title: The apical polarity determinant Crumbs 2 is a novel regulator of ESC-derived neural progenitors.
abstract: ESCs undergoing neural differentiation in vitro display an intrinsic heterogeneity with a large subset of the cells forming polarized neural rosettes that maintain the neural progenitor microenvironment. This heterogeneity is not only necessary for normal development but also causes substantial technical challenges for practical applications. Here, we report a novel regulator of early neural progenitors, the apical polarity protein Crb2 (Crumbs homologue 2). Employing monolayer differentiation of mouse ESCs to model neurogenesis in vitro, we find that Crb2 is upregulated with Sox1 and Musashi at the onset of neuroepithelial specification and localizes to the apical side of neural rosettes. Stable Crb2-knockdown (KD) lines die at the onset of neural specification and fail to stabilize several apical polarity proteins. However, these cells are able to proliferate under self-renewing conditions and can be differentiated into mesodermal and endodermal lineages. Conversely, Crb2 overexpression during neural differentiation results in elevated levels of other apical polarity proteins and increases proliferation. Additionally, sustained overexpression of Crb2 reduces terminal differentiation into TuJ1-positive neurons. Furthermore, we demonstrate that Crb2 overexpression under self-renewing conditions increases glycogen synthase kinase (GSK)-3β inhibition, correlating with an increase in clonogenicity. To confirm the importance of GSK-3β inhibition downstream of Crb2, we show that Crb2-KD cells can be forced into neural lineages by blocking GSK-3β function and supplementing Epidermal Growth Factor (EGF) and basic Fibroblast Growth Factor (bFGF). Thus, this is the first demonstration that a member of the Crumbs family is essential for survival and differentiation of ESC-derived neural progenitors.
authors: Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield, S10 2TN United Kingdom.
authors: Thorsten
authors: T
authors: Boroviak
authors: Penny
authors: P
authors: Rashbass
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D016764:Cell Polarity; D049109:Cell Proliferation; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors; D000071679:Glycogen Synthase Kinase 3 beta; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics; D058953:Neural Stem Cells / Q000166:cytology / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D012397:Rosette Formation; D055748:SOXB1 Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D014404:Tubulin / Q000096:biosynthesis
chemical_list: C568885:Crb2 protein, mouse; D008565:Membrane Proteins; C104786:Msi1h protein, mouse; D009419:Nerve Tissue Proteins; D016601:RNA-Binding Proteins; D055748:SOXB1 Transcription Factors; C098693:Sox1 protein, mouse; D014404:Tubulin; C462941:beta3 tubulin, mouse; D016222:Fibroblast Growth Factor 2; D004815:Epidermal Growth Factor; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D038362:Glycogen Synthase Kinase 3