Wnt/β-Catenin Signaling Blockade Promotes Neuronal Induction and Dopaminergic Differentiation in Embryonic Stem Cells
Tóm tắt
Embryonic stem cells (ESCs) represent not only a promising source of cells for cell replacement therapy, but also a tool to study the molecular mechanisms underlying cellular signaling and dopaminergic (DA) neuron development. One of the main regulators of DA neuron development is Wnt signaling. Here we used mouse ESCs (mESCs) lacking Wnt1 or the low-density lipoprotein receptor-related protein 6 (LRP6) to decipher the action of Wnt/β-catenin signaling on DA neuron development in mESCs. We provide evidence that the absence of LRP6 abrogates responsiveness of mESCs to Wnt ligand stimulation. Using two differentiation protocols, we show that the loss of Wnt1 or LRP6 increases neuroectodermal differentiation and the number of mESC-derived DA neurons. These effects were similar to those observed following treatment of mESCs with the Wnt/β-catenin pathway inhibitor Dickkopf1 (Dkk1). Combined, our results show that decreases in Wnt/β-catenin signaling enhance neuronal and DA differentiation of mESCs. These findings suggest that: 1) Wnt1 or LRP6 are not strictly required for the DA differentiation of mESCs in vitro, 2) the levels of morphogens and their activity in ESC cultures need to be optimized to improve DA differentiation, and 3) by enhancing the differentiation and number of ESC-derived DA neurons with Dkk1, the application of ESCs for cell replacement therapy in Parkinson's disease may be improved.
Disclosure of potential conflicts of interest is found at the end of this article.
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Tài liệu tham khảo
Parish, 2007, Stem-cell-based strategies for the treatment of Parkinson's disease, Neurodegener Dis, 4, 339, 10.1159/000101892
Ciani, 2005, WNTs in the vertebrate nervous system: From patterning to neuronal connectivity, Nat Rev Neurosci, 6, 351, 10.1038/nrn1665
Ille, 2005, Wnt signaling: Multiple functions in neural development, Cell Mol Life Sci, 62, 1100, 10.1007/s00018-005-4552-2
Gaulden, 2008, Neur-ons and neur-offs: Regulators of neural induction in vertebrate embryos and embryonic stem cells, Hum Mol Genet, 17, R60, 10.1093/hmg/ddn119
Castelo-Branco, 2006, Function of Wnts in dopaminergic neuron development, Neurodegener Dis, 3, 5, 10.1159/000092086
Castelo-Branco, 2003, Differential regulation of midbrain dopaminergic neuron development by Wnt-1, Wnt-3a, and Wnt-5a, Proc Natl Acad Sci U S A, 100, 12747, 10.1073/pnas.1534900100
Parish, 2008, Wnt5a-treated midbrain neural stem cells improve dopamine cell replacement therapy in parkinsonian mice, J Clin Invest, 118, 149, 10.1172/JCI32273
Panhuysen, 2004, Effects of Wnt1 signaling on proliferation in the developing mid-/hindbrain region, Mol Cell Neurosci, 26, 101, 10.1016/j.mcn.2004.01.011
McMahon, 1990, The Wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain, Cell, 62, 1073, 10.1016/0092-8674(90)90385-R
Thomas, 1990, Targeted disruption of the murine int-1 proto-oncogene resulting in severe abnormalities in midbrain and cerebellar development, Nature, 346, 847, 10.1038/346847a0
Joksimovic, 2009, Wnt antagonism of Shh facilitates midbrain floor plate neurogenesis, Nat Neurosci, 10.1038/nn.2243
Prakash, 2006, A Wnt1-regulated genetic network controls the identity and fate of midbrain-dopaminergic progenitors in vivo, Development, 133, 89, 10.1242/dev.02181
Kohn, 2005, Wnt and calcium signaling: beta-catenin-independent pathways, Cell Calcium, 38, 439, 10.1016/j.ceca.2005.06.022
Seifert, 2007, Frizzled/PCP signalling: A conserved mechanism regulating cell polarity and directed motility, Nat Rev Genet, 8, 126, 10.1038/nrg2042
Semenov, 2007, SnapShot: Noncanonical Wnt Signaling Pathways, Cell, 131, 1378, 10.1016/j.cell.2007.12.011
Andersson, 2008, Wnt5a regulates ventral midbrain morphogenesis and the development of A9-A10 dopaminergic cells in vivo, Plos One, 3, e3517, 10.1371/journal.pone.0003517
Kawano, 2003, Secreted antagonists of the Wnt signalling pathway, J Cell Sci, 116, 2627, 10.1242/jcs.00623
Bryja, 2006, An efficient method for the derivation of mouse embryonic stem cells, Stem Cells, 24, 844, 10.1634/stemcells.2005-0444
Parisi, 2003, Nodal-dependent Cripto signaling promotes cardiomyogenesis and redirects the neural fate of embryonic stem cells, J Cell Biol, 163, 303, 10.1083/jcb.200303010
Barberi, 2003, Neural subtype specification of fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice, Nat Biotechnol, 21, 1200, 10.1038/nbt870
Ying, 2003, Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture, Nat Biotechnol, 21, 183, 10.1038/nbt780
