title: ProNodal acts via FGFR3 to govern duration of Shh expression in the prechordal mesoderm.
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.
authors.affiliation: The Bateson Centre and Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
authors.forename: Pamela S
authors.initials: PS
authors.lastname: Ellis
authors.affiliation: The Bateson Centre and Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
authors.forename: Sarah
authors.initials: S
authors.lastname: Burbridge
authors.affiliation: The Bateson Centre and Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Soubes
authors.affiliation: The Bateson Centre and Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
authors.forename: Kyoji
authors.initials: K
authors.lastname: Ohyama
authors.affiliation: ISREC, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Epalinges CH 1066, Switzerland.
authors.forename: Nadav
authors.initials: N
authors.lastname: Ben-Haim
authors.affiliation: Departments of Medicine and Genetics & Development, Columbia University Medical Center, Herbert Irving Comprehensive Cancer Center, 1130 St. Nicholas Avenue, New York, NY 10032, USA Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673, Singapore.
authors.forename: Canhe
authors.initials: C
authors.lastname: Chen
authors.affiliation: College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.
authors.forename: Kim
authors.initials: K
authors.lastname: Dale
authors.affiliation: Departments of Medicine and Genetics & Development, Columbia University Medical Center, Herbert Irving Comprehensive Cancer Center, 1130 St. Nicholas Avenue, New York, NY 10032, USA.
authors.forename: Michael M
authors.initials: MM
authors.lastname: Shen
authors.affiliation: ISREC, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Epalinges CH 1066, Switzerland.
authors.forename: Daniel
authors.initials: D
authors.lastname: Constam
authors.affiliation: The Bateson Centre and Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield S10 2TN, UK m.placzek@sheffield.ac.uk.
authors.forename: Marysia
authors.initials: M
authors.lastname: Placzek
mesh_terms: D000818:Animals; D002642:Chick Embryo; D018274:Electroporation; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D053823:Hedgehog Proteins / Q000378:metabolism*; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D017403:In Situ Hybridization; D008648:Mesoderm / Q000196:embryology* / Q000378:metabolism; D055457:Nodal Protein / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016548:Prosencephalon / Q000196:embryology*; D034741:RNA, Small Interfering / Q000235:genetics; D051498:Receptor, Fibroblast Growth Factor, Type 3 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D051785:Smad Proteins / Q000378:metabolism
chemical_list: D053823:Hedgehog Proteins; D055457:Nodal Protein; D034741:RNA, Small Interfering; D051785:Smad Proteins; D051498:Receptor, Fibroblast Growth Factor, Type 3
keywords: BMP; Forebrain ventral midline; Nodal; Prechordal mesoderm; Sonic hedgehog