Daple基因在鼠脑内及体内的空间分布表达
摘要
研究目的：
Daple又称为含有高亮氨酸残基的Dvl相关蛋白。Daple是Girdin家族的基因(Girdin, Daple, FLJ004354)。其中Daple和Girdin在N端有很大的同源性,(54%的氨基酸相同),意味着两个基因功能相似,而C末端则与Girdin较少的相似性,只有27%,这意味着Daple具有不同于Girdin的特殊功能。Daple在Wnt经典途径中抑制β-catenin聚集和T细胞因子(Tcf)的转录活性,起负调节作用。而在非经典Wnt通路中增加蛋白激酶C(PKC)与Dvl互相作用激活Rac蛋白,起正调节作用。目前已知Daple在出生后期细胞迁移中起着重要的作用,Daple可能会影响胚胎发育、血管发生和肿瘤发展。目前哺乳动物Daple的分布表达的仍不明确。因此,全面了解Daple蛋白的功能及分布对于进一步了解相关疾病的发病机制及预后有重要意义。本研究重点是Daple基因的表达分布。
材料与方法：
通过SA剪接受体技术制作的Daple基因敲除的老鼠购自transgenic公司。出生7天的老鼠(p7；雌性)和8周老鼠(p56；雄性)(野生型老鼠(+／+)同源性老鼠(-／-))用4%PFA灌注,解剖出组织、冰冻、冰冻切片,解冻安装在载玻片上。Daple分布可以通过Lac Z染色显示并可以应用原位杂交技术验证LacZ染色的结果。用33P或地高辛标记的Daple cRNA探针(外显子30)。根据daple基因在脑中的分布,进一步对神经进行免疫组化染色,用PGP9.5, GFAP标记,判定daple基因是在神经元或是胶质细胞表达。根据其分布的特点对脑内可能出现的异常酶进行PCR和RT-PCR分析。
结果：
我们发现Lac Z染色的p7和p56的同源性Daple敲除的小鼠在纹状体、脑室室管膜细胞、小脑(大概在皮质上的浦肯野细胞)、海马、嗅球、脊髓、气管、膀胱、毛囊、舌、胃、脾、胰腺、心肌细胞、视网膜、肾乳头和门静脉,beta-geo(Daple启动子介导Lac Z和抗新霉素基因融合)表达而野生型小鼠不表达。原位杂交技术也证实了这个结果。我们发现在脉络丛和海马CA3区,Daple在神经胶质细胞中表达,而在纹状体中,Daple同时在胶质细胞和神经细胞中表达。酪氨酸羟化酶(TH)RT-PCR和eal time PCR的结果显示同源性Daple敲除小鼠和野生型小鼠在酪氨酸羟化酶表达中并没有显著性差异。
结论：
我们成功的描述出Daple的基因表达。Daple在脑、脊髓、眼、膀胱上皮及平滑肌、支气管上皮、胃、肾脏、睾丸、输精管、毛囊、舌、门静脉表达显著。Daple在小鼠脑中的分布主要在纹状体、海马、齿状回、皮质、黑质和室管膜细胞上。在脉络丛和海马CA3区,Daple在神经胶质细胞中表达,在纹状体中Daple同时在胶质细胞和神经细胞中表达。通过酪氨酸羟化酶PCR的结果显示同源性Daple敲除小鼠和野生型小鼠在酪氨酸羟化酶表达中并没有显著性差异,提示同源型敲除小鼠中多巴胺含量与野生型小鼠区别不大。希望通过了解Daple在小鼠全身器官、中枢神经系统分布情况,可以更清楚地了解Wnt信号通路的作用,并为以后的神经功能实验打下基础。
Objectives
Daple is named as Dishevelled-associating protein with a high frequency of leucine residues. Daple belongs to Girdin-family genes (Girdin, Daple, FLJ004354) Among those, Daple and Girdin show considerably high homology in N-terminal domains (54%amino acids identity), which implies the functional analogy between two genes in mice, however C-terminal domains lacks similarities(only27%amino acids identity) which means Daple has its own distinctive function. Daple plays a negative control over Wnt canonical pathway, inhibiting β-catenin aggregation and Tcf transcription. By contrast, Daple has a positive impact on Wnt non-canonical pathway by enhancing PKC/Dvl interaction and activation of Rac protein. Daple is known to play an important role in postnatal cell migration. It could possibly influence fetus development, angiogenesis and tumor development. Daple's distribution is still unknown. For the purpose of understanding related mechanisms of diseases, Daple's function and distribution should be under study. Here we performed preliminary study to know the distribution of Daple gene expression.
