Small molecule valproic acid enhances ventral patterning of human neural tube organoids by regulating Wnt and Shh signalling

Abstract

Valproic acid (VPA), a clinically approved small molecule, has been reported to activate Wnt signalling that is critical for dorsal–ventral (DV) patterning of neural tube. However, little is known about the impact of VPA on DV patterning process. Here, we show that even though VPA has a negative impact on the early formation of human neural tube organoids (hNTOs), it significantly enhances the efficiency of ventrally patterned hNTOs, when VPA is added during the entire differentiation process. RNA sequencing and RT-qPCR analysis demonstrates VPA activates endogenous Wnt signalling in hNTOs. Surprisingly, transcriptome analysis also identifies upregulation of genes for degradation of GLI2 and GLI3 proteins, whose truncated fragment are transcriptional repressors of Shh signalling. The Western-blot analysis confirms the increase of GLI3R proteins after VPA treatment. Thus, VPA might enhance ventral patterning of hNTOs through both activating Wnt, which can antagonise Shh signalling by inducing GLI3 expression, and/or inhibiting Shh signalling by inducing GLI protein degradation. We further obtain results to show that VPA still increases patterning efficiency of hNTOs with a weak influence on their early formation when the initiation time of VPA is delayed and its duration is reduced. Taken together, this study demonstrates that VPA enhances the generation of more reproducible hNTOs with ventral patterning, opening the avenues for the applications of hNTOs in developmental biology and regenerative medicine.

References

[1] Sullivan-BrownJ, Goldstein B. Neural tube closure: the curious case of shrinking junctions. Curr Biol. 2012;22:R574-R576.

[2] NikolopoulouE, GaleaGL, RoloA, Greene ND, CoppAJ. Neural tube closure: cellular, molecular and biomechanical mechanisms. Development. 2017;144:552-566.

[3] Le DreauG, MartiE. Dorsal-ventral patterning of the neural tube: a tale of three signals. Dev Neurobiol. 2012;72:1471-1481.

[4] LiemKF Jr, TremmlG, RoelinkH, Jessell TM. Dorsal differentiation of neural plate cells induced by BMP-mediated signals from epidermal ectoderm. Cell. 1995;82:969-979.

[5] ChizhikovVV, MillenKJ. Roof plate-dependent patterning of the vertebrate dorsal central nervous system. Dev Biol. 2005;277:287-295.

[6] TimmerJR, WangC, NiswanderL. BMP signaling patterns the dorsal and intermediate neural tube via regulation of homeobox and helix-loop-helix transcription factors. Development. 2002;129:2459-2472.

[7] JessellTM. Neuronal specification in the spinal cord: inductive signals and transcriptional codes. Nat Rev Genet. 2000;1:20-29.

[8] Fakhraei LahijiS, Seo SH, KimS, et al. Transcutaneous implantation of valproic acid-encapsulated dissolving microneedles induces hair regrowth. Biomaterials. 2018;167:69-79.

[9] WangL, LiuY, LiS, LongZY, WuYM. Wnt signaling pathway participates in valproic acid-induced neuronal differentiation of neural stem cells. Int J Clin Exp Pathol. 2015;8:578-585.

[10] ParkG, JangWE, KimS, et al. Dysregulation of the Wnt/β-catenin signaling pathway via Rnf146 upregulation in a VPA-induced mouse model of autism spectrum disorder. Exp Mol Med. 2023;55:1783-1794.

[11] PeukertD, WeberS, LumsdenA, Scholpp S. Lhx2 and Lhx9 determine neuronal differentiation and compartition in the caudal forebrain by regulating Wnt signaling. PLoS Biol. 2011;9:e1001218.

[12] CuiK, WangY, ZhuY, et al. Neurodevelopmental impairment induced by prenatal valproic acid exposure shown with the human cortical organoid-on-a-chip model. Microsyst Nanoeng. 2020;6:49.

[13] ZhengY, XueX, Resto-IrizarryAM, et al. Dorsal-ventral patterned neural cyst from human pluripotent stem cells in a neurogenic niche. Sci Adv. 2019;5:eaax5933.

[14] TangC, WangX, D’UrsoM, van der PuttenC, Kurniawan NA. 3D interfacial and spatiotemporal regulation of human Neuroepithelial organoids. Adv Sci. 2022;9:e2201106.

[15] Abdel FattahAR, DazaB, RustandiG, et al. Actuation enhances patterning in human neural tube organoids. Nat Commun. 2021;12:3192.

[16] XuJ, FangS, DengS, et al. Generation of neural organoids for spinal-cord regeneration via the direct reprogramming of human astrocytes. Nat Biomed Eng. 2023;7:253-269.

