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MicroRNA Mirn140 modulates Pdgf signaling during palatogenesis

Nature Genetics volume 40, pages 290–298 (2008)Cite this article

Abstract

Disruption of signaling pathways such as those mediated by sonic hedgehog (Shh) or platelet-derived growth factor (Pdgf) causes craniofacial abnormalities, including cleft palate. The role that microRNAs play in modulating palatogenesis, however, is completely unknown. We show that, in zebrafish, the microRNA Mirn140 negatively regulates Pdgf signaling during palatal development, and we provide a mechanism for how disruption of Pdgf signaling causes palatal clefting. The pdgf receptor alpha (pdgfra) 3′ UTR contained a Mirn140 binding site functioning in the negative regulation of Pdgfra protein levels in vivo. pdgfra mutants and Mirn140-injected embryos shared a range of facial defects, including clefting of the crest-derived cartilages that develop in the roof of the larval mouth. Concomitantly, the oral ectoderm beneath where these cartilages develop lost pitx2 and shha expression. Mirn140 modulated Pdgf-mediated attraction of cranial neural crest cells to the oral ectoderm, where crest-derived signals were necessary for oral ectodermal gene expression. Mirn140 loss of function elevated Pdgfra protein levels, altered palatal shape and caused neural crest cells to accumulate around the optic stalk, a source of the ligand Pdgfaa. These results suggest that the conserved regulatory interactions of mirn140 and pdgfra define an ancient mechanism of palatogenesis, and they provide candidate genes for cleft palate.

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Figure 1: Overexpression of Mirn140 phenocopies pdgfra mutants.
Figure 2: The oral ectoderm is similarly disrupted in Mirn140 duplex–injected embryos and pdgfra mutants.
Figure 3: Mirn140 regulates Pdgfra levels.
Figure 4: mirn140 overlaps with pdgfra during crest cell migration.
Figure 5: Pdgf signaling, modulated by Mirn140, guides palatal skeleton precursors to the oral ectoderm.
Figure 6: Pdgfra is required in neural crest for neural crest cell migration, palatal skeleton development and proper oral ectoderm specification.
Figure 7: Loss of Mirn140 function alters palatal skeleton morphology and neural crest cell migration.
Figure 8: Model of how Mirn140 modulates Pdgf signaling during palatogenesis.

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EMBL/GenBank/DDBJ

GenBank/EMBL/DDBJ

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Acknowledgements

We thank R. BreMiller for help with histology and J. Dowd and the University of Oregon Zebrafish Facility for animal care. Morpholinos were provided by J. Moulton (Gene Tools) as part of Multi-Blocker field testing. This work was supported by the US National Center for Research Resources (5R01RR020833 to J.H.P.) and US National Institutes of Health (P01 HD22486 to C.B.K. and J.H.P. and 5 K99 DE018088 to J.K.E.); the contents of this study are solely the responsibility of the authors and do not necessarily represent the official views of NCRR or NIH.

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Author notes

  1. Macie B Walker

    Present address: Present address: Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, Missouri 64110, USA.,

  2. Johann K Eberhart and Xinjun He: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Neuroscience, 1254 University of Oregon, Eugene, 97403, Oregon, USA

    Johann K Eberhart, Xinjun He, Mary E Swartz, Yi-Lin Yan, Hao Song, Taylor C Boling, Allison K Kunerth, Charles B Kimmel & John H Postlethwait

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Contributions

J.K.E. performed functional characterization of pdgfra mutants, assisted with mirn140 analyses and wrote the manuscript. X.H. performed functional characterization of mirn140, assisted with analyses on pdgfra mutants and assisted with manuscript preparation. M.E.S. genetically mapped the b1059 locus, assisted with pdgfra mutant characterizations and assisted with manuscript preparation. Y.-L.Y. assisted with mirn140 analyses and assisted with manuscript preparation. H.S. assisted with immunoblotting. T.C.B. sequenced the b1059 lesion and assisted with cloning of zebrafish pdgf family members. A.K.K. assisted with in situ hybridization of Pdgf family members. M.B.W. isolated pdgfrab1059 from our forward genetic screen. J.H.P. and C.B.K. supervised the project and assisted with manuscript preparation.

Corresponding author

Correspondence to Johann K Eberhart.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Tables 1–2 (PDF 3183 kb)

Supplementary Movie 1

Crest cell migration in a pdgfra+ embryo. (MOV 1662 kb)

Supplementary Movie 2

Crest cell migration in a pdgfra−/− embryo. (MOV 1719 kb)

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Eberhart, J., He, X., Swartz, M. et al. MicroRNA Mirn140 modulates Pdgf signaling during palatogenesis. Nat Genet 40, 290–298 (2008). https://doi.org/10.1038/ng.82

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