东亚三角涡虫肌球蛋白必需轻链（DjElc）基因的表达及功能分析
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摘要
东亚三角涡虫是扁形动物门的代表动物(三肠目、无体腔),它具有明确定义的前后轴(AP)和背腹轴(DV)以及大脑和腹神经索组成的相对简单的中枢神经系统,更有着非凡的再生能力。在本研究中,为了鉴定涡虫肌球蛋白必需轻链DjElc基因在再生中的功能,我们克隆了DjElc基因,并研究了DjElc基因在成虫和胚胎发育早期各阶段的表达模式。系统发育分析表明：肌球蛋白必需轻链DjElc具有高度保守的序列特征,有EF-Hand结构域和钙离子结合位点,与脊椎动物和无脊椎动物都有相当高的同源性,这可能是肌球蛋白必需轻链DjElc发挥作用的关键。本文运用相应的技术探索DjElc基因在涡虫生长发育和再生过程中的生物学功能,为进一步深入研究涡虫胚胎发育和再生机制、神经起源奠定基础。
肌球蛋白必需轻链是构成肌球蛋白结构的关键元素,其作用主要是通过α-螺旋杠杆臂来维持肌球蛋白结构的稳定性。从涡虫cDNA文库中鉴定的DjElc cDNA为肌球蛋白必需轻链基因。整体原位杂交研究发现胚胎发育的第一阶段并没有检测到阳性信号；信号最初出现在Stage2,卵裂球均匀的分散在合胞体上,胚胎咽和胚胎表皮分化完成,这时信号定位在胚胎咽上；Stage3时胚胎摄取外卵黄,信号在胚胎咽及胚胎外壁上表达；Stage4,信号定位在胚带和胚胎咽上；Stage5、Stage6阶段,胚胎开始拉伸,早期器官初步形成信号点状分布于腹神经索、真正的咽和外围实质组织上；Stage7,肌肉组织分化、大脑缩合,胚胎丌始向幼虫变化,这时DjElc基因的表达与成虫和再生整体原位杂交结果显示一致均在大脑和中枢神经上表达并沿腹神经索延伸。以上所有结果说明该基因与肌球蛋白Ⅱ在轴突生长中所提到的功能相一致,暗示DjElc基因潜在的功能也许参与涡虫中枢神经系统CNS的再生
为了进一步检测肌球蛋白必需轻链基因彻底沉默后是否导致中枢神经系统的缺失,我们筛选出干扰再生后不同时期代表性的片段,用特异的神经marker anti-SYNORF1 (3C11)进行免疫组化实验,检测发现干扰后涡虫能再生它们的神经回路,但是大脑侧枝不能正常再生、神经元轴突缺陷,特别是尾部再生头部的缺失最为明显。
综上所述肌球蛋白必需轻链基因不仅对于大脑再生中轴突的延伸是必需的而且对维持神经元和神经生长相一致也是必需的。
Dugesia japonica Planarian is representative animals of Platyhelminthes (Triclada、acoelomate), It have extraordinary regenerative ability, simple relatively nervous system and well-defined anteroposterior (AP) and dorsoventral (DV) axes. In this study, to identify the function of DjElc gene in Planarian regeneration, we cloning planarian DjElc gene, and study DjElc gene in the expression patterns in embryonic development and regeneration, Phylogenetic analysis showed that DjElc gene had an EF-Hand domain and Ca2+site which indicated a high degree of identity compared with Vertebrates and invertebrates.This may be the key to its roles. In this paper Explore DjElc gene in planarian growth and regeneration of biological functions, lay the foundation for further study of embryonic development regeneration mechanisms and neural origin in planarians.
The essential light chain of myosin is a key element in myosin structure that is known to be important for structural stability of the a-helical lever arm domain. The cDNA DjElc, encoding a planarian essential light chain of myosin, was identified from the planarian Dugesia japonica cDNA library. Whole mount in situ hybridization studies show that DjElc transcripts were not detectable in the embryos of stage 1, but were detected from stage 2 to juvenile. At satge 2, cleavage had finished and blastomeres were dispersed within syncytium, and the embryonic epidermis and embryo pharynx had differentiated the expression of DjElc could be observed in embryonic pharynx. In stage3 the embryos ingested the yolk contents, the signal of DjElc displayed in the embryonic pharynx and embryonic wall. In stage 4 embryos, DjElc transcripts were apparently localized in the germ band and embryonic pharynx. During the embryonic stretching and early organogenesis formed at stage 5 and 6, the expression of DjElc was distributed in a dotted pattern along the VNCs, parenchyma, and definitive pharynx. Then the embryo becomes worm-shaped due to muscular differentiation and brain condensation by stage 7, DjElc is expressed in nervous system, and continued to be detected in brain and VNCs at juvenile, as well as observed in the adult and regeneration. These data consistent with the established role of myosin II in axon outgrowth, Imply a potential involvement of DjElc in the formation of CNS in planarian.
