In this study, we investigated the involvement of Wnt/β-catenin signaling in the anterior-posterior patterning during human ES cell-derived DA neuron development and the activation of Wnt signaling by small molecule glycogen synthase kinase-3 (GSK-3) inhibitor to obtain the midbrain specific characteristics.
Also, we have shown that Wnt/β-catenin signal can increase expression of En1 by direct interaction with each other, and involvement of FGF8 signal. Although previous studies revealed the relationship between Wnt1 and En1, the role which En1 played during the brain development was poorly understood. However, using human ES cell-derived NPCs, we have revealed Wnt/β-catenin signal controls the increase of En1 expression during AP patterning as well as the involvement of FGF8 signals to the increase of the expression. Based on my study, I conclude that the small molecule GSK-3 ihibitor can modulate Wnt signaling pathway and destine regional characters in AP patterning of the brain development.
REFERENCES
1. Lang AE, Lozano AM. Parkinson’s Disease: Second of Two Part. New Engl J Med 1998;339:1130-43
2. Echemarria D, Vieira C, Gimeno L, Martinez S. Neuroepithelial secondary organizersand cell fate specification in the developing brain. Brain Res Rev.
2004;43:179-91
3. Dorsky RI, Moon RT, Raible EW. Control of neural crest cell fate by the Wnt signaling pathway. Nature 1998;396:370-3
4. Megason SG, McMahon AP. A mitogen gradient of dorsal midline Wnts organizes growth in the CNS. Development 2002;129:2087-98
5. McMahon AP, Bradley A. The Wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain. Cell 1990;62:1073-85
6. Thomas KR, Capecchi MR. Targeted disruption of the murine int-1 proto-oncogene resulting in severe abnormalities in midbrain and cerebellar development.
Nature 1990; 346:846-50
7. Danielian PS, McMahon AP. Engrailed-1 as a target of the Wnt-1 signalling pathway in vertebrate midbrain development pathway. Nature 1996;396:370-73
8. Huelsken J, Behrens J. The Wnt signaling pathway. Cell Sci. 2002;115:3977-78
9. Logan CY, Nusse R. The Wnt signaling pathway in development and disease.
Annu Rev Cell Dev Biol. 2004;20:781-810
10. Moon RT, Brown JD, Torres M. WNTs modulate cell fate and behavior during
vertebrate development. Trends Genet. 1997;13:157-62
11. Chenn A, Walsh CA. Increased neuronal production, enlarged forebrains and cytoarchitectural distortions in beta-catenin overexpressing transgenic mice. Cereb Cortex 2003;13:599-606
12. Cilov D, Sinjushina N, Saarimaki-Vire J, Taketo MM, Partanen J. Beta-catenin regulates intercellular signaling networks and cell-type specific trasnctiption in the developing mouse midbrain-rhombomere 1 region. PLoS One 2010;5:e10881
13. Veeman MT, Axelrod JD, Moon RT. A second canon. Functions and mechanisms of beta-catenin-independent Wnt signaling. Dev Cell 2003;5:367-77
14. Cohen P, Frame S. The renaissance of GSK3. Nat Rev Mol Cell Biol.
2001;2:769-76
15. Bain J, Mclauchlan H, Elliott M, Cohen P. The specificities of protein kinase inhibitors: an update. Biochem J. 2002; 371:199-204
16. Bennette CN, Ross SE, Longo KA, Bajnok L, Hemati N, Johnson KW, et al.
Regulation of Wnt signaling During adipogenesis. J Biol Chem. 2002;277:30998-1004
17. Meijer L, Skaltsounis A-L, Magiatis P, Polychronopoulos P, Knockaert M, Leost M, et al., GSK-3-selective inhibitors derived from tyrian purple indirutins. Chem Biol. 2003;10:1255-66
18. Sato N, Meijer L, Skaltsounis L, Greengard P, Brivanlou AH. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3 specific inhibitor. Nat Med. 2004;10:55-63
19. Kim D-S, Lee JS, Leem JW, Huh YJ, Kim JY, Kim H-S, et al. Robust enhancement of neural differentiation from human ES and iPS cells regardless of their innate difference in differentiation propensity. Stem Cell Rev and Rep.
