title: Identification of Noncanonical Wnt Receptors Required for Wnt-3a-Induced Early Differentiation of Human Neural Stem Cells.
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.
authors.affiliation: Cell Biology and Stem Cells Unit, CIC bioGUNE, Bilbao, Spain.|Department of Physiology, Anatomy and Genetics, Oxford University, Oxford, UK.
authors.forename: Nora
authors.initials: N
authors.lastname: Bengoa-Vergniory
authors.affiliation: Cell Biology and Stem Cells Unit, CIC bioGUNE, Bilbao, Spain.
authors.forename: Irantzu
authors.initials: I
authors.lastname: Gorroño-Etxebarria
authors.affiliation: Cell Biology and Stem Cells Unit, CIC bioGUNE, Bilbao, Spain.
authors.forename: Inmaculada
authors.initials: I
authors.lastname: López-Sánchez
authors.affiliation: Cell Biology and Stem Cells Unit, CIC bioGUNE, Bilbao, Spain.
authors.forename: Michele
authors.initials: M
authors.lastname: Marra
authors.affiliation: Cell Biology and Stem Cells Unit, CIC bioGUNE, Bilbao, Spain.
authors.forename: Pierluigi
authors.initials: P
authors.lastname: Di Chiaro
authors.affiliation: Cell Biology and Stem Cells Unit, CIC bioGUNE, Bilbao, Spain. rkypta@cicbiogune.es.|Department of Surgery and Cancer, Imperial College London, London, UK. rkypta@cicbiogune.es.
authors.forename: Robert
authors.identifier: http://orcid.org/0000-0002-2389-310X
authors.initials: R
authors.lastname: Kypta
mesh_terms: D006801:Humans; D058953:Neural Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D055495:Neurogenesis / Q000187:drug effects / Q000502:physiology*; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D060490:Receptors, Wnt / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D060509:Wnt3A Protein / Q000494:pharmacology*
chemical_list: D060490:Receptors, Wnt; D060509:Wnt3A Protein
keywords: Neural stem cells; Wnt signaling pathways