Abstract
The secreted glycoprotein sonic hedgehog (Shh) is expressed in the prechordal mesoderm, where it plays a crucial role in induction and patterning of the ventral forebrain. Currently little is known about how Shh is regulated in prechordal tissue. Here we show that in the embryonic chick, Shh is expressed transiently in prechordal mesoderm, and is governed by unprocessed 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. Further, Shh expression can be maintained by a recombinant Nodal cleavage mutant, proNodal, but not by purified mature Nodal. A number of lines of evidence suggest that proNodal acts via FGFR3. ProNodal and FGFR3 co-immunoprecipitate and proNodal increases FGFR3 tyrosine phosphorylation. In microcultures, soluble FGFR3 abolishes Shh without affecting Gsc expression. Further, prechordal mesoderm cells in which Fgfr3 expression is reduced by Fgfr3 siRNA fail to bind to proNodal. Finally, 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. Our studies suggest that proNodal/FGFR3 signalling governs Shh duration by repressing canonical BMP signalling, and that local BMPs rapidly silence Shh once endogenous Nodal-FGFR3 signalling is downregulated.
Highlights
Nodal signals via canonical and non-canonical routes in prechordal mesoderm The transcription factor Gsc is a well-characterised marker of chick prechordal mesoderm, where it is expressed from gastrula stages (Izpisúa-Belmonte et al, 1993)
This, and the correlation in expression patterns that we detect in the chick, led us to ask whether Nodal signalling governs the temporal duration of both Gsc and sonic hedgehog (Shh) expression within the chick prechordal mesoderm
Shh is required through stages at which the prechordal mesoderm induces RDVM cells, and initiates patterning of the telencephalon, pre-optic regions and anterior/tuberal hypothalamus (Dale et al, 1999; Pearson and Placzek, 2013; Seifert et al, 1993) before being downregulated
Summary
In the developing vertebrate embryo, ventral midline cells of the neural tube are underlain by a cellular rod of axial mesoderm, composed of prechordal mesoderm beneath the prospective. Studies of isolated tissue explants, in which the role of prechordal mesoderm-derived Shh can be tested, reveal that Shh from the prechordal mesoderm is required to induce RDVM-like cells (Dale et al, 1997; Patten et al, 2003) and pattern ventral forebrain-like cells (Ohyama et al, 2005)
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