2010;6:270-81
20. Papalopulu N, Kintner C. A posteriorising factor, retinoic acid, reveals that anteroposterior patterning controls the timing of neuronal differentiation in Xenopus neuroectoderm. Development 1996;122: 3409–18
21. Blumberg B, Bolado J Jr, Moreno TA, Kintner C, Evans RM, Papalopulu N. An essential role for retinoid signalling in anteroposterior neural patterning.
Development 1997;124:373–79
22. Maden M. Retinoid signaling in the development of the central nervous system.
Nat Rev Neurosci. 2002;3:843-53
23. Ohmachi S, Watanabe Y, Mikami T, Kusu N, Ibi T, Akaike A, et al. FGF-20, a Novel Neurotrophic Factor, Preferentially Expressed in the Substantia Nigra Pars Compacta of Rat Brain. Biochem Biophys Res Commun. 2000;277:355–60
24. Correia AS, Anisimov SV, Roybon L, Li JY, Brundin P. Fibroblast frowth factor -20 increase the yield of midbrain dopaminergic neurons derived from human embryonic stem cells. Front Neuroanat. 2007; 1:1-9
25. Ye W, Shimamura K, Rubenstein JLR, Hynes MA, Rosenthal A. FGF and Shh signals control dopaminergic and serotonergic cell fate in the anterior neural plate.
Cell 1998;93:755-66.
26. Kimura J, Sato-Maeda M, Noji S, Ide H. Synergistic effects of FGF and non-ridge ectoderm on gene expression involved in the formation of the anteroposterior axis of the chick limb bud in cell culture. Dev Growth Differ. 2000;42:219-27.
27. Yan Y, Yang D, Zarnowska ED, Du Z, Werbel B, Valliere C, et al. Directed differentiation of dopaminergic neuronal subtypes from human embryonic stem cells. Stem Cells 2005;23:781–90.
28. Liu A, Losos K, Joyner AL. FGF8 can activate Gbx2 and transform regions of the rostral mouse brain into a hindbrain fate. Development 1999;126:4827-38
29. Castelo-Branco G, Wagner J, Rodrigue FJ, Kele J, Sousa K, Rawal N, et al.
Differential regulation of midbrain dopaminergic neuron development by Wnt-1, Wnt-3a, and Wnt-5a. Proc Natl Acad Sci. USA 2003;100:12747-52
30. McMahon AP, Joyner AL, Bradley A, McMahon JA. The midbrain-hindbrain phenotype of Wnt-1-/Wnt-1-mice results from stepwise deletion of engrailed-expressing cells by 9.5 days postcoitum. Cell 1992;69:581-95
31. Chung S, Sonntag KC, Andersson T, Bjorklund LM, Park JJ, Kim DW, et al.
Genetic engineering of mouse embryonic stem cells by Nurr1 enhances differentiation and maturation into dopaminergic neurons. Eur J Neurosci.
2002;16:1829–38.
32. Kawasaki H, Mizuseki K, Nishikawa S, Kaneko S, Kuwana Y, Nakanishi S, et al. Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity. Neuron 2000;28:31–40.
33. Ang SL. Transcriptional control of midbrain dopaminergic neuron development.
Development 2006;133:3499-506
34. Smidt MP, Asbreuk CH, Cox JJ, Chen H, Johnson RL, Burbach JP. A second independent pathway for development of mesencephalic dopaminergic neurons requires Lmx1b. Nat Neurosci. 2000;3:337-41
35. Crossley PH, Martines S, Martin GR. Midbrain development induced by FGF8 in the chick embryo. Nature 1996;380:66-8
36. Lumsden A, Krumlauf R. Patterning the vertebrate neuraxis. Science 1996;274:1109-14
37. Shimamura K, Hartigan DJ, Martinez S, Puelles L, Rubenstein JLR.
Longitudinal organization of the anterior neural plate and neural tube. Development 1995;121:3923-33
38. Castelo-Brancko G, Rawal N, Arenas E. GSK-3β inhibition/β-catenin stabilization in ventral midbrain precursors increases differentiation into dopamine neurons. J Cell Sci. 2004;117:5731-7
39. Prakash N, Brodsi C, Naserke T, Puelles E, Gogoi R, Hall A, et al. A Wnt1-regulated genetic network controls the identity and fate of midbrain-dopaminergic progenitors in vivo. Dvelopment 2006;133:89-98
40. Wittmann DM, Blöchl F, Trümbach D, Wurst W, Prakash N, Theis FJ. Spatial analysis of expression patterns predicts genetic interactions at the mid-hindbrain boundary. PLoS Comput Biol. 2009;5:e1000569.
