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Identification of Noncanonical Wnt Receptors Required for Wnt-3a-Induced Early Differentiation of Human Neural Stem Cells

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Abstract

Wnt proteins preferentially activate either β-catenin-dependent or β-catenin-independent signals, but the activity of a particular Wnt also depends on cellular context and receptor availability. We previously reported that Wnt-3a induces neural differentiation of human embryonic stem cell-derived neural stem cells (NSCs) in a β-catenin-independent manner by activating a signal involving JNK and the AP-1 family member ATF-2. Here, we report the results of a gene silencing approach to identify the Wnt receptors that mediate this response to Wnt-3a. Silencing of ROR2 increased neuronal differentiation, as measured by expression of the genes DCX, NEUROD1, and NGN1, suggesting ROR2 signals normally prevent differentiation. Silencing of the other Wnt receptors singly did not affect Wnt-3a-induced neuronal differentiation. However, pairwise silencing of ROR1 and FZD4 or FZD5 and of LRP6 and FZD4 or FZD5 inhibited neuronal differentiation, as detected by reductions in the expression of neuronal genes and immunocytochemical detection of DCX, NEUROD1 and DCX. Ectopic expression of these receptors in HEK 293 cells increased ATF2-dependent transcription. In addition, ROR1 coimmunoprecipitated with FZD4 and LRP6 in transfected HEK 293 cells and colocalized with FZD4 and with LRP6 at the cell surface of transfected L cells. Wnt-3a did not appear to affect these interactions but did alter the interactions between LRP6 and FZD4/5. Together, these observations highlight roles for ROR1, LRP6, FZD4, and FZD5 in neural stem cell differentiation and provide support for a model in which dynamic interactions among these receptors mediate Wnt-3a activation of ATF2 signaling.

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Acknowledgments

This work was facilitated by funding from the Spanish Ministry of Education and Science (SAF2014-51966-R, IJCI-2014-21255) and the Government of the Autonomous Community of the Basque Country Departments of Education, Industry, Tourism and Trade (Elkartek) and Innovation Technology. We thank Maria Vivanco for critical reading of the manuscript.

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  1. Nora Bengoa-Vergniory

    Present address: Department of Physiology, Anatomy and Genetics, Oxford University, Oxford, UK

Authors and Affiliations

  1. Cell Biology and Stem Cells Unit, CIC bioGUNE, Bilbao, Spain

    Nora Bengoa-Vergniory, Irantzu Gorroño-Etxebarria, Inmaculada López-Sánchez, Michele Marra, Pierluigi Di Chiaro & Robert Kypta

  2. Department of Surgery and Cancer, Imperial College London, London, UK

    Robert Kypta

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  1. Nora Bengoa-Vergniory
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Correspondence to Robert Kypta.

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Bengoa-Vergniory, N., Gorroño-Etxebarria, I., López-Sánchez, I. et al. Identification of Noncanonical Wnt Receptors Required for Wnt-3a-Induced Early Differentiation of Human Neural Stem Cells. Mol Neurobiol 54, 6213–6224 (2017). https://doi.org/10.1007/s12035-016-0151-5

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  • DOI: https://doi.org/10.1007/s12035-016-0151-5

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