原钙粘附蛋白（Protocadherin）18b在斑马鱼胚胎神经系统发育中的作用
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摘要
原钙粘附蛋白(protocadherin)是钙粘蛋白超家族中一个最大的亚族。钙粘附蛋白家族(cadherin family)为钙离子依赖型粘附分子,均为单链跨膜糖蛋白,介导特定组织或器官同型细胞间粘附,对胚胎发育中细胞识别、迁移、通讯和组织分化及中枢神经系统中神经回路的形成具有重要作用。原钙粘附蛋白在神经元发育和突触形成中有重要的作用。
原钙粘附蛋白18(protocadherinl 8,Pcdh18)基因表达主要在小鼠的前脑、后脑脑室管膜区、嗅泡、大脑皮层、丘脑、及小脑中。斑马鱼Pcdhl8b则在神经管和中枢神经系统表达,其在神经系统发育中的作用及其分子机制尚不清楚。新兴模式生物斑马鱼胚胎透明、体外发育、个体小、繁殖率高、产卵周期长以及鱼种发育遗传背景清晰等优点使其正成为研究脊椎动物胚胎发育过程中基因功能的理想模式系统。2000年,Nasevicius等首次用吗啡啉修饰性寡核苷酸(MO)获得了目的基因表达下调的斑马鱼。从而建立了特异、快速、高效下调斑马鱼基因表达的重要技术,加快了基因功能研究的步伐。我们利用这种技术研究斑马鱼胚胎发育中pcdhl8b基因的功能,显示了独特优势。
本文第一部分：利用生物信息学的方法证明在脊椎动物进化过程中Pcdh18分子的功能结构域保守。用反义RNA探针-全胚胎原位杂交和RT-PCR的方法研究pcdhl8b基因在斑马鱼胚胎发育早期的时空表达谱。
本文第二部分：用靶向pcdhl8bmRNA的MO (pcdh18b-MO)干扰Pcdhl8b翻译起始,建立了pcdhl8b基因表达下调的斑马鱼模型。为了验证pcdh18b-MO的有效性,我们将其与编码pcdh18b-EGFP蛋白质的质粒DNA共注射,结果显示pcdh18b-MO抑制了pcdhl8b-EGFP蛋白的表达,从而也验证了pcdh18b-MO能够有效抑制内源性的pcdhl8b表达；并显示了pcdh18b-MO的特异性。
本文第三部分：在胚胎整体发育水平上,我们运用胚胎整体原位杂交方法分析pcdh18基因表达下调后神经系统标志基因的表达以及脑部的表型,同时利用丫叮橙染色的方法检测脑部细胞凋亡情况。pcdhl8b下调使神经前体细胞的标志基因neurogl..神经元标志基因elav13和神经胶质细胞标志基因gfap的表达均出现下调,中后脑边界的标志基因pax2a和wnt1表达减弱并出现神经管分叉现象,同时与后脑分节相关的基因krox20表达减少。丫叮橙染色显示pcdhl8b下调后斑马鱼中脑、后脑及中后脑边界细胞凋亡增多.这些结果表明pcdhl8b基因表达下降导致斑马鱼神经系统发育的异常.
本文第四部分：本部分利用J. Keith Joung采用的方式建立锌指库,设计并构建针对斑马鱼pcdhl8b基因的锌指核酸酶(pcdh18b-ZFN)载体,同时体外转录了pcdhl8b-ZFN mRNA。
Protocadherins constitute the largest subgroup within the cadherin family of calcium-dependent cell-cell adhesion molecules. Cadherins associate laterally within the same plasma membrane to form parallel cis dimers, and cadherins protruding from adjacent plasma membranes associate in an anti-parallel fashion to form transdimers. in particular, primary cadherins (classic cadherins) were identified as synaptic components, and roles for them in neuronal circuitry, synaptic junction formation, and synaptic plasticity have been suggested. Interestingly, many of theprotocadherins in mammals are highly expressed in the central nervous system. Roles in tissue morphogenesis and formation of neuronal circuits during early vertebrate development have been inferred. In the postnatal brain, protocadherins are possibly involved in the modulation of synaptic transmission and the generation of specific synaptic connections.