[17] YangY, FanY, ZhangH, et al. Small molecules combined with collagen hydrogel direct neurogenesis and migration of neural stem cells after spinal cord injury. Biomaterials. 2021;269:120479.

[18] LeeJ-Y, HongS, ChoiK-A, Jeong H, ParkH-S. Generation of induced pluripotent stem cells without genetic defects by small molecules. Biomaterials. 2015;47-58:47-58.

[19] PhielCJ, ZhangF, HuangEY, Guenther MG, LazarMA, KleinPS. Histone deacetylase is a direct target of valproic acid, a potent anticonvulsant, mood stabilizer, and teratogen. J Biol Chem. 2001;276:36734-36741.

[20] ZhangX, HuangCT, ChenJ, et al. Pax6 is a human neuroectoderm cell fate determinant. Cell Stem Cell. 2010;7:90-100.

[21] PesceM, Scholer HR. Oct-4: gatekeeper in the beginnings of mammalian development. Stem Cells. 2001;19:271-278.

[22] ZanattaG, SulaA, MilesAJ, et al. Valproic acid interactions with the NavMs voltage-gated sodium channel. Proc Natl Acad Sci U S A. 2019;116:26549-26554.

[23] ZechnerD, MullerT, WendeH, et al. Bmp and Wnt/beta-catenin signals control expression of the transcription factor Olig3 and the specification of spinal cord neurons. Dev Biol. 2007;303:181-190.

[24] MooreS, RibesV, TerrienteJ, Wilkinson D, RelaixF, BriscoeJ. Distinct regulatory mechanisms act to establish and maintain Pax3 expression in the developing neural tube. PLoS Genet. 2013;9:e1003811.

[25] BangAG, Papalopulu N, GouldingMD, KintnerC. Expression of Pax-3 in the lateral neural plate is dependent on a Wnt-mediated signal from posterior nonaxial mesoderm. Dev Biol. 1999;212:366-380.

[26] YuW, McDonnell K, TaketoMM, BaiCB. Wnt signaling determines ventral spinal cord cell fates in a time-dependent manner. Development. 2008;135:3687-3696.

[27] UlloaF, MartiE. Wnt won the war: antagonistic role of Wnt over Shh controls dorso-ventral patterning of the vertebrate neural tube. Dev Dyn. 2010;239:69-76.

[28] SlenterDN, KutmonM, HanspersK, et al. WikiPathways: a multifaceted pathway database bridging metabolomics to other omics research. Nucleic Acids Res. 2018;46:D661-D667.

[29] KanehisaM, Furumichi M, TanabeM, SatoY, Morishima K. KEGG: new perspectives on genomes, pathways, diseases and drugs. Nucleic Acids Res. 2017;45:D353-D361.

[30] ZhangY, Alexander PB, WangX-F. TGF-βfamily signaling in the control of cell proliferation and survival. Csh Perspect Biol. 2017;9:a022145.

[31] ShiX, WangJ, LeiY, CongC, TanD, ZhouX. Research progress on the PI3K/AKT signaling pathway in gynecological cancer (review). Mol Med Rep. 2019;19:4529-4535.

[32] FuM, HuY, LanT, GuanKL, LuoT, LuoM. The Hippo signalling pathway and its implications in human health and diseases. Signal Transduct Target Ther. 2022;7:376.

[33] Borreguero-MunozN, Fletcher GC, Aguilar-AragonM, ElbediwyA, Vincent-Mistiaen ZI, ThompsonBJ. The Hippo pathway integrates PI3K-Akt signals with mechanical and polarity cues to control tissue growth. PLoS Biol. 2019;17:e3000509.

[34] SubramanianA, TamayoP, MoothaVK, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005;102:15545-15550.

[35] WuJ, YangJ, KleinPS. Neural crest induction by the canonical Wnt pathway can be dissociated from anterior-posterior neural patterning in Xenopus. Dev Biol. 2005;279:220-232.

[36] DorskyRI, MoonRT, RaibleDW. Control of neural crest cell fate by the Wnt signalling pathway. Nature. 1998;396:370-373.

[37] MaczkowiakF, MateosS, WangE, Roche D, HarlandR, Monsoro-BurqAH. The Pax3 and Pax7 paralogs cooperate in neural and neural crest patterning using distinct molecular mechanisms, in Xenopus laevis embryos. Dev Biol. 2010;340:381-396.

[38] WangZ, ShuW, LuMM, Morrisey EE. Wnt7b activates canonical signaling in epithelial and vascular smooth muscle cells through interactions with Fzd1, Fzd10, and LRP5. Mol Cell Biol. 2005;25:5022-5030.