41. Parr BA, Shea MJ, Vassileva G, McMahon AP. Mouse Wnt genes exhibit discrete domains of expression in the early embryonic CNS and limb buds.
Development 1993;119:247-61.
42. Crossley PH, Martin GR. The mouse Fgf8 gene encodes a family of polypeptides and is expressed in regions that direct outgrowth and patterning in the developing embryo. Development 1995;11:439-51
43. Li JY, Joyner AL (2001) Otx2 and gbx2 are required for refinement and not induction of mid hindbrain gene expression. Development 128: 4979–4991.
44. Simeone A (2000) Positioning the isthmic organizer: where otx2 and gbx2 meet.
Trends Genet 16: 237–40.
45. Hirsch C, Campano LM, Wöhrle S, Hecht A. Canonical Wnt signaling transiently stimulates proliferation and enhances neurogenesis in neonatal neural progenitor cultures. Exp Cell Res. 2007;313:572-87.
46. Joksimovic M, Yun BA, Kittappa R, Anderegg AM, Chang WW, Taketo MM, et al. Wnt antagonism of Shh facilitates midbrain floor plate neurogenesis. Nat Neurosci. 2009;12:125-31.
47. Chung S, Leung A, Han B-S, Chang M-Y, Moon J-I, Kim CH, et al. Wnt1-lmx1a form a novel autoregulatory loop and controls midbrain dopaminergic differentiation synergistically with the SHH-FoxA2 pathway. Cell Stem Cell 2009;5:646-58
48. Simon HH, Saueressig H, Wurst W, Goulding MD, O’Leary DD. Fate of midbrain dopaminergic neurons controlled by the engrailed genes. J Neurosci.
2001;21:3126–34.
49. Davis CA, Joyner AL. Expression patterns of the homeo box-containing genes En-1 and En-2 and the proto-oncogene int-1 diverge during mouse development.
Genes Dev. 1988;2:1736–44.
50. Gardner CA, Barlad KF. Expression patterns of engrailed-like proteins in the chick embryo. Dev Dyn. 1992;193:370–88.
51. Liu A, Jouner AL. EN and GBX2 play essential roles downstream of FGF8 in
patterning the mouse mid/hindbrain region. Development 2001;128:181–91.
52. Danielian P, McMahon A. Engrailed-1 as a target of the Wnt-1 signalling pathway in vertebrate midbrain development. Nature 1996;383:332–4.
53. Gemel J, Jacobsen C, MacArthur C. Fibroblast Growth Factor-8 Expression Is Regulated by Intronic Engrailed and Pbx1-binding Sites. J Biol Chem.
1999;274:6020–26.
54. Rowitch D, McMahon A. Pax-2 expression in the murine neural plate precedes and encompasses the expression domains of Wnt-1 and En-1. Mech Dev.
1995;52:3–8.
55. Broccoli V, Boncinelli E, Wurst W. The caudal limit of Otx2 expression positions the isthmic organizer. Nature 1999;401:164–8.
56. Heemskerk J, DiNardo S, Kostriken R, O’Farrell PH. Multiple modes of engrailed regulation in the progression towards cell fate determination. Nature 1991;352:404-10
57. DiNardo S, Sher E, Heemskerk-Jongens J, kassis JA, O’Farrell PH. Two-tiered regulation of spatially patterned engrailed gene expression during. Nature 1988;332:604-9
58. Araki I, Nakamura H. Engailed defines the position of dorsal di-mesencephalic boundary by repressing diencephalic fate. Development 1999;126:5127-35
59. Millet S, Campbell K, Epstein DJ, Losos K, Harris E, Joyner AL. A role for Gbx2 in repression of Otx2 and positioning the mid/hindbrain organizer. Nature 2000;401:161–4.
60. Joyner AL, Herrup K, Auerbach BA, Davis CA, Rossant J. Subtle cerebellar phenotype in mice homozygous for a targeted deletion of the En-2 homeobox.
Science 1991;251:1239–43.