Mouse Pcdhl8(mPcdhl8), which consists of four exons similar to other protocadherin family members, maps to chromosome3. The amino acid sequence of mPcdhl8 contains six extracellular cadherin motifs, a single transmembrane region, and a large intracellular domain. Although Pcdhl8 was described in mice, not much is known about its role in embryonic development. Mouse Pcdhl8 is present throughout the embryo, in particular in the ventricular zone in the forebrain and midbrain, in the olfactory bulb, cerebral cortex, thalamus, cerebellum, and in additional organs of the adult. The expression pattern of pcdhl8 in zebrafish embryos seemed to be quite different from that in mice; pcdhl8 expression is rather systemic in rodents whereas it was limited to the CNS and head structures in zebrafish. Nonetheless, further comparative studies on the functional diversity of pcdhl8 in zebrafish are required.
In recent years, zebrafish has emerged as an exciting animal model system for studying vertebrate organ development, in particular, the development of centre neural system, as zebrafish embryos are optically transparent, easily manipulated. Owing to its optical clarity, genetics, and ease of manipulation, zebrafish may be an ideal model system to obtain a comprehensive understanding of pcdhl8b in vivo. In the present study, we turn to the zebrafish embryos to pursue the role of pcdhl8b during embryonic neurogenesis.
To study the role of pcdhl8b on embryonic neurogenesis in zebrafish, we injected one type of well designed antisense morpholino oligonucleotide into one or two-cell stage embryos to block the translation of pcdhl8b. After injection, the phenotypes of nervous system were monitored by whole mount in situ hybridization and acridine orange staining. Whole-mount in situ hybridization with neurogl, elav13, gfap and krox20 RNA probes showed that the expression of neural precursor cells, neurons, neuroglia cells and rhombencephalon3,5 was strikingly affected; meanwhile, the expression of MHB (midbrain-hindbrain boundary) markers pax2a and wntl was significantly compromised and showed duplication of neural tube in pcdh18b down regulation group. Acridine orange staining pointed out that down regulation of pcdhl8b increased cell apoptosis in midbrain, hindbrain and MHB region. These results suggest that pcdhl8b plays an important role in the zabrafish neurogenesis.
Several platforms for constructing artificial zinc finger arrays using'modular assembly'have been described by J Keith Joung, standardized reagents and protocols that permit rapid, cross-platform'mixing-and-matching'of the various zinc finger modules are available. Meanwhile, we constructed zinc finger nuclease expression vector for knockouting pcdhl8b, and transcribed mRNA in vitro.
原钙粘附蛋白18(protocadherinl 8,Pcdh18)基因表达主要在小鼠的前脑、后脑脑室管膜区、嗅泡、大脑皮层、丘脑、及小脑中。斑马鱼Pcdhl8b则在神经管和中枢神经系统表达,其在神经系统发育中的作用及其分子机制尚不清楚。新兴模式生物斑马鱼胚胎透明、体外发育、个体小、繁殖率高、产卵周期长以及鱼种发育遗传背景清晰等优点使其正成为研究脊椎动物胚胎发育过程中基因功能的理想模式系统。2000年,Nasevicius等首次用吗啡啉修饰性寡核苷酸(MO)获得了目的基因表达下调的斑马鱼。从而建立了特异、快速、高效下调斑马鱼基因表达的重要技术,加快了基因功能研究的步伐。我们利用这种技术研究斑马鱼胚胎发育中pcdhl8b基因的功能,显示了独特优势。
