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

Pamela S. Ellis, Sarah Burbridge, Sandrine Soubes, Kyoji Ohyama, Nadav Ben-Haim, Canhe Chen, Kim Dale, Michael M. Shen, Daniel Constam, Marysia Placzek (Lead / Corresponding author)

Research output: Contribution to journalArticle

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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 BMPmediated 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.

Original languageEnglish
Pages (from-to)3821-3832
Number of pages12
JournalDevelopment
Volume142
Issue number22
Early online date28 Sep 2015
DOIs
Publication statusPublished - 15 Nov 2015

Fingerprint

Mesoderm
Down-Regulation
Small Interfering RNA
Electroporation
Conditioned Culture Medium
Prosencephalon
Tyrosine
Glycoproteins
Phosphorylation

Keywords

  • BMP
  • Forebrain ventral midline
  • Nodal
  • Prechordal mesoderm
  • Sonic hedgehog

Cite this

Ellis, P. S., Burbridge, S., Soubes, S., Ohyama, K., Ben-Haim, N., Chen, C., ... Placzek, M. (2015). Pronodal acts via FGFR3 to govern duration of Shh expression in the prechordal mesoderm. Development, 142(22), 3821-3832. https://doi.org/10.1242/dev.119628
Ellis, Pamela S. ; Burbridge, Sarah ; Soubes, Sandrine ; Ohyama, Kyoji ; Ben-Haim, Nadav ; Chen, Canhe ; Dale, Kim ; Shen, Michael M. ; Constam, Daniel ; Placzek, Marysia. / Pronodal acts via FGFR3 to govern duration of Shh expression in the prechordal mesoderm. In: Development. 2015 ; Vol. 142, No. 22. pp. 3821-3832.
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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 BMPmediated 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.",
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Ellis, PS, Burbridge, S, Soubes, S, Ohyama, K, Ben-Haim, N, Chen, C, Dale, K, Shen, MM, Constam, D & Placzek, M 2015, 'Pronodal acts via FGFR3 to govern duration of Shh expression in the prechordal mesoderm', Development, vol. 142, no. 22, pp. 3821-3832. https://doi.org/10.1242/dev.119628

Pronodal acts via FGFR3 to govern duration of Shh expression in the prechordal mesoderm. / Ellis, Pamela S.; Burbridge, Sarah; Soubes, Sandrine; Ohyama, Kyoji; Ben-Haim, Nadav; Chen, Canhe; Dale, Kim; Shen, Michael M.; Constam, Daniel; Placzek, Marysia (Lead / Corresponding author).

In: Development, Vol. 142, No. 22, 15.11.2015, p. 3821-3832.

Research output: Contribution to journalArticle

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AU - Ellis, Pamela S.

AU - Burbridge, Sarah

AU - Soubes, Sandrine

AU - Ohyama, Kyoji

AU - Ben-Haim, Nadav

AU - Chen, Canhe

AU - Dale, Kim

AU - Shen, Michael M.

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Ellis PS, Burbridge S, Soubes S, Ohyama K, Ben-Haim N, Chen C et al. Pronodal acts via FGFR3 to govern duration of Shh expression in the prechordal mesoderm. Development. 2015 Nov 15;142(22):3821-3832. https://doi.org/10.1242/dev.119628