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研究生:呂璦竹
研究生(外文):Ai-Chu Lu
論文名稱:探討Protogenin於早期胚胎中的功能
論文名稱(外文):Functional Study of Protogenin in Early Embryos
指導教授:范明基
指導教授(外文):Ming-Ji Fann
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:神經科學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:英文
論文頁數:49
中文關鍵詞:Protogenin神經發生Sox2-Cre上胚葉三胚層分化Nodal
外文關鍵詞:ProtogeninneurogenesisSox2-CreepiblastgastrulationNodal
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Protogenin (PRTG) 是一個未被分析完全的受體,具有四個免疫球蛋白區域 (immunoglobulin domain) 與五個第三型纖維蛋白連結區域 (fibronectin type III domain)。PRTG表現在小鼠胚胎第7天的早期定向細胞 (early committed cells)中,而在胚胎第10天、細胞正在進行分化時,PRTG的表現量會漸漸消失。這表示PRTG可能參與在調控細胞增生或細胞分化的過程中。由之前的研究成果已知,PRTG在可誘導成神經細胞的P19胚胎癌細胞株與雞胚胎的神經管中會抑制過早發育的神經細胞分化。為了更進一步研究PRTG在早期胚胎發育中的功能,我們建立了一隻雙基因轉殖 (double transgenic) 小鼠,Sox2-Cre/+; PRTGetSTOP-flox/+,並證明牠會經由Sox2啟動子轉譯出來的Cre的作用,過度表現顯性抑制 (dominant negative) 的PRTG在胚胎第6.5天的上胚葉 (epiblast) 細胞中。結果發現,此雙基因轉殖小鼠會因為無法與胎盤相連而死於胚胎期的第11.5天左右。此突變胚胎最早在第7天時的外觀就不同於正常的胚胎,其胚胎外組織 (extra-embryonic tissues) 的比例變大,而胚胎本體變小。原位雜交實驗 (in situ hybridization) 中,由Brachyury,FoxA2與Fgf8的結果顯示,此現象是因為在三胚層分化 (gastrulation) 的過程中出現異常而導致。Hesx1的結果可佐證:發育7.5天後的突變胚胎,其前神經外胚層 (anterior neural ectoderm) 與較晚期的前腦 (prosencephalon) 發育皆不正常。從Brachyury及FoxA2在突變胚胎中的表現模式得知,第8.5天的突變胚胎沒有脊索前板 (prechordal plate),而9.5天時則發現其脊索 (notochord) 發育異常。此外,幾乎全部的8.5天突變胚胎有前部截斷 (anterior truncation) 的問題,而有些具有異常的、與卵黃囊 (yolk sac) 相連的前端突出組織。第9.5天時,突變胚胎會出現軸旋轉 (axis-rotation)異常與心環 (heart-looping) 形成的缺陷,或是尿囊 (allantois) 無法與絨毛膜 (chorion) 連接而膨大的現象。整體來說,根據此雙基因轉殖小鼠在胚胎時期的突變表現型而言,我們推測顯性抑制的PRTG在上胚葉細胞中的過度表現會干擾Nodal的訊息傳遞,而導致早期胚胎的三胚層分化異常。另一方面,我們利用TuJ1的免疫螢光染色研究顯性抑制的PRTG對突變胚胎的神經細胞發育是否有影響。結果顯示,在小鼠胚胎中,可能還有其它機制調控神經細胞的產生。總結而言,結果指出PRTG的訊息傳遞可能對於早期胚胎的三胚層分化,軸中胚層 (axial mesoderm) 的發育,以及脊索前板與前神經管 (anterior neural tube) 的形成相當重要。
Protogenin (PRTG) is an uncharacterized receptor with four immunoglobulin (Ig) domains and five fibronectin type III domains. Expression of PRTG begins at mouse embryonic day 7 (E7) in early committed cells, and is sharply down-regulated at E10 when cells differentiate. This implies that PRTG may be involved in the regulation of cell proliferation and differentiation. To examine whether PRTG inhibits cell differentiation, an assay based on neuronal differentiation of P19 cells and in ovo electroporation have been adopted in our laboratory. The results suggest that PRTG suppresses precocious neuronal differentiation in both P19 embryonic carcinoma cells and in the chick neural tube. To further probe the function of PRTG in mouse embryos, we generated a double transgenic mouse, Sox2-Cre/+; PRTGetSTOP-flox/+, in which the dominant-negative PRTG is over-expressed in epiblast cells beginning from E6.5 by Sox2 promoter-driven Cre. The double transgenic embryos die by E11.5 due to failure of connection to the placenta. Mutant embryos are first recognizable by E7 with the larger portion of extra-embryonic tissues and smaller size of embryo proper. This is caused by gastrulation defect as revealed by in situ hybridization results of Brachyury, FoxA2 and Fgf8 that exhibit defects in the primitive streak. The head folds of mutant embryos from E7.5 to E8 are not prominent, displayed by reduced Hesx1 pattern in the anterior neural ectoderm and prosencephalon. There is no prechordal plate as no expression of Brachyury and FoxA2, and almost all E8.5 mutants show anterior truncation. Some mutants also exhibit ectopic anterior extra protruding tissues linked to the visceral yolk sac. When examined at E9.5, double transgenics have laterality defects, axis-rotation and heart-looping defects, and hydropic allantois that are not fused to the chorion. Moreover, through the expression patterns of Brachyury and FoxA2, there are rostral notochord defects in the E9.5 mutants. Overall, these data implicate that dominant-negative PRTG expression in the double transgenics affects Nodal signaling beginning from gastrulation, as many patterning defects are similar to those of mice deficient in the target genes of Nodal pathway. It is likely that dominant-negative PRTG interferes with the activity of Nodal signaling in the epiblast and disrupts the patterning of early mouse embryos. When examined by immunofluorescence staining of TuJ1, there is normal temporal progression of neuronal differentiation in the double transgenics, which is different from the findings in P19 differentiation assay and in the chick neural tube. This implies that there are other pathways, in addition to PRTG, that regulate neurogenesis. Collectively, these results demonstrate that dominant-negative PRTG interferes the processes of gastrulation, midline development, and formation of the prechordal mesoderm and anterior neural tube in early mouse embryos.
摘要 …………………………………………………………………I