61. Wurst W, Auerbach AB, Joyner AL. Multiple developmental defects in Engrailed-1 mutant mice: an early mid-hindbrain deletion and patterning defects in forelimbs and sternum. Development 1994;120:2065-75
ABSTRACT (IN KOREAN)
Wnt/β-catenin 신호전달체계 조절에 의한 인간 배아줄기세포로부터
생성된 신경전구세포의 중뇌 특이적 분화
<지도교수 : 김 동 욱>
연세대학교 대학원 의과학과 김 지 영
인간 배아줄기세포(human embryonic stem cells, hESCs)로부터 중뇌 특이적인 도파민 신경세포로의 효율적인 분화는 파킨슨 병에 세포치료를 적용하기 위하여 아주 중요한 과정이며, 이를 위하여 중뇌 특이적 도파민 신경세포의 발생과정을 완전히 이해하는 것이 필요하다. 이 연구에서는 인간 배아줄기세포로부터 도파민 신경세포로 만들어낼 때 중뇌 특이적 세포로의 특징을 가진 세포로 유도하는 것에 초점을 두었다. 이전의 연구 들에서 척추동물의 발생과정 중 앞-뒤 축 형성 과정에서 Wnt/β-catenin 신 호가 관여하고 있다는 보고가 여러 차례 있었고, 이 연구에서 Wnt/β-catenin 신호전달체계를 이용하여 중뇌 특이적 세포로의 형성을 할 수 있 음을 확인할 수 있었다. 본인은 인간 배아줄기세포로부터 유도된 신경전 구세포에 저분자 화합물
glycogen synthase kinase-3(
GSK-3) 저해제인 6-bromoindirubin-3'-oxime (BIO) 를 처리하였을 때 Wnt/β-catenin 신호전달체 계를 활성화시킬 수 있고, 전뇌 특이적 마커인 Bf1 이나 후뇌 특이적 마 커인 Gbx2 의 발현은 감소시키면서, 중뇌 특이적 마커인 En1의 발현만을 증가시키는 결과를 얻을 수 있었다. BIO 이 외에 또 다른 GSK-3 저해제 인 1-Azakenpaullene 과 LiCl 을 신경전구세포에 처리해 보았을 때도 BIO를 처리했을 때와 같은 결과를 얻을 수 있었다. 이어서 Wnt1을 처리해서 세포막의 수용체로부터 신호전달을 시작했을 때도 En1 의 발현이 증가하 는 것을 확인할 수 있었다. 한 편, Wnt 신호체계의 antagonist 인 DKK-1과 Frizzled-5 를 처리해서 이들의 길항작용을 확인해본 결과, 모두 En1 의 발현을 유의하게 감소시킴을 확인하였다. 뿐만 아니라 β-catenin 에 대한 shRNA 를 신경전구세포에 도입하였을 때, β-catenin 의 축적량이 감소하였 고, En1 의 발현양도 감소하였다. 이를 통해 BIO 에 의한 중뇌 특이적 마 커의 증가가 Wnt/β-catenin 신호전달체계에 의해 조절됨을 알 수 있다. 이 어서, 중뇌의 특징을 발현하는데 Wnt/β-catenin 신호 이외에 다른 신호전 달체계가 관여하고 있음을 확인하기 위하여 FGF8 신호의 관여 여부를 확인하였다. 신경전구세포에 BIO 를 처리함과 동시에, FGF8 를 첨가하거 나 FGF 신호전달체계의 억제제인 SU5402 를 처리하여 그 결과를 확인하 였다. BIO 와 FGF8을 함께 처리한 그룹에서는 BIO 만 처리한 그룹보다 En1 발현을 더 증가시켰고, 반대로 BIO 와 SU5402 를 함께 처리한 그룹 에서는 En1 의 발현을 BIO 만 처리했을 때보다 더 감소시켰다. 따라서, 중뇌 특이적 세포 형성에 Wnt/β-catenin 신호뿐만 아니라 FGF8 신호도 관 여하고 있다는 것을 알 수 있었다. 이 후, BIO 에 의해 중뇌 특이적으로 형성된 신경전구세포를 성숙한 도파민 신경세포로 분화시켜서 이들의 특 징을 확인해 보았다. 이들 신경세포는 신경세포 생성, 도파민 신경세포를 높은 수율로 발현하였고 중뇌 특이적 마커를 잘 발현함을 확인하였다.
핵심되는 말 : 인간 배아줄기세포, Wnt/β-catenin 신호전달체계, GSK-3 저 해제, 앞-뒤 축 형성, 신경전구세포, 중뇌 특이적 도파민 신경세포