Materials and methods
Daple knock mice generated by SA (Splicing acceptor) trap vector techniques were purchased from TransGenic Co.,ltd. Seven-day-old (P7; female) and8-week-old mice (P56; male)(wild-type mice (+/+) and homozygous mice (-/-)) were perfused with4%PFA, then tissues were dissected, frozen, cryosectioned, and thaw-mounted on the glass slide. Daple expression was visualized using LacZ staining. To confirm LacZ staining results, in situ hybridization (ISH) was carried out using33P or Digoxigenin (DIG) labeled cRNA probe targeting Daple specific sequences (exon30). According to the distribution of Daple in the brain, we did immunohistochemistry to the brain tissue by using PGP9.5and GFAP probe. We could then determine whether Daple expresses in neuron or glial cells. We also intended to examine the possible abnormal enzyme by PCR and RT-PCR.
Results
LacZ staining of P7and P56brain exhibited significant expression of beta-geo (fusion of LacZ and Neomycin resistant gene driven by Daple promoter) in caudate putamen (striatum), ependymal cells of ventricles, cerebellum (cortex and presumably in Purkinje cells), hippocampus, olfactory bulb, bronchi, bladder, hair, tongue, stomach, intestine, spleen, spinal cord, pancrease, caridial muscle, retina, renal papillary and portal tract only in Daple (-/-) mice. ISH results confirmed the findings in LacZ staining.We found that in choroid plexus and hippocampus CA3area, daple expresses in glial cells whereas in striatum, daple expresses in both glial cells and neuron cells. Tyrosine hydroxylase (TH) RT-PCR and real time PCR results show that TH expression has no difference between homo daple knock out mice and wildtype mice.
Conclusion
We succeeded in depicting the preliminary map of Daple expression. Daple exhibited significant expression in brain, spinal cord, bronchi,bladder, hair, tongue, stomach, intestine, spleenpancrease, caridial muscle, retina, renal papillary and portal tract. Daple expresses most prominently in brain is that striatum, ependymal cells of ventricles, hippocampus, dentate gyrus, substantia nigra. In choroid plexus and hippocampus CA3area, daple expresses in glial cells whereas in striatum, daple expresses in both glial cells and neuron cells. Tyrosine hydroxylase (TH) RT-PCR and real time PCR results show that TH expression has no difference between homo daple knock out mice and wildtype mice, indicating Dopamine concentration no difference between them. We hope through understanding the distribution of Daple in systematic organs and CNS, we could more clearly figure out the way that Wnt pathway works and play an fundamental basis for the future neurological function study of Daple knock out mice.