To test whether DjElc depletion by RNAi caused the loss of CNS structures, we screened a representative sample of regenerating fragments in different times by immunostaining with anti-SYNORF1(3C11).The results revealed that RNAi-treated planarians could regenerate their neural circuits, but the lateral branch of the brain could not. formed normally axon defect, especially in regenerated head from tail pieces.
In sum, all of these results suggested that DjElc may be required not only for the axonal extension at the brain regeneration after patterning, but also for maintenance of neurons consistent with the nerve outgrowth required myosin II activity to variable degree.
肌球蛋白必需轻链是构成肌球蛋白结构的关键元素,其作用主要是通过α-螺旋杠杆臂来维持肌球蛋白结构的稳定性。从涡虫cDNA文库中鉴定的DjElc cDNA为肌球蛋白必需轻链基因。整体原位杂交研究发现胚胎发育的第一阶段并没有检测到阳性信号；信号最初出现在Stage2,卵裂球均匀的分散在合胞体上,胚胎咽和胚胎表皮分化完成,这时信号定位在胚胎咽上；Stage3时胚胎摄取外卵黄,信号在胚胎咽及胚胎外壁上表达；Stage4,信号定位在胚带和胚胎咽上；Stage5、Stage6阶段,胚胎开始拉伸,早期器官初步形成信号点状分布于腹神经索、真正的咽和外围实质组织上；Stage7,肌肉组织分化、大脑缩合,胚胎丌始向幼虫变化,这时DjElc基因的表达与成虫和再生整体原位杂交结果显示一致均在大脑和中枢神经上表达并沿腹神经索延伸。以上所有结果说明该基因与肌球蛋白Ⅱ在轴突生长中所提到的功能相一致,暗示DjElc基因潜在的功能也许参与涡虫中枢神经系统CNS的再生
为了进一步检测肌球蛋白必需轻链基因彻底沉默后是否导致中枢神经系统的缺失,我们筛选出干扰再生后不同时期代表性的片段,用特异的神经marker anti-SYNORF1 (3C11)进行免疫组化实验,检测发现干扰后涡虫能再生它们的神经回路,但是大脑侧枝不能正常再生、神经元轴突缺陷,特别是尾部再生头部的缺失最为明显。
综上所述肌球蛋白必需轻链基因不仅对于大脑再生中轴突的延伸是必需的而且对维持神经元和神经生长相一致也是必需的。
Dugesia japonica Planarian is representative animals of Platyhelminthes (Triclada、acoelomate), It have extraordinary regenerative ability, simple relatively nervous system and well-defined anteroposterior (AP) and dorsoventral (DV) axes. In this study, to identify the function of DjElc gene in Planarian regeneration, we cloning planarian DjElc gene, and study DjElc gene in the expression patterns in embryonic development and regeneration, Phylogenetic analysis showed that DjElc gene had an EF-Hand domain and Ca2+site which indicated a high degree of identity compared with Vertebrates and invertebrates.This may be the key to its roles. In this paper Explore DjElc gene in planarian growth and regeneration of biological functions, lay the foundation for further study of embryonic development regeneration mechanisms and neural origin in planarians.
The essential light chain of myosin is a key element in myosin structure that is known to be important for structural stability of the a-helical lever arm domain. The cDNA DjElc, encoding a planarian essential light chain of myosin, was identified from the planarian Dugesia japonica cDNA library. Whole mount in situ hybridization studies show that DjElc transcripts were not detectable in the embryos of stage 1, but were detected from stage 2 to juvenile. At satge 2, cleavage had finished and blastomeres were dispersed within syncytium, and the embryonic epidermis and embryo pharynx had differentiated the expression of DjElc could be observed in embryonic pharynx. In stage3 the embryos ingested the yolk contents, the signal of DjElc displayed in the embryonic pharynx and embryonic wall. In stage 4 embryos, DjElc transcripts were apparently localized in the germ band and embryonic pharynx. During the embryonic stretching and early organogenesis formed at stage 5 and 6, the expression of DjElc was distributed in a dotted pattern along the VNCs, parenchyma, and definitive pharynx. Then the embryo becomes worm-shaped due to muscular differentiation and brain condensation by stage 7, DjElc is expressed in nervous system, and continued to be detected in brain and VNCs at juvenile, as well as observed in the adult and regeneration. These data consistent with the established role of myosin II in axon outgrowth, Imply a potential involvement of DjElc in the formation of CNS in planarian.
