Characterization of Functional Primary Cilia in Human Induced Pluripotent Stem Cell-Derived Neurons

Miki, Daisuke; Kobayashi, Yuki; Okada, Tomoya; Miyamoto, Tatsuo; Takei, Nobuyuki; Sekino, Yuko; Koganezawa, Noriko; Shirao, Tomoaki; Saito, Yumiko

doi:10.1007/s11064-019-02806-4

Cited by 17 publications

(8 citation statements)

References 35 publications

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“…We describe the presence of cilia on human NES and differentiating neuronal cells. This is in accordance with previous studies describing the occurrence of cilia in human neural progenitor cells (NPCs), differentiating cells in rosettes and organoids [51][52][53], hiPSC-derived neurons [54], and in the adult brain [55]. The hedgehog effector smoothened localizes to primary cilia in NPCs in maturing rosettes indicating that similar processes might be active in human neural cells as in rodents [51].…”

Section: Discussionsupporting

confidence: 92%

Dyslexia Candidate Gene and Ciliary Gene Expression Dynamics During Human Neuronal Differentiation

et al. 2020

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Developmental dyslexia (DD) is a neurodevelopmental condition with complex genetic mechanisms. A number of candidate genes have been identified, some of which are linked to neuronal development and migration and to ciliary functions. However, expression and regulation of these genes in human brain development and neuronal differentiation remain uncharted. Here, we used human long-term self-renewing neuroepithelial stem (lt-NES, here termed NES) cells derived from human induced pluripotent stem cells to study neuronal differentiation in vitro. We characterized gene expression changes during differentiation by using RNA sequencing and validated dynamics for selected genes by qRT-PCR. Interestingly, we found that genes related to cilia were significantly enriched among upregulated genes during differentiation, including genes linked to ciliopathies with neurodevelopmental phenotypes. We confirmed the presence of primary cilia throughout neuronal differentiation. Focusing on dyslexia candidate genes, 33 out of 50 DD candidate genes were detected in NES cells by RNA sequencing, and seven candidate genes were upregulated during differentiation to neurons, including DYX1C1 (DNAAF4), a highly replicated DD candidate gene. Our results suggest a role of ciliary genes in differentiating neuronal cells and show that NES cells provide a relevant human neuronal model to study ciliary and DD candidate genes. Keywords Cilia. Ciliopathies. Reading disorder. Human neuroepithelial stem cells. RNA sequencing Abbreviations BBS Bardet-Biedl syndrome CAGE Cap analysis of gene expression DAVID Database for Annotation, Visualization and Integrated Discovery DCDC2 Doublecortin domain containing 2 DCG Dyslexia candidate gene DCX Doublecortin DD Developmental dyslexia DNAAF Dynein axonemal assembly factor DYX1C1 Dyslexia susceptibility 1 candidate 1 EGF Epidermal growth factor FANTOM Functional annotation of the mammalian genome FDR False discovery rate FGF Fibroblast growth factor Andrea Bieder and Masahito Yoshihara contributed equally to this work.

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Section: Discussionsupporting

confidence: 92%

Dyslexia Candidate Gene and Ciliary Gene Expression Dynamics During Human Neuronal Differentiation

et al. 2020

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“…However, after Haus6 conditional knockout loss of luminal HAUS6 required up to nine days of in vitro culture (Supplementary Fig. 2e-g), whereas ciliogenesis occurs much earlier 63,64 . Moreover, Haus6 conditional knockout in neural progenitors of the developing mouse brain is embryonic lethal 65 .…”

Section: Resultsmentioning

confidence: 99%

Sub-centrosomal mapping identifies augmin-γTuRC as part of a centriole-stabilizing scaffold

et al. 2021

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Centriole biogenesis and maintenance are crucial for cells to generate cilia and assemble centrosomes that function as microtubule organizing centers (MTOCs). Centriole biogenesis and MTOC function both require the microtubule nucleator γ-tubulin ring complex (γTuRC). It is widely accepted that γTuRC nucleates microtubules from the pericentriolar material that is associated with the proximal part of centrioles. However, γTuRC also localizes more distally and in the centriole lumen, but the significance of these findings is unclear. Here we identify spatially and functionally distinct subpopulations of centrosomal γTuRC. Luminal localization is mediated by augmin, which is linked to the centriole inner scaffold through POC5. Disruption of luminal localization impairs centriole integrity and interferes with cilium assembly. Defective ciliogenesis is also observed in γTuRC mutant fibroblasts from a patient suffering from microcephaly with chorioretinopathy. These results identify a non-canonical role of augmin-γTuRC in the centriole lumen that is linked to human disease.