Daple又称为含有高亮氨酸残基的Dvl相关蛋白。Daple是Girdin家族的基因(Girdin, Daple, FLJ004354)。其中Daple和Girdin在N端有很大的同源性,(54%的氨基酸相同),意味着两个基因功能相似,而C末端则与Girdin较少的相似性,只有27%,这意味着Daple具有不同于Girdin的特殊功能。Daple在Wnt经典途径中抑制β-catenin聚集和T细胞因子(Tcf)的转录活性,起负调节作用。而在非经典Wnt通路中增加蛋白激酶C(PKC)与Dvl互相作用激活Rac蛋白,起正调节作用。目前已知Daple在出生后期细胞迁移中起着重要的作用,Daple可能会影响胚胎发育、血管发生和肿瘤发展。目前哺乳动物Daple的分布表达的仍不明确。因此,全面了解Daple蛋白的功能及分布对于进一步了解相关疾病的发病机制及预后有重要意义。本研究重点是Daple基因的表达分布。
材料与方法：
通过SA剪接受体技术制作的Daple基因敲除的老鼠购自transgenic公司。出生7天的老鼠(p7；雌性)和8周老鼠(p56；雄性)(野生型老鼠(+／+)同源性老鼠(-／-))用4%PFA灌注,解剖出组织、冰冻、冰冻切片,解冻安装在载玻片上。Daple分布可以通过Lac Z染色显示并可以应用原位杂交技术验证LacZ染色的结果。用33P或地高辛标记的Daple cRNA探针(外显子30)。根据daple基因在脑中的分布,进一步对神经进行免疫组化染色,用PGP9.5, GFAP标记,判定daple基因是在神经元或是胶质细胞表达。根据其分布的特点对脑内可能出现的异常酶进行PCR和RT-PCR分析。
结果：
我们发现Lac Z染色的p7和p56的同源性Daple敲除的小鼠在纹状体、脑室室管膜细胞、小脑(大概在皮质上的浦肯野细胞)、海马、嗅球、脊髓、气管、膀胱、毛囊、舌、胃、脾、胰腺、心肌细胞、视网膜、肾乳头和门静脉,beta-geo(Daple启动子介导Lac Z和抗新霉素基因融合)表达而野生型小鼠不表达。原位杂交技术也证实了这个结果。我们发现在脉络丛和海马CA3区,Daple在神经胶质细胞中表达,而在纹状体中,Daple同时在胶质细胞和神经细胞中表达。酪氨酸羟化酶(TH)RT-PCR和eal time PCR的结果显示同源性Daple敲除小鼠和野生型小鼠在酪氨酸羟化酶表达中并没有显著性差异。
结论：
我们成功的描述出Daple的基因表达。Daple在脑、脊髓、眼、膀胱上皮及平滑肌、支气管上皮、胃、肾脏、睾丸、输精管、毛囊、舌、门静脉表达显著。Daple在小鼠脑中的分布主要在纹状体、海马、齿状回、皮质、黑质和室管膜细胞上。在脉络丛和海马CA3区,Daple在神经胶质细胞中表达,在纹状体中Daple同时在胶质细胞和神经细胞中表达。通过酪氨酸羟化酶PCR的结果显示同源性Daple敲除小鼠和野生型小鼠在酪氨酸羟化酶表达中并没有显著性差异,提示同源型敲除小鼠中多巴胺含量与野生型小鼠区别不大。希望通过了解Daple在小鼠全身器官、中枢神经系统分布情况,可以更清楚地了解Wnt信号通路的作用,并为以后的神经功能实验打下基础。
Objectives
Daple is named as Dishevelled-associating protein with a high frequency of leucine residues. Daple belongs to Girdin-family genes (Girdin, Daple, FLJ004354) Among those, Daple and Girdin show considerably high homology in N-terminal domains (54%amino acids identity), which implies the functional analogy between two genes in mice, however C-terminal domains lacks similarities(only27%amino acids identity) which means Daple has its own distinctive function. Daple plays a negative control over Wnt canonical pathway, inhibiting β-catenin aggregation and Tcf transcription. By contrast, Daple has a positive impact on Wnt non-canonical pathway by enhancing PKC/Dvl interaction and activation of Rac protein. Daple is known to play an important role in postnatal cell migration. It could possibly influence fetus development, angiogenesis and tumor development. Daple's distribution is still unknown. For the purpose of understanding related mechanisms of diseases, Daple's function and distribution should be under study. Here we performed preliminary study to know the distribution of Daple gene expression.
Materials and methods
Daple knock mice generated by SA (Splicing acceptor) trap vector techniques were purchased from TransGenic Co.,ltd. Seven-day-old (P7; female) and8-week-old mice (P56; male)(wild-type mice (+/+) and homozygous mice (-/-)) were perfused with4%PFA, then tissues were dissected, frozen, cryosectioned, and thaw-mounted on the glass slide. Daple expression was visualized using LacZ staining. To confirm LacZ staining results, in situ hybridization (ISH) was carried out using33P or Digoxigenin (DIG) labeled cRNA probe targeting Daple specific sequences (exon30). According to the distribution of Daple in the brain, we did immunohistochemistry to the brain tissue by using PGP9.5and GFAP probe. We could then determine whether Daple expresses in neuron or glial cells. We also intended to examine the possible abnormal enzyme by PCR and RT-PCR.