To test whether DjElc depletion by RNAi caused the loss of CNS structures, we screened a representative sample of regenerating fragments in different times by immunostaining with anti-SYNORF1(3C11).The results revealed that RNAi-treated planarians could regenerate their neural circuits, but the lateral branch of the brain could not. formed normally axon defect, especially in regenerated head from tail pieces.
In sum, all of these results suggested that DjElc may be required not only for the axonal extension at the brain regeneration after patterning, but also for maintenance of neurons consistent with the nerve outgrowth required myosin II activity to variable degree.
引文
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10.王晓安,平角涡虫早期胚胎发育的观察(山东省烟台师范学院生物学系264025)
11. Cebria, F., Regenerating the central nervous system:how easy for planarians! Development Genes and Evolution,2007.217(11-12):p.733-748.
12. Keiji okamoto,kosei takeuchi and kiyokazu agata.Neural projections in Planarian brain revealed by fluorescent dye tracing. Zoological science 22:535-546(2005)
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19. Brown, J. and P.C. Bridgman, Role of myosin Ⅱ in axon outgrowth. Journal of Histochemistry & Cytochemistry,2003.51(4):p.421-428.
20. Diefenbach, T.J., et al., Myosin 1c and myosin ⅡB serve opposing roles in lamellipodial dynamics of the neuronal growth cone. Journal of Cell Biology,2002.158(7):p. 1207-1217.
21. Walker, A., et al., Non-muscle myosin Ⅱ regulates survival threshold of pluripotent stem cells. Nature Communications,2010.1:p.-
22. Kwon, Y.C., et al., Nonmuscle myosin Ⅱ localization is regulated by JNK during Drosophila larval wound healing. Biochemical and Biophysical Research Communications,2010.393(4):p.656-661.
23. Escudero, L.M., M. Bischoff, and M. Freeman, Myosin Ⅱ regulates complex cellular arrangement and epithelial architecture in drosophila. Developmental Cell,2007.13(5):p. 717-729.
24. Barros, C.S., C.B. Phelps, and A.H. Brand, Drosophila nonmuscle myosin II promotes the asymmetric segregation of cell fate determinants by cortical exclusion rather than active transport. Developmental Cell,2003.5(6):p.829-840.
25. Lenz, S., et al., The alkali light chains of human smooth and nonmuscle myosins are encoded by a single gene. Tissue-specific expression by alternative splicing pathways. Journal of Biological Chemistry,1989.264(15):p.9009-15.
26. Ikura, M., Calcium binding and conformational response in EF-hand proteins. Trends in Biochemical Sciences,1996.21(1):p.14-17.
27. Bridgman, P.C. and L.L. Elkin, Axonal myosins. Journal of Neurocytology,2000. 29(11-12):p.831-841.
28. Park I, H.C., Jin S, Lee B, Choi H, Kwon JT, Kim D, Kim J, Lifirsu E, Park WJ, Park ZY, Kim do H, Cho C., Myosin regulatory light chains are required to maintain the stability of myosin Ⅱ and cellular integrity. Biochem J,2011.434(1):p.171-80.
29. Ushakov, D.S., Structure and function of the essential light chain of myosin. Biofizika, 2008.53(6):p.950-5.
30. Newmark, P.A. and A.S. Alvarado, Not your father's planarian:A classic model enters the era of functional genomics. Nature Reviews Genetics,2002.3(3):p.210-219.
31. Cardona, A., et al., An in situ hybridization protocol for planarian embryos:monitoring myosin heavy chain gene expression. Development Genes and Evolution,2005.215(9):p. 482-488.
32. Sakai, T., et al., Planarian pharynx regeneration revealed by the expression of myosin heavy chain-A. International Journal of Developmental Biology,2002.46(3):p.329-32.
33. Han, F., Z. Wang, and X. Wang, Characterization of myosin light chain in shrimp hemocytic phagocytosis. Fish & Shellfish Immunology,2010.29(5):p.875-883.
34. Altschul S.F., Madden T.L., Schaffer A.A., Zhang J.,Zhang Z., Miller W., Lipman D.J. 1997. Gapped BLAST and PSI_BLAST:A new generation of protein database search programs. Nucleic Acids Res.25,3389-3402.