本文第一部分：利用生物信息学的方法证明在脊椎动物进化过程中Pcdh18分子的功能结构域保守。用反义RNA探针-全胚胎原位杂交和RT-PCR的方法研究pcdhl8b基因在斑马鱼胚胎发育早期的时空表达谱。
本文第二部分：用靶向pcdhl8bmRNA的MO (pcdh18b-MO)干扰Pcdhl8b翻译起始,建立了pcdhl8b基因表达下调的斑马鱼模型。为了验证pcdh18b-MO的有效性,我们将其与编码pcdh18b-EGFP蛋白质的质粒DNA共注射,结果显示pcdh18b-MO抑制了pcdhl8b-EGFP蛋白的表达,从而也验证了pcdh18b-MO能够有效抑制内源性的pcdhl8b表达；并显示了pcdh18b-MO的特异性。
本文第三部分：在胚胎整体发育水平上,我们运用胚胎整体原位杂交方法分析pcdh18基因表达下调后神经系统标志基因的表达以及脑部的表型,同时利用丫叮橙染色的方法检测脑部细胞凋亡情况。pcdhl8b下调使神经前体细胞的标志基因neurogl..神经元标志基因elav13和神经胶质细胞标志基因gfap的表达均出现下调,中后脑边界的标志基因pax2a和wnt1表达减弱并出现神经管分叉现象,同时与后脑分节相关的基因krox20表达减少。丫叮橙染色显示pcdhl8b下调后斑马鱼中脑、后脑及中后脑边界细胞凋亡增多.这些结果表明pcdhl8b基因表达下降导致斑马鱼神经系统发育的异常.
本文第四部分：本部分利用J. Keith Joung采用的方式建立锌指库,设计并构建针对斑马鱼pcdhl8b基因的锌指核酸酶(pcdh18b-ZFN)载体,同时体外转录了pcdhl8b-ZFN mRNA。
Protocadherins constitute the largest subgroup within the cadherin family of calcium-dependent cell-cell adhesion molecules. Cadherins associate laterally within the same plasma membrane to form parallel cis dimers, and cadherins protruding from adjacent plasma membranes associate in an anti-parallel fashion to form transdimers. in particular, primary cadherins (classic cadherins) were identified as synaptic components, and roles for them in neuronal circuitry, synaptic junction formation, and synaptic plasticity have been suggested. Interestingly, many of theprotocadherins in mammals are highly expressed in the central nervous system. Roles in tissue morphogenesis and formation of neuronal circuits during early vertebrate development have been inferred. In the postnatal brain, protocadherins are possibly involved in the modulation of synaptic transmission and the generation of specific synaptic connections.
Mouse Pcdhl8(mPcdhl8), which consists of four exons similar to other protocadherin family members, maps to chromosome3. The amino acid sequence of mPcdhl8 contains six extracellular cadherin motifs, a single transmembrane region, and a large intracellular domain. Although Pcdhl8 was described in mice, not much is known about its role in embryonic development. Mouse Pcdhl8 is present throughout the embryo, in particular in the ventricular zone in the forebrain and midbrain, in the olfactory bulb, cerebral cortex, thalamus, cerebellum, and in additional organs of the adult. The expression pattern of pcdhl8 in zebrafish embryos seemed to be quite different from that in mice; pcdhl8 expression is rather systemic in rodents whereas it was limited to the CNS and head structures in zebrafish. Nonetheless, further comparative studies on the functional diversity of pcdhl8 in zebrafish are required.
In recent years, zebrafish has emerged as an exciting animal model system for studying vertebrate organ development, in particular, the development of centre neural system, as zebrafish embryos are optically transparent, easily manipulated. Owing to its optical clarity, genetics, and ease of manipulation, zebrafish may be an ideal model system to obtain a comprehensive understanding of pcdhl8b in vivo. In the present study, we turn to the zebrafish embryos to pursue the role of pcdhl8b during embryonic neurogenesis.