[39] ZengY, KongY, LiaoL, Zhu H. Involvement of RUVBL1 in WNT/β-catenin signaling in Oral squamous cell carcinoma. Dis Markers. 2022;2022:1-12.

[40] FilaliM, ChengN, AbbottD, Leontiev V, EngelhardtJF. Wnt-3A/β-catenin signaling induces transcription from the LEF-1 promoter. J Biol Chem. 2002;277:33398-33410.

[41] HaoY-H, Lafita-Navarro MC, ZachariasL, et al. Induction of LEF1 by MYC activates the WNT pathway and maintains cell proliferation. Cell Commun Signal. 2019;17:1-16.

[42] BrafmanD, Willert K. Wnt/β-catenin signaling during early vertebrate neural development. Dev Neurobiol. 2017;77:1239-1259.

[43] FiskeB. Wnt signals lead cells down the caudal path. Nat Neurosci. 2002;5:510.

[44] JacobJ, Briscoe J. Gli proteins and the control of spinal-cord patterning. EMBO Rep. 2003;4:761-765.

[45] CarballoGB, Honorato JR, de LopesGPF, SpohrT. A highlight on sonic hedgehog pathway. Cell Commun Signal. 2018;16:1-15.

[46] TeglundS, Toftgard R. Hedgehog beyond medulloblastoma and basal cell carcinoma. Biochim Biophys Acta. 2010;1805:181-208.

[47] ChaudhryP, SinghM, TricheTJ, Guzman M, MerchantAA. GLI3 repressor determines Hedgehog pathway activation and is required for response to SMO antagonist glasdegib in AML. Blood. 2017;129:3465-3475.

[48] MeyerNP, Roelink H. The amino-terminal region of Gli3 antagonizes the Shh response and acts in dorsoventral fate specification in the developing spinal cord. Dev Biol. 2003;257:343-355.

[49] BondAM, Bhalala OG, KesslerJA. The dynamic role of bone morphogenetic proteins in neural stem cell fate and maturation. Dev Neurobiol. 2012;72:1068-1084.

[50] LumsdenA, GrahamA. Neural patterning: a forward role for hedgehog. Curr Biol. 1995;5:1347-1350.

[51] GaoJ, LiaoY, QiuM, ShenW. Wnt/β-catenin signaling in neural stem cell homeostasis and neurological diseases. Neuroscientist. 2021;27:58-72.

[52] WuY, PengS, FinnellRH, Zheng Y. Organoids as a new model system to study neural tube defects. FASEB J. 2021;35:e21545.

[53] BalmerNV, WengMK, ZimmerB, et al. Epigenetic changes and disturbed neural development in a human embryonic stem cell-based model relating to the fetal valproate syndrome. Hum Mol Genet. 2012;21:4104-4114.

[54] SahniG, ChangSY, TeoJCM, et al. A micropatterned human-specific Neuroepithelial tissue for modeling gene and Drug-induced neurodevelopmental defects. Adv Sci. 2021;8:e2101786.

[55] LeeSK, PfaffSL. Transcriptional networks regulating neuronal identity in the developing spinal cord. Nat Neurosci. 2001;4:1183-1191.

[56] PalmerAJ, SaveryD, MassaV, Copp AJ, GreeneNDE. Genetic interaction of Pax3 mutation and canonical Wnt signaling modulates neural tube defects and neural crest abnormalities. Genesis. 2021;59:e23445.

[57] ZangZ, YinH, DuZ, et al. Valproic acid exposure decreases neurogenic potential of outer radial glia in human brain organoids. Front Mol Neurosci. 2022;15:1023765.

[58] UlloaF, Itasaki N, BriscoeJ. Inhibitory Gli3 activity negatively regulates Wnt/beta-catenin signaling. Curr Biol. 2007;17:545-550.

[59] TaniguchiK, Anderson AE, SutherlandAE, WottonD. Loss of Tgif function causes holoprosencephaly by disrupting the SHH signaling pathway. PLoS Genet. 2012;8:e1002524.

[60] DuZW, ChenH, LiuH, et al. Generation and expansion of highly pure motor neuron progenitors from human pluripotent stem cells. Nat Commun. 2015;6:6626.

[61] HuangfuD, MaehrR, GuoW, et al. Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds. Nat Biotechnol. 2008;26:795-797.

[62] FattahARA, Grebenyuk S, de RooijLP, SalmonI, RangaA. Neuroepithelial organoid patterning is mediated by a neighborhood watch mechanism. Cell Rep. 2023;42:113334.

[63] ShaoY, Taniguchi K, TownshendRF, MikiT, Gumucio DL, FuJ. A pluripotent stem cell-based model for post-implantation human amniotic sac development. Nat Commun. 2017;8:208.

RIGHTS & PERMISSIONS

2024 The Author(s). Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.