early development

In addition to our work on multipotent neural crest stem cells in midface development development, we are studying signaling pathways that regulate five stem cell types that can be isolated from the early mouse embryo. These include Embryonic Stem (ES) Cells, Trophoblast Stem (TS) cells, Extraembryonic Endoderm (XEN) cells, Epiblast-Like Cells (EpiLCs) and Epiblast Stem Cells (EpiSCs). These stem cells have significantly different differentiation potentials, and their establishment and maintenance are governed by multiple factors, among which fibroblast growth factor (FGF) signaling plays a prominent role. In the mouse, 18 FGF ligands signal through 4 FGF receptors, of which only FGFR1 and FGFR2 have critical functions in early development. In the embryo, FGFR1 is known to be important for epiblast differentiation and during gastrulation, whereas FGFR2 orchestrates the development of extraembryonic structures required to pattern and support the growth of the embryo proper. In vitro studies have suggested that FGFR1 and FGFR2 exert their activity mainly through a relay mechanism involving FRS2 and FRS3 adapter proteins and subsequent ERK1/2 induction, with signaling through CRK, PLCγ, SHB and GRB14 playing more minor roles (Brewer et al. 2016). However knock-in mutations of the FRS2/3 binding sites at each receptor loci indicate that there are numerous FRS independent FGFR activities (e.g. Hoch and Soriano, 2006; Brewer et al. 2015), suggesting that multiple signaling pathways engaged by the FGFRs function cooperatively in vivo.

To determine the function of different FGF-induced signaling pathways in establishing stem cell identity, we  have established allelic series at the Fgfr1 and Fgfr2 loci, carrying point mutations that prevent binding of the FRS2, CRK, PLCγ, SHB and GRB14 effectors, alone or in combination. For Fgfr1, disruption of FRS2 binding to the receptor leads to the most pleiotropic phenotypes in development, but CRK proteins and PLCγ also contribute to ERK1/2 activation, affecting axis elongation and craniofacial and limb development. Disruption of all binding sites failed to recapitulate the Fgfr1 null mutant phenotype, suggesting that ERK1/2 independent pathways are functionally important in vivo (Brewer et al., 2015). On the 129S4 co-isogenic background on which all of these studies were performed, we have also identified a critical role for Fgfr1 in regulating development of the primitive endoderm (Brewer et al., 2015), suggesting that FGF signaling may play a critical role in XEN cell establishment and maintenance.

We have generated fluorescent reporter knock-in lines that demonstrate Fgfr1 expression in all cell populations of the blastocyst, while Fgfr2 expression becomes restricted to extraembryonic lineages, including the PrE. We further showed that loss of both receptors prevents the development of the PrE and demonstrate that FGFR1 plays a more prominent role in this process than FGFR2. Last, we demonstrated an essential role for FGFRs in embryonic stem cell (ESC) differentiation, with FGFR1 again having a greater influence than FGFR2 in ESC exit from the pluripotent state. Collectively, these results identify mechanisms through which FGF signaling regulates inner cell mass lineage restriction and cell commitment during preimplantation development (Molotkov et al., 2017). We are also using our allelic series of Fgfr1 and Fgfr2  signaling mutants to identify the signaling pathways that play critical roles in the establishment and maintenance of TS and XEN stem cells.