Results
LacZ staining of P7and P56brain exhibited significant expression of beta-geo (fusion of LacZ and Neomycin resistant gene driven by Daple promoter) in caudate putamen (striatum), ependymal cells of ventricles, cerebellum (cortex and presumably in Purkinje cells), hippocampus, olfactory bulb, bronchi, bladder, hair, tongue, stomach, intestine, spleen, spinal cord, pancrease, caridial muscle, retina, renal papillary and portal tract only in Daple (-/-) mice. ISH results confirmed the findings in LacZ staining.We found that in choroid plexus and hippocampus CA3area, daple expresses in glial cells whereas in striatum, daple expresses in both glial cells and neuron cells. Tyrosine hydroxylase (TH) RT-PCR and real time PCR results show that TH expression has no difference between homo daple knock out mice and wildtype mice.
Conclusion
We succeeded in depicting the preliminary map of Daple expression. Daple exhibited significant expression in brain, spinal cord, bronchi,bladder, hair, tongue, stomach, intestine, spleenpancrease, caridial muscle, retina, renal papillary and portal tract. Daple expresses most prominently in brain is that striatum, ependymal cells of ventricles, hippocampus, dentate gyrus, substantia nigra. In choroid plexus and hippocampus CA3area, daple expresses in glial cells whereas in striatum, daple expresses in both glial cells and neuron cells. Tyrosine hydroxylase (TH) RT-PCR and real time PCR results show that TH expression has no difference between homo daple knock out mice and wildtype mice, indicating Dopamine concentration no difference between them. We hope through understanding the distribution of Daple in systematic organs and CNS, we could more clearly figure out the way that Wnt pathway works and play an fundamental basis for the future neurological function study of Daple knock out mice.
引文
1. Lijam, N. and D.J. Sussman, Organization and promoter analysis of the mouse dishevelled-1 gene. Genome Res,1995.5(2):p.116-24.
2. Chen, W., et al., beta-Arrestinl modulates lymphoid enhancer factor transcriptional activity through interaction with phosphorylated dishevelled proteins. Proc Natl Acad Sci U S A,2001.98(26):p.14889-94.
3. Hino, S., et al., Inhibition of the Wnt signaling pathway by Idax, a novel Dvl-binding protein. Mol Cell Biol,2001.21(1):p.330-42.
4. Li, L., et al., Axin and Fratl interact with dvl and GSK, bridging Dvl to GSK in Wnt-mediated regulation of LEF-1. EMBO J,1999.18(15):p.4233-40.
5. Yan, D., et al., Cell autonomous regulation of multiple Dishevelled-dependent pathways by mammalian Nkd. Proc Natl Acad Sci U S A,2001.98(7):p. 3802-7.
6. Cook, D., et al., Wingless inactivates glycogen synthase kinase-3 via an intracellular signalling pathway which involves a protein kinase C. EMBO J, 1996.15(17):p.4526-36.
7. Xing, Y., et al., Crystal structure of a beta-catenin/axin complex suggests a mechanism for the beta-catenin destruction complex. Genes Dev,2003.17(22): p.2753-64.
8. Giles, R.H., J.H. van Es, and H. Clevers, Caught up in a Wnt storm:Wnt signaling in cancer. Biochim Biophys Acta,2003.1653(1):p.1-24.
9. Kuhl, M., et al., The Wnt/Ca2+ pathway:a new vertebrate Wnt signaling pathway takes shape. Trends Genet,2000.16(7):p.279-83.
10. Pandur, P., D. Maurus, and M. Kuhl, Increasingly complex:new players enter the Wnt signaling network. Bioessays,2002.24(10):p.881-4.
11. Enomoto, A., et al., Akt/PKB regulates actin organization and cell motility via Girdin/APE. Dev Cell,2005.9(3):p.389-402.