Abstract ……………………………….………………………… II

Table of contents ……………………………………………… IV

Introduction ………………………………………………………1
Development of mouse embryos following the onset of gastrulation
Critical roles of the allantois during early embryogenesis
Nodal signaling in patterning the early embryos
Characterization of mouse Protogenin (PRTG)
Ex vivo functions of PRTG in neuronal development
In vivo roles of PRTG in early embryogenesis

Materials and Methods ………………………………………… 6
Animals and reagents
Preparation of the fusion protein
Antiserum production
Western blotting analysis
In ovo electroporation and BrdU incorporation assay
Cryostat section
Antibodies
Immunofluorescence staining
Embryo dissection
X-Gal staining of mouse embryos
Preparation of in situ hybridization probes
Whole mount in situ hybridization

Results …………………………………………………………… 12
Characterization of the antiserum against chick PRTG
Expression of PRTG is primarily present in committed cells of early chick embryos
Characterization of PRTGet transgenic mice
PRTGet disrupts the early patterning in double transgenics
Neurogenesis occurs at the correct timing but with abnormal patterning in double transgenics
PRTG is required for gastrulation and development of axial mesoderm and anterior brain
Defects in gastrulation in Sox2-Cre/+; PRTGetSTOP-flox/+ embryos
Prechordal mesoderm and notochord are severely affected in double transgenics
Failure of anterior neural ectoderm development in double transgenics

Discussion ………………………………………………………… 21
Redundant pathways of neurogenesis in the mouse embryos
PRTG signaling is involved in the Nodal pathway for proper early patterning
PRTG signaling is required for normal progression of gastrulation
PRTG activity is essential for regulation of development of APS and anterior brain
PRTG signal is necessary for establishment of the left-right axis