To study the role of pcdhl8b on embryonic neurogenesis in zebrafish, we injected one type of well designed antisense morpholino oligonucleotide into one or two-cell stage embryos to block the translation of pcdhl8b. After injection, the phenotypes of nervous system were monitored by whole mount in situ hybridization and acridine orange staining. Whole-mount in situ hybridization with neurogl, elav13, gfap and krox20 RNA probes showed that the expression of neural precursor cells, neurons, neuroglia cells and rhombencephalon3,5 was strikingly affected; meanwhile, the expression of MHB (midbrain-hindbrain boundary) markers pax2a and wntl was significantly compromised and showed duplication of neural tube in pcdh18b down regulation group. Acridine orange staining pointed out that down regulation of pcdhl8b increased cell apoptosis in midbrain, hindbrain and MHB region. These results suggest that pcdhl8b plays an important role in the zabrafish neurogenesis.
Several platforms for constructing artificial zinc finger arrays using'modular assembly'have been described by J Keith Joung, standardized reagents and protocols that permit rapid, cross-platform'mixing-and-matching'of the various zinc finger modules are available. Meanwhile, we constructed zinc finger nuclease expression vector for knockouting pcdhl8b, and transcribed mRNA in vitro.
引文
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2. Yagi, T. and M. Takeichi, Cadherin superfamily genes:functions, genomic organization, and neurologic diversity. Genes Dev,2000.14(10):p.1169-80.
3. Hatta, K., et al., Cloning and expression of cDNA encoding a neural calcium-dependent cell adhesion molecule:its identity in the cadherin gene family. J Cell Biol,1988.106(3):p.873-81.
4. Morishita, H. and T. Yagi, Protocadherin family:diversity, structure, and function. Curr Opin Cell Biol,2007.19(5):p.584-92.
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7. Cronin, K.D. and A.A. Capehart, Gamma protocadherin expression in the embryonic chick nervous system. Int J Biol Sci,2007.3(1):p.8-11.
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9. Sano, K., et al., Protocadherins:a large family of cadherin-related molecules in central nervous system. EMBO J,1993.12(6):p.2249-56.
10. Washbourne, P., et al., Cell adhesion molecules in synapse formation. J Neurosci,2004.24(42):p.9244-9.
11. Bass, T., et al., Differential expression of four protocadherin alpha and gamma clusters in the developing and adult zebrafish:DrPcdh2gamma but not DrPcdhl gamma is expressed in neuronal precursor cells, ependymal cells and non-neural epithelia. Dev Genes Evol,2007.217(5):p.337-51.
12. Nasevicius, A. and S.C. Ekker, Effective targeted gene'knockdown'in zebrafish. Nat Genet,2000.26(2):p.216-20.
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14. Kimmel, C.B., et al., Stages of embryonic development of the zebrafish. Dev Dyn,1995.203(3):p.253-310.
15. Homayouni, R., D.S. Rice, and T. Curran, Disabled-1 interacts with a novel developmentally regulated protocadherin. Biochem Biophys Res Commun, 2001.289(2):p.539-47.
16. Wolverton, T. and M. Lalande, Identification and characterization of three members of a novel subclass of protocadherins. Genomics,2001.76(1-3):p. 66-72.
17. Cau, E., S. Casarosa, and F. Guillemot, Mashl and Ngnl control distinct steps of determination and differentiation in the olfactory sensory neuron lineage. Development,2002.129(8):p.1871-80.
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18. Urnov, F.D., et al., Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature,2005.435(7042):p.646-51.
19. Nazari, R. and S. Joshi, CCR5 as target for HIV-1 gene therapy. Curr Gene Ther,2008.8(4):p.264-72.
20. Perez, E.E., et al., Establishment of HIV-1 resistance in CD4+T cells by genome editing using zinc-finger nucleases. Nat Biotechnol,2008.26(7):p-808-16.
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23. Doyon, Y., et al., Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nat Biotechnol,2008.26(6):p.702-8.
2. Yagi, T. and M. Takeichi, Cadherin superfamily genes:functions, genomic organization, and neurologic diversity. Genes Dev,2000.14(10):p.1169-80.
3. Hatta, K., et al., Cloning and expression of cDNA encoding a neural calcium-dependent cell adhesion molecule:its identity in the cadherin gene family. J Cell Biol,1988.106(3):p.873-81.