Parish, 2005, Cripto as a target for improving embryonic stem cell-based therapy in Parkinson's disease, Stem Cells, 23, 471, 10.1634/stemcells.2004-0294
Bryja, 2005, Abnormal development of mouse embryoid bodies lacking p27Kip1 cell cycle regulator, Stem Cells, 23, 965, 10.1634/stemcells.2004-0174
Kawasaki, 2000, Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity, Neuron, 28, 31, 10.1016/S0896-6273(00)00083-0
Danielian, 1996, Engrailed-1 as a target of the Wnt-1 signalling pathway in vertebrate midbrain development, Nature, 383, 332, 10.1038/383332a0
Andersson, 2006, Identification of intrinsic determinants of midbrain dopamine neurons, Cell, 124, 393, 10.1016/j.cell.2005.10.037
Kittappa, 2007, The foxa2 gene controls the birth and spontaneous degeneration of dopamine neurons in old age, Plos Biol, 5, e325, 10.1371/journal.pbio.0050325
He, 2004, LDL receptor-related proteins 5 and 6 in Wnt/beta-catenin signaling: arrows point the way, Development, 131, 1663, 10.1242/dev.01117
Young, 1998, Wnt-1 induces growth, cytosolic beta-catenin, and Tcf/Lef transcriptional activation in Rat-1 fibroblasts, Mol Cell Biol, 18, 2474, 10.1128/MCB.18.5.2474
Bryja, 2007, Wnt-3a utilizes a novel low dose and rapid pathway that does not require casein kinase 1-mediated phosphorylation of Dvl to activate beta-catenin, Cell Signal, 19, 610, 10.1016/j.cellsig.2006.08.011
Gonzalez-Sancho, 2004, Wnt proteins induce dishevelled phosphorylation via an LRP5/6-independent mechanism, irrespective of their ability to stabilize beta-catenin, Mol Cell Biol, 24, 4757, 10.1128/MCB.24.11.4757-4768.2004
Jho, 2002, Wnt/beta-catenin/Tcf signaling induces the transcription of Axin2, a negative regulator of the signaling pathway, Mol Cell Biol, 22, 1172, 10.1128/MCB.22.4.1172-1183.2002
Yamaguchi, 1999, T (Brachyury) is a direct target of Wnt3a during paraxial mesoderm specification, Genes Dev, 13, 3185, 10.1101/gad.13.24.3185
Arnold, 2000, Brachyury is a target gene of the Wnt/beta-catenin signaling pathway, Mech Dev, 91, 249, 10.1016/S0925-4773(99)00309-3
Castelo-Branco, 2004, GSK-3beta inhibition/beta-catenin stabilization in ventral midbrain precursors increases differentiation into dopamine neurons, J Cell Sci, 117, 5731, 10.1242/jcs.01505
Pinson, 2000, An LDL-receptor-related protein mediates Wnt signalling in mice, Nature, 407, 535, 10.1038/35035124
Tamai, 2000, LDL-receptor-related proteins in Wnt signal transduction, Nature, 407, 530, 10.1038/35035117
Wehrli, 2000, Arrow encodes an LDL-receptor-related protein essential for Wingless signalling, Nature, 407, 527, 10.1038/35035110
McMahon, 1989, Ectopic expression of the proto-oncogene int-1 in Xenopus embryos leads to duplication of the embryonic axis, Cell, 58, 1075, 10.1016/0092-8674(89)90506-0
Sokol, 1991, Injected Wnt RNA induces a complete body axis in Xenopus embryos, Cell, 67, 741, 10.1016/0092-8674(91)90069-B
Otero, 2004, Beta-catenin signaling is required for neural differentiation of embryonic stem cells, Development, 131, 3545, 10.1242/dev.01218
Aubert, 2002, Functional gene screening in embryonic stem cells implicates Wnt antagonism in neural differentiation, Nat Biotechnol, 20, 1240, 10.1038/nbt763
Verani, 2007, Expression of the Wnt inhibitor Dickkopf-1 is required for the induction of neural markers in mouse embryonic stem cells differentiating in response to retinoic acid, J Neurochem, 100, 242, 10.1111/j.1471-4159.2006.04207.x
Semënov, 2001, Head inducer Dickkopf-1 is a ligand for Wnt coreceptor LRP6, Curr Biol, 11, 951, 10.1016/S0960-9822(01)00290-1
Bafico, 2001, Novel mechanism of Wnt signalling inhibition mediated by Dickkopf-1 interaction with LRP6/Arrow, Nat Cell Biol, 3, 683, 10.1038/35083081
Hayashi, 2008, Meningeal cells induce dopaminergic neurons from embryonic stem cells, Eur J Neurosci, 27, 261, 10.1111/j.1460-9568.2008.06027.x
Veeman, 2003, A second canon. Functions and mechanisms of beta-catenin-independent Wnt signaling, Dev Cell, 5, 367, 10.1016/S1534-5807(03)00266-1
Topol, 2003, Wnt-5a inhibits the canonical Wnt pathway by promoting GSK-3-independent beta-catenin degradation, J Cell Biol, 162, 899, 10.1083/jcb.200303158
Tahinci, 2007, Lrp6 is required for convergent extension during Xenopus gastrulation, Development, 134, 4095, 10.1242/dev.010272
Bryja, 2009, The extracellular domain of Lrp5/6 inhibits noncanonical Wnt signaling in vivo, Mol Biol Cell, 20, 924, 10.1091/mbc.e08-07-0711
Clevers, 2006, Wnt/beta-Catenin Signaling in Development and Disease, Cell, 127, 469, 10.1016/j.cell.2006.10.018
White, 2007, Wnt signaling: It gets more humorous with age, Curr Biol, 17, R923, 10.1016/j.cub.2007.08.062