12. Anai, M., et al., A novel protein kinase B (PKB)/AKT-binding protein enhances PKB kinase activity and regulates DNA synthesis. J Biol Chem,2005.280(18): p.18525-35.
13. Zhang, B., et al., Reduction of Akt2 inhibits migration and invasion of glioma cells. Int J Cancer,2009.125(3):p.585-95.
14. Jiang, P., et al., An actin-binding protein Girdin regulates the motility of breast cancer cells. Cancer Res,2008.68(5):p.1310-8.
15. Kitamura, T., et al., Regulation of VEGF-mediated angiogenesis by the Akt/PKB substrate Girdin. Nat Cell Biol,2008.10(3):p.329-37.
16. Enomoto, A., et al., Roles of disrupted-in-schizophrenia 1-interacting protein girdin in postnatal development of the dentate gyrus. Neuron,2009.63(6):p. 774-87.
17. Oshita, A., et al., Identification and characterization of a novel Dvl-binding protein that suppresses Wnt signalling pathway. Genes Cells,2003.8(12):p. 1005-17.
18. Sato, A., et al., Wnt5a regulates distinct signalling pathways by binding to Frizzled2. EMBO J,2010.29(1):p.41-54.
19. Ishida-Takagishi, M., et al., The Dishevelled-associating protein Daple controls the non-canonical Wnt/Rac pathway and cell motility. Nat Commun,2012.3:p. 859.
20. Moon, R.T. and D. Kimelman, From cortical rotation to organizer gene expression:toward a molecular explanation of axis specification in Xenopus. Bioessays,1998.20(7):p.536-45.
21. Kobayashi, H., et al., Novel Daple-like protein positively regulates both the Wnt/beta-catenin pathway and the Wnt/JNK pathway in Xenopus. Mech Dev, 2005.122(10):p.1138-53.
22. Weeraratna, A.T., et al., Wnt5a signaling directly affects cell motility and invasion of metastatic melanoma. Cancer Cell,2002.1(3):p.279-88.
23. Kurayoshi, M., et al., Expression of Wnt-5a is correlated with aggressiveness of gastric cancer by stimulating cell migration and invasion. Cancer Res,2006. 66(21):p.10439-48.
24. Katoh, M., WNT/PCP signaling pathway and human cancer (review). Oncol Rep,2005.14(6):p.1583-8.
25. Yano, K., Gene expression correlation analysis predicts involvement of high-and low-confidence risk genes in different stages of prostate carcinogenesis. Prostate,2010.70(16):p.1746-59.
26. Le-Niculescu, H., et al., Identification and characterization of GIV, a novel Galpha i/s-interacting protein found on COPI, endoplasmic reticulum-Golgi transport vesicles. J Biol Chem,2005.280(23):p.22012-20.
27. Simpson, F., et al., A novel hook-related protein family and the characterization of hook-related protein 1. Traffic,2005.6(6):p.442-58.
28. Enomoto, A., J. Ping, and M. Takahashi, Girdin, a novel actin-binding protein, and its family of proteins possess versatile functions in the Akt and Wnt signaling pathways. Ann N Y Acad Sci,2006.1086:p.169-84.
2. Chen, W., et al., beta-Arrestinl modulates lymphoid enhancer factor transcriptional activity through interaction with phosphorylated dishevelled proteins. Proc Natl Acad Sci U S A,2001.98(26):p.14889-94.
3. Hino, S., et al., Inhibition of the Wnt signaling pathway by Idax, a novel Dvl-binding protein. Mol Cell Biol,2001.21(1):p.330-42.
4. Li, L., et al., Axin and Fratl interact with dvl and GSK, bridging Dvl to GSK in Wnt-mediated regulation of LEF-1. EMBO J,1999.18(15):p.4233-40.
5. Yan, D., et al., Cell autonomous regulation of multiple Dishevelled-dependent pathways by mammalian Nkd. Proc Natl Acad Sci U S A,2001.98(7):p. 3802-7.
6. Cook, D., et al., Wingless inactivates glycogen synthase kinase-3 via an intracellular signalling pathway which involves a protein kinase C. EMBO J, 1996.15(17):p.4526-36.