4. Morishita, H. and T. Yagi, Protocadherin family:diversity, structure, and function. Curr Opin Cell Biol,2007.19(5):p.584-92.
5. Angst, B.D., C. Marcozzi, and A.I. Magee, The cadherin superfamily: diversity inform and function. J Cell Sci,2001.114(Pt 4):p.629-41.
6. Frank, M. and R. Kemler, Protocadherins. Curr Opin Cell Biol,2002.14(5):p. 557-62.
7. Cronin, K.D. and A.A. Capehart, Gamma protocadherin expression in the embryonic chick nervous system. Int J Biol Sci,2007.3(1):p.8-11.
8. Yoshida, K., et al., BH-protocadherin-c, a member of the cadherin superfamily, interacts with protein phosphatase 1 alpha through its intracellular domain. FEBS Lett,1999.460(1):p.93-8.
9. Sano, K., et al., Protocadherins:a large family of cadherin-related molecules in central nervous system. EMBO J,1993.12(6):p.2249-56.
10. Washbourne, P., et al., Cell adhesion molecules in synapse formation. J Neurosci,2004.24(42):p.9244-9.
11. Bass, T., et al., Differential expression of four protocadherin alpha and gamma clusters in the developing and adult zebrafish:DrPcdh2gamma but not DrPcdhl gamma is expressed in neuronal precursor cells, ependymal cells and non-neural epithelia. Dev Genes Evol,2007.217(5):p.337-51.
12. Nasevicius, A. and S.C. Ekker, Effective targeted gene'knockdown'in zebrafish. Nat Genet,2000.26(2):p.216-20.
13. Aamar, E. and I.B. Dawid, Protocadherin-18a has a role in cell adhesion, behavior and migration in zebrafish development. Dev Biol,2008.318(2):p. 335-46.
14. Kimmel, C.B., et al., Stages of embryonic development of the zebrafish. Dev Dyn,1995.203(3):p.253-310.
15. Homayouni, R., D.S. Rice, and T. Curran, Disabled-1 interacts with a novel developmentally regulated protocadherin. Biochem Biophys Res Commun, 2001.289(2):p.539-47.
16. Wolverton, T. and M. Lalande, Identification and characterization of three members of a novel subclass of protocadherins. Genomics,2001.76(1-3):p. 66-72.
17. Cau, E., S. Casarosa, and F. Guillemot, Mashl and Ngnl control distinct steps of determination and differentiation in the olfactory sensory neuron lineage. Development,2002.129(8):p.1871-80.
18. Kim, C.H., et al., Zebrafish elav/HuC homologue as a very early neuronal marker. Neuroscience Letters,1996.216(2):p.109-112.
19. Marusich, M.F., et al., Hu Neuronal Proteins Are Expressed in Proliferating Neurogenic Cells. Journal of Neurobiology,1994.25(2):p.143-155.
20. Bernardos, R.L. and P.A. Raymond, GFAP transgenic zebrafish. Gene Expr Patterns,2006.6(8):p.1007-13.
21. Krauss, S., et al., Expression of the zebrafish paired box gene pax[zf-b] during early neurogenesis. Development,1991.113(4):p.1193-206.
22. Pfeffer, P.L., et al., Characterization of three novel members of the zebrafish Pax2/5/8 family:dependency of Pax5 and Pax8 expression on the Pax2.1 (noi) function. Development,1998.125(16):p.3063-74.
23. Guo, C., et al., Lmxlb is essential for Fgf8 and Wntl expression in the isthmic organizer during tectum and cerebellum development in mice. Development, 2007.134(2):p.317-25.
24. Seitanidou, T., et al., Krox-20 is a key regulator of rhombomere-specific gene expression in the developing hindbrain. Mech Dev,1997.65(1-2):p.31-42.
25. Porteus, M.H. and D. Carroll, Gene targeting using zinc finger nucleases. Nat Biotechnol,2005.23(8):p.967-73.
26. Wright, D.A., et al., High-frequency homologous recombination in plants mediated by zinc-finger nucleases. Plant J,2005.44(4):p.693-705.
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