35. Rehm B.H.2001. Bioinformatic tools for DNA/protein sequence analysis, functional assignment of genes and protein classification. Appl. Microbiol. Biotechnol.57,579-592.
36. Qu X., Wang Y, Geng W., Zhao B.2008. Construction of cDNA library and trial EST analysis from planaria (Dujesia japonica). Sichuan J. Zool.127,2
37. Bresnick, A. R.,1999. Molecular mechanisms of nonmuscle myosin-Ⅱ regulation. Current Opinion in Cell Biology.11,26-33.
38. Hernandez, O. M., Jones, M., Guzman, G., Szczesna-Cordary, D.,2007. Myosin essential light chain in health and disease. American Journal of Physiology-Heart and Circulatory Physiology.292, H1643-H1654.
39. Timson, D. J.,2003. Fine tuning the myosin motor:the role of the essential light chain in striated muscle myosin. Biochimie.85,639-645.
40. Falkenthal, S., Parker, V. P., Mattox, W. W., Davidson, N.,1984. Drosophila melanogaster has only one myosin alkali light-chain gene which encodes a protein with considerable amino acid sequence homology to chicken myosin alkali light chains. Molecular and cellular biology.4,956-65.
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2. Salo, E. and J. Baguna, Regeneration in Planarians and other worms:New findings, new tools, and new perspectives. Journal of Experimental Zoology,2002.292(6):p.528-539.
3. Agata, K., et al., Intercalary regeneration in planarians. Developmental Dynamics,2003. 226(2):p.308-316.
4. Reddien, P.W. and A.S. Alvarado, Fundamentals of planarian regeneration. Annual Review of Cell and Developmental Biology,2004.20:p.725-757.
5. Inoue, T., et al., Morphological and functional recovery of the planarian photosensing system during head regeneration. Zoological Science,2004.21(3):p.275-283.
6. Reddien, P.W., et al., Identification of genes needed for regeneration, stem cell function, and tissue homeostasis by systematic gene perturbation in planaria. Developmental Cell, 2005.8(5):p.635-649.
7. Agata, K. and Y. Umesono, Brain regeneration from pluripotent stem cells in planarian. Philosophical Transactions of the Royal Society B-Biological Sciences,2008.363(1500): p.2071-2078.
8. Cebria, F. and P.A. Newmark, Morphogenesis defects are associated with abnormal nervous system regeneration following roboA RNAi in planarians. Development,2007. 134(5):p.833-837.
9. Gentile L, C.F., and Bartscherer K, The planarian flatworm:an in vivo model for stem cell biology and nervous system regeneration, in Dis Model Mech 2011. p.12-19.
10.王晓安,平角涡虫早期胚胎发育的观察(山东省烟台师范学院生物学系264025)
11. Cebria, F., Regenerating the central nervous system:how easy for planarians! Development Genes and Evolution,2007.217(11-12):p.733-748.
12. Keiji okamoto,kosei takeuchi and kiyokazu agata.Neural projections in Planarian brain revealed by fluorescent dye tracing. Zoological science 22:535-546(2005)
13. Holland, L.Z., et al., Sequence and Expression of Amphioxus Alkali Myosin Light-Chain (Amphimlc-Alk) Throughout Development-Implications for Vertebrate Myogenesis. Developmental Biology,1995.171(2):p.665-676.
14. Falkenthal, S., et al., Drosophila melanogaster has only one myosin alkali light-chain gene which encodes a protein with considerable amino acid sequence homology to chicken myosin alkali light chains. Molecular and cellular biology,1984.4(5):p.956-65.
15. Goodwin, E.B., A.G. Szent-Gyorgyi, and L.A. Leinwand, Cloning and characterization of the scallop essential and regulatory myosin light chain cDNAs. Journal of Biological Chemistry,1987.262(23):p.11052-6.
16. Clark, K., et al., Myosin Ⅱ and mechanotransduction:a balancing act. Trends in Cell Biology,2007.17(4):p.178-186.
17. Vicente-Manzanares, M., et al., Non-muscle myosin Ⅱ takes centre stage in cell adhesion and migration. Nature Reviews Molecular Cell Biology,2009.10(11):p.778-790.
18. Franke, J.D., R.A. Montague, and D.P. Kiehart, Nonmuscle myosin Ⅱ is required for cell proliferation, cell sheet adhesion and wing hair morphology during wing morphogenesis. Developmental Biology,2010.345(2):p.117-132.
