Selective activation of FZD2 and FZD7 reveals non-redundant function during mesoderm differentiation

Wnt-ßcatenin signalling controls stem cell self-renewal and cell fate decisions and therefore its precise control could influence differentiation protocols efficiency and cell therapy efforts. During gastrulation, Wnt-ßcatenin signalling dictates lineage bifurcation choices leading from pluripotency to distinct primitive streak patterning, ultimately resulting in various mesendoderm cell types. However, the nature of the Wnt receptors involved in this process and how their signaling dynamics impact mesoderm specification remain elusive. Here, we used selective Frizzled and LRP5/6 antibody-based agonists (FLAgs) to investigate the function and activation kinetics of Frizzled receptor complexes during directed mesoderm differentiation of hPSCs. We found that FZD2 and FZD7 are expressed at the surface of hPSCs and that their activation triggers ßcatenin signaling with different kinetics thereby influencing mesoderm patterning choices. Using bulk and single-cell RNA sequencing, we showed that FZD7 activation promotes lineage commitment towards both paraxial and lateral mesoderm whereas FZD2 engagement preferentially induces paraxial mesoderm. Mechanistically, our results demonstrate that although the short-term response following FZD2 and FZD7 activation is similar, ßcatenin signaling kinetics differs. FZD2 activation results in a sustained Wnt activation profile that drives hPSCs differentiation to paraxial mesoderm while blocking lateral mesoderm. In contrast, FZD7 activation kinetics consists of a similar initial activation followed by dampening of the response to a low level of ßcatenin signaling that is permissive for lateral mesoderm induction in addition to paraxial mesoderm specification. Our results suggest that the function of FZD2 and FZD7 during mesoderm specification is non-redundant and that their selective activation to temporally modulate Wnt-ßcatenin signaling can be leveraged for precise directed differentiation outcomes.