ProNodal acts via FGFR3 to govern duration of Shh expression in the prechordal mesoderm

The secreted glycoprotein Sonic hedgehog (Shh) is expressed in the prechordal mesoderm, where it plays a critical role in induction and patterning of the ventral forebrain. As yet, little is known about how Shh expression and duration are regulated there. Here we show that in the embryonic chick, Shh is expressed transiently in prechordal mesoderm, closely correlating with expression of Nodal. Exposure of prechordal mesoderm microcultures to Nodal conditioned-medium, the Nodal inhibitor CerS, or to an ALK4/5/7 inhibitor reveals that Nodal is required to maintain both Shh and Gsc expression, but whereas Gsc is largely maintained through canonical signalling, Nodal signals through a non-canonical route to maintain Shh expression. Further, Shh expression appears to be governed by unprocessed Nodal, since a recombinant Nodal cleavage mutant, proNodal, but not purified mature Nodal, maintains its expression. proNodal appears to act via FGFR3: FgfR3 is expressed in prechordal mesoderm and prechordal mesoderm cells in which FgfR3 expression is reduced by FgfR3 siRNA fail to bind to proNodal. Further, proNodal and FGFR3 co-immunoprecipitate and proNodal increases FGFR3 tyrosine phosphorylation. In microcultures, soluble FGFR3 abolishes Shh without affecting Gsc expression. Similarly, targeted electroporation of FgfR3 siRNA to prechordal mesoderm in vivo results in premature Shh downregulation without affecting Gsc. We report an inverse correlation between proNodal/FGFR3 signalling and pSmad1//5/8 and show that proNodal/FGFR3 signalling antagonises BMP-mediated pSmad1/5/8 signalling, which is poised to downregulate Shh. Together, our studies show that the essential role of Nodal in prechordal mesoderm development does not operate through one simple mechanism. Our studies suggest that proNodal/FGFR3 signalling governs Shh duration by repressing canonical BMP signalling, and are consistent with a model in which local BMPs are poised to rapidly silence Shh once endogenous Nodal/FGFR3 signalling is downregulated