19. Brown, J. and P.C. Bridgman, Role of myosin Ⅱ in axon outgrowth. Journal of Histochemistry & Cytochemistry,2003.51(4):p.421-428.
20. Diefenbach, T.J., et al., Myosin 1c and myosin ⅡB serve opposing roles in lamellipodial dynamics of the neuronal growth cone. Journal of Cell Biology,2002.158(7):p. 1207-1217.
21. Walker, A., et al., Non-muscle myosin Ⅱ regulates survival threshold of pluripotent stem cells. Nature Communications,2010.1:p.-
22. Kwon, Y.C., et al., Nonmuscle myosin Ⅱ localization is regulated by JNK during Drosophila larval wound healing. Biochemical and Biophysical Research Communications,2010.393(4):p.656-661.
23. Escudero, L.M., M. Bischoff, and M. Freeman, Myosin Ⅱ regulates complex cellular arrangement and epithelial architecture in drosophila. Developmental Cell,2007.13(5):p. 717-729.
24. Barros, C.S., C.B. Phelps, and A.H. Brand, Drosophila nonmuscle myosin II promotes the asymmetric segregation of cell fate determinants by cortical exclusion rather than active transport. Developmental Cell,2003.5(6):p.829-840.
25. Lenz, S., et al., The alkali light chains of human smooth and nonmuscle myosins are encoded by a single gene. Tissue-specific expression by alternative splicing pathways. Journal of Biological Chemistry,1989.264(15):p.9009-15.
26. Ikura, M., Calcium binding and conformational response in EF-hand proteins. Trends in Biochemical Sciences,1996.21(1):p.14-17.
27. Bridgman, P.C. and L.L. Elkin, Axonal myosins. Journal of Neurocytology,2000. 29(11-12):p.831-841.
28. Park I, H.C., Jin S, Lee B, Choi H, Kwon JT, Kim D, Kim J, Lifirsu E, Park WJ, Park ZY, Kim do H, Cho C., Myosin regulatory light chains are required to maintain the stability of myosin Ⅱ and cellular integrity. Biochem J,2011.434(1):p.171-80.
29. Ushakov, D.S., Structure and function of the essential light chain of myosin. Biofizika, 2008.53(6):p.950-5.
30. Newmark, P.A. and A.S. Alvarado, Not your father's planarian:A classic model enters the era of functional genomics. Nature Reviews Genetics,2002.3(3):p.210-219.
31. Cardona, A., et al., An in situ hybridization protocol for planarian embryos:monitoring myosin heavy chain gene expression. Development Genes and Evolution,2005.215(9):p. 482-488.
32. Sakai, T., et al., Planarian pharynx regeneration revealed by the expression of myosin heavy chain-A. International Journal of Developmental Biology,2002.46(3):p.329-32.
33. Han, F., Z. Wang, and X. Wang, Characterization of myosin light chain in shrimp hemocytic phagocytosis. Fish & Shellfish Immunology,2010.29(5):p.875-883.
34. Altschul S.F., Madden T.L., Schaffer A.A., Zhang J.,Zhang Z., Miller W., Lipman D.J. 1997. Gapped BLAST and PSI_BLAST:A new generation of protein database search programs. Nucleic Acids Res.25,3389-3402.
35. Rehm B.H.2001. Bioinformatic tools for DNA/protein sequence analysis, functional assignment of genes and protein classification. Appl. Microbiol. Biotechnol.57,579-592.
36. Qu X., Wang Y, Geng W., Zhao B.2008. Construction of cDNA library and trial EST analysis from planaria (Dujesia japonica). Sichuan J. Zool.127,2
37. Bresnick, A. R.,1999. Molecular mechanisms of nonmuscle myosin-Ⅱ regulation. Current Opinion in Cell Biology.11,26-33.
38. Hernandez, O. M., Jones, M., Guzman, G., Szczesna-Cordary, D.,2007. Myosin essential light chain in health and disease. American Journal of Physiology-Heart and Circulatory Physiology.292, H1643-H1654.
39. Timson, D. J.,2003. Fine tuning the myosin motor:the role of the essential light chain in striated muscle myosin. Biochimie.85,639-645.
40. Falkenthal, S., Parker, V. P., Mattox, W. W., Davidson, N.,1984. Drosophila melanogaster has only one myosin alkali light-chain gene which encodes a protein with considerable amino acid sequence homology to chicken myosin alkali light chains. Molecular and cellular biology.4,956-65.
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