7. Xing, Y., et al., Crystal structure of a beta-catenin/axin complex suggests a mechanism for the beta-catenin destruction complex. Genes Dev,2003.17(22): p.2753-64.
8. Giles, R.H., J.H. van Es, and H. Clevers, Caught up in a Wnt storm:Wnt signaling in cancer. Biochim Biophys Acta,2003.1653(1):p.1-24.
9. Kuhl, M., et al., The Wnt/Ca2+ pathway:a new vertebrate Wnt signaling pathway takes shape. Trends Genet,2000.16(7):p.279-83.
10. Pandur, P., D. Maurus, and M. Kuhl, Increasingly complex:new players enter the Wnt signaling network. Bioessays,2002.24(10):p.881-4.
11. Enomoto, A., et al., Akt/PKB regulates actin organization and cell motility via Girdin/APE. Dev Cell,2005.9(3):p.389-402.
12. Anai, M., et al., A novel protein kinase B (PKB)/AKT-binding protein enhances PKB kinase activity and regulates DNA synthesis. J Biol Chem,2005.280(18): p.18525-35.
13. Zhang, B., et al., Reduction of Akt2 inhibits migration and invasion of glioma cells. Int J Cancer,2009.125(3):p.585-95.
14. Jiang, P., et al., An actin-binding protein Girdin regulates the motility of breast cancer cells. Cancer Res,2008.68(5):p.1310-8.
15. Kitamura, T., et al., Regulation of VEGF-mediated angiogenesis by the Akt/PKB substrate Girdin. Nat Cell Biol,2008.10(3):p.329-37.
16. Enomoto, A., et al., Roles of disrupted-in-schizophrenia 1-interacting protein girdin in postnatal development of the dentate gyrus. Neuron,2009.63(6):p. 774-87.
17. Oshita, A., et al., Identification and characterization of a novel Dvl-binding protein that suppresses Wnt signalling pathway. Genes Cells,2003.8(12):p. 1005-17.
18. Sato, A., et al., Wnt5a regulates distinct signalling pathways by binding to Frizzled2. EMBO J,2010.29(1):p.41-54.
19. Ishida-Takagishi, M., et al., The Dishevelled-associating protein Daple controls the non-canonical Wnt/Rac pathway and cell motility. Nat Commun,2012.3:p. 859.
20. Moon, R.T. and D. Kimelman, From cortical rotation to organizer gene expression:toward a molecular explanation of axis specification in Xenopus. Bioessays,1998.20(7):p.536-45.
21. Kobayashi, H., et al., Novel Daple-like protein positively regulates both the Wnt/beta-catenin pathway and the Wnt/JNK pathway in Xenopus. Mech Dev, 2005.122(10):p.1138-53.
22. Weeraratna, A.T., et al., Wnt5a signaling directly affects cell motility and invasion of metastatic melanoma. Cancer Cell,2002.1(3):p.279-88.
23. Kurayoshi, M., et al., Expression of Wnt-5a is correlated with aggressiveness of gastric cancer by stimulating cell migration and invasion. Cancer Res,2006. 66(21):p.10439-48.
24. Katoh, M., WNT/PCP signaling pathway and human cancer (review). Oncol Rep,2005.14(6):p.1583-8.
25. Yano, K., Gene expression correlation analysis predicts involvement of high-and low-confidence risk genes in different stages of prostate carcinogenesis. Prostate,2010.70(16):p.1746-59.
26. Le-Niculescu, H., et al., Identification and characterization of GIV, a novel Galpha i/s-interacting protein found on COPI, endoplasmic reticulum-Golgi transport vesicles. J Biol Chem,2005.280(23):p.22012-20.
27. Simpson, F., et al., A novel hook-related protein family and the characterization of hook-related protein 1. Traffic,2005.6(6):p.442-58.
28. Enomoto, A., J. Ping, and M. Takahashi, Girdin, a novel actin-binding protein, and its family of proteins possess versatile functions in the Akt and Wnt signaling pathways. Ann N Y Acad Sci,2006.1086:p.169-84.