Endogenous FGFs drive ERK-dependent cell fate patterning in 2D human gastruloids.

The role of FGF is the least understood of the morphogens driving mammalian gastrulation. Here we investigated the function of FGF in a stem cell model for human gastrulation known as a 2D gastruloid. We found a ring of FGF-dependent ERK activity that closely follows the emergence of primitive streak (PS)-like cells but expands further inward. We showed that this ERK activity pattern is required for PS-like differentiation and that loss of PS-like cells upon FGF receptor inhibition can be rescued by directly activating ERK. We further demonstrated that the ERK-ring depends on localized activation of basolaterally positioned FGF receptors (FGFR) by endogenous FGF gradients. We confirmed and extended previous studies in analyzing expression of FGF pathway components, showing FGFR1 is the main receptor, FGF2 is highly expressed across several cell types, and FGF4/17 are the main FGF ligands expressed in the PS-like cells, similar to the human and monkey embryo but different from the mouse. We found that knockdown of FGF4 greatly reduced PS-like differentiation while FGF17 knockdown primarily affected subsequent mesoderm differentiation. FGF8 expression was spatially displaced from PS-markers and FGF4 expression and peaked earlier, while knockdown led to an expansion in PS-like cells, suggesting FGF8 may counteract FGF4 to limit PS-like differentiation. Thus, we have identified a previously unknown role for FGF-dependent ERK signaling in 2D gastruloids and possibly the human embryo, driven by a mechanism where FGF4 and FGF17 signal through basally localized FGFR1 to induce PS-like cells and their derivatives, potentially restricted by FGF8.