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“…To identify new components involved in Hh signaling transduction, we isolated the mouse embryonic fibroblasts (MEFs) and seeded them in different densities to make MEFs deciliated or ciliated and biochemically extracted total membrane proteins from the deciliated MEFs in low densities as well as the ciliated MEFs in high densities (Figure 1 A-B). Data of liquid chromatography-tandem mass spectrometry (LC-MS/MS) with normalization to protein content revealed that, compared with the deciliated MEFs, the ciliated MEFs are enriched in acetylated tubulin (Ac-Tub) and Arl13b, two markers for primary cilia 23 , 24 , and IFT88 and KIF3A as well, two molecules pivotal to the maintenance and function of primary cilia 25 , 26 (Figure 1 C). Intriguingly, among the three members in Rho family of small GTPases 27 , only enrichment of Rac1 but not Cdc42 or RhoA was observed in the ciliated MEFs, compared with that in deciliated MEFs (Figure 1 C), and the enrichment of Rac1 in MEF primary cilia was additionally verified by immunnofluorescent staining (Figure 1 D), indicating that Rac1 may affect Hh signaling.…”

Section: Resultsmentioning

confidence: 99%

Hedgehog signaling is controlled by Rac1 activity

Tang¹,

Wu²,

Ren³

et al. 2022

Theranostics

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Rationale: The nuclear translocation of transcriptional factor Gli is indispensable for Hedgehog (Hh) signaling activation, whose deregulation causes cancer progressions; however, the mechanisms governing Gli nuclear translocation are poorly understood. Here, we report that the Gli translocation in response to Hh requires Rac1 activation. Methods: C3H10T1/2 cell line and mouse embryonic fibroblasts were used to explore the molecular mechanisms underlying Rac1 activity in regulation of Hh signaling transduction. Transgenic mouse strains and human medulloblastoma (MB) tissue samples were utilized to examine the role of Rac1 in Hh-directed limb bud development and MB progression. Results: We show that upon the binding of Hh to receptor Patched1 (Ptch1), receptor Smoothened (Smo) dissociates from Ptch1 and binds to Vav2, resulting in the increased phosphorylation levels of Vav2 at Y172, which further activates Rac1. The role of Rac1 is dependent on the regulation of phosphorylation levels of KIF3A at S689 and T694, which in turn affects IFT88 stability and subsequently dampens SuFu-Gli complex formation, leading to the release of Gli from the complex and the consequent translocation of Gli into the nucleus. Moreover, Vav2 phospho-Y172 levels are up-regulated in GFAP-Cre;SmoM2 +/- mouse cerebellum and human Shh type MB tissues, whereas deficiency of Rac1 in mouse embryonic limb bud ectoderm ( Prx1-Cre;Rac1 f/f ) impedes Hh activation by disruption of Gli nuclear translocation. Conclusion: Together, our results uncover the Rac1 activation and the subsequent Gli translocation as a hitherto uncharacterized mechanism controlling Hh signaling and may provide targets for therapeutic intervention of this signaling pathway.

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Characterization of Functional Primary Cilia in Human Induced Pluripotent Stem Cell-Derived Neurons

Cited by 17 publications

References 35 publications

Dyslexia Candidate Gene and Ciliary Gene Expression Dynamics During Human Neuronal Differentiation

Dyslexia Candidate Gene and Ciliary Gene Expression Dynamics During Human Neuronal Differentiation

Sub-centrosomal mapping identifies augmin-γTuRC as part of a centriole-stabilizing scaffold

Hedgehog signaling is controlled by Rac1 activity

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