, Venus expression increased and expression of the pluripotency-associated marker NANOG decreased in response to increasing doses of FGF, consistent with FGF/ERK inducing ESC differentiation (Kunath et al., 2007). However, we did not observe a negative correlation between Venus and NANOG levels within individual cells (Fig. S1E-G), hence the majority of the Spry4 signal observed within ESCs was associated with FGF/ERK activity below the threshold needed to trigger differentiation. FGF signaling plays a multitude of roles in development and disease. Although genetic mutations and inhibitor experiments have yielded valuable information regarding the functions of FGF signaling, many open questions remain such as when this signaling pathway is first active in development; which cells within a tissue or organism respond to FGF; if the response is synchronous across a population of cells; how signaling changes over time. A relatively simple system to address many of these questions is the pre-implantation mouse embryo. While there is evidence for a role of FGF signaling in the TE at pre-implantation stages (Lu et al., 2008, Nichols et al., 1998, Tanaka, 2006), its function is unclear (Sasaki, 2010) and we did not observe Spry4 fluorescence within the TE. Conversely, the role of FGF signaling in the ICM of the blastocyst, to regulate PrE specification and Epi maturation, has been well characterized (Chazaud et al., 2006, Frankenberg et al., 2011, Kang et al., 2013, Nichols et al., 2009, Yamanaka et al., 2010, Krawchuk et al., 2013, Ohnishi et al., 2014, Guo et al., 2010, Feldman et al., 1995). Here we observed heterogeneous Spry4 expression within the ICM (Fig. 3A-D), though there was no association of Venus fluorescence with a particular cell state (GATA6 and NANOG double positive – DP cells; GATA6+ PrE; NANOG+ Epi; GATA6 and NANOG double negative – DN cells). This suggests that all cells transduce the FGF4 signal, but that individual ICM cells of all lineages experience varying levels of FGF/ERK activity, and hence the Spry4 reporter represents a useful tool to probe the effect of signaling dynamics on early lineage specification. We additionally observed previously unreported sites of Spry4 expression, such as within cells of the VE of the early post-implantation embryo. Throughout early post-implantation development (E5.5–7.5) we observed Spry4 expression within the Epi and VE of the embryo (Fig. 6, Fig. 7, Fig. 8). Although in situ hybridization labeling of Spry4 mRNA does not display a clear signal in either of these cell types (Fig. S7A,B) (Minowada et al., 1999), the Epi and VE Venus signal was responsive to inhibition of signaling through FGFR and/or ERK and therefore does not represent reporter perdurance. FGF pathway components and targets are expressed within the Epi and VE, including Fgf4 (Niswander and Martin, 1992, Sakaki-Yumoto et al., 2006, Wright et al., 2003), Fgf5 (Haub and Goldfarb, 1991; Hebert et al., 1991), Fgf8 (Crossley and Martin, 1995), Ets2 (Donnison et al., 2015), Eras (Zhao et al., 2015) and Etv5 (Chotteau-Lelievre et al., 2001). Furthermore, although FGF/ERK activity has not been described within the VE, abnormal development of the VE and its derivatives has been noted in mouse embryos and in vitro cell lines with mutations in the FGF pathway receptor Fgfr1 (Deng et al., 1994, Yamaguchi et al., 1994, Esner et al., 2002). Hence, FGF/ERK signaling may play a role in the VE that has yet to be fully elucidated. Spry4 expression within the VE may also be stimulated by non-FGF ligands, such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF) or WNT, which can also regulate the expression of Sprouty family members (Gross et al., 2001, Katoh and Katoh, 2006, Reich et al., 1999, Taniguchi et al., 2007, Winn et al., 2005). Nevertheless, as the Spry4 construct contains a NEO selection cassette, which could affect expression (Pan et al., 2016, Scarff et al., 2003), further transcriptional validation of novel Spry4 domains identified by this reporter may be required or excision of the selection cassette using a Dre recombinase mouse line should be performed (Anastassiadis et al., 2009).