Reference ………………………………………………………… 25
Tables ……………………………………………………………… 28
Figures and Legends …………………………………………… 32
Appendix …………………………………………………………… 41
1 Arnold, S.J. & Robertson, E.J., Making a commitment: cell lineage allocation and axis patterning in the early mouse embryo. Nat Rev Mol Cell Biol 10 (2), 91-103 (2009).
2 Constam, D.B. & Robertson, E.J., Tissue-specific requirements for the proprotein convertase furin/SPC1 during embryonic turning and heart looping. Development 127 (2), 245-254 (2000).
3 Lawson, K.A., Fate mapping the mouse embryo. Int J Dev Biol 43 (7), 773-775 (1999).
4 Camus, A., Perea-Gomez, A., Moreau, A., & Collignon, J., Absence of Nodal signaling promotes precocious neural differentiation in the mouse embryo. Dev Biol 295 (2), 743-755 (2006).
5 Di-Gregorio, A. et al., BMP signalling inhibits premature neural differentiation in the mouse embryo. Development 134 (18), 3359-3369 (2007).
6 Kimura-Yoshida, C. et al., Crucial roles of Foxa2 in mouse anterior-posterior axis polarization via regulation of anterior visceral endoderm-specific genes. Proc Natl Acad Sci U S A 104 (14), 5919-5924 (2007).
7 Kaufman, M.H., The Atlas of Mouse Development. Academic Press, London. (1992).
8 Downs, K.M., Gifford, S., Blahnik, M., & Gardner, R.L., Vascularization in the murine allantois occurs by vasculogenesis without accompanying erythropoiesis. Development 125 (22), 4507-4520 (1998).
9 Tremblay, K.D., Dunn, N.R., & Robertson, E.J., Mouse embryos lacking Smad1 signals display defects in extra-embryonic tissues and germ cell formation. Development 128 (18), 3609-3621 (2001).
10 Gurtner, G.C. et al., Targeted disruption of the murine VCAM1 gene: essential role of VCAM-1 in chorioallantoic fusion and placentation. Genes Dev 9 (1), 1-14 (1995).
11 Beddington, R.S. & Robertson, E.J., Axis development and early asymmetry in mammals. Cell 96 (2), 195-209 (1999).
12 Schier, A.F. & Shen, M.M., Nodal signalling in vertebrate development. Nature 403 (6768), 385-389 (2000).
13 Whitman, M., Nodal signaling in early vertebrate embryos: themes and variations. Dev Cell 1 (5), 605-617 (2001).
14 Ang, S.L. et al., A targeted mouse Otx2 mutation leads to severe defects in gastrulation and formation of axial mesoderm and to deletion of rostral brain. Development 122 (1), 243-252 (1996).
15 Toyoda, R., Nakamura, H., & Watanabe, Y., Identification of protogenin, a novel immunoglobulin superfamily gene expressed during early chick embryogenesis. Gene Expr Patterns 5 (6), 778-785 (2005).
16 Vesque, C., Anselme, I., Couve, E., Charnay, P., & Schneider-Maunoury, S., Cloning of vertebrate Protogenin (Prtg) and comparative expression analysis during axis elongation. Dev Dyn 235 (10), 2836-2844 (2006).
17 Yu-Hui Wong, Y.-C.W., Ai-Chu Lu, Celia Chang, Po-Hao Chen, Hsu-Chen Cheng, Jenn-Yah Yu and Ming-Ji Fann, A membrane receptor protein Protogenin defines a transition stage between pluripotent epiblasts and committed neural progenitor cells and prevents precocious neurogenesis. (unpublished data).
18 Keino-Masu, K. et al., Deleted in Colorectal Cancer (DCC) encodes a netrin receptor. Cell 87 (2), 175-185 (1996).
19 Wigg, K.G. et al., Association of ADHD and the Protogenin gene in the chromosome 15q21.3 reading disabilities linkage region. Genes Brain Behav 7 (8), 877-886 (2008).
20 Yu, M., Haslam, R.H., & Haslam, D.B., HEDJ, an Hsp40 co-chaperone localized to the endoplasmic reticulum of human cells. J Biol Chem 275 (32), 24984-24992 (2000).
21 Hayashi, S., Lewis, P., Pevny, L., & McMahon, A.P., Efficient gene modulation in mouse epiblast using a Sox2Cre transgenic mouse strain. Mech Dev 119 Suppl 1, S97-S101 (2002).
22 Kwee, L. et al., Defective development of the embryonic and extraembryonic circulatory systems in vascular cell adhesion molecule (VCAM-1) deficient mice. Development 121 (2), 489-503 (1995).
23 Luo, J. et al., Placental abnormalities in mouse embryos lacking the orphan nuclear receptor ERR-beta. Nature 388 (6644), 778-782 (1997).
24 Herrmann, B.G., Expression pattern of the Brachyury gene in whole-mount TWis/TWis mutant embryos. Development 113 (3), 913-917 (1991).
25 Lowe, L.A., Yamada, S., & Kuehn, M.R., Genetic dissection of nodal function in patterning the mouse embryo. Development 128 (10), 1831-1843 (2001).
26 Kinder, S.J. et al., The organizer of the mouse gastrula is composed of a dynamic population of progenitor cells for the axial mesoderm. Development 128 (18), 3623-3634 (2001).
27 Sasaki, H. & Hogan, B.L., Differential expression of multiple fork head related genes during gastrulation and axial pattern formation in the mouse embryo. Development 118 (1), 47-59 (1993).
28 Crossley, P.H. & Martin, G.R., The mouse Fgf8 gene encodes a family of polypeptides and is expressed in regions that direct outgrowth and patterning in the developing embryo. Development 121 (2), 439-451 (1995).
29 Norris, D.P., Brennan, J., Bikoff, E.K., & Robertson, E.J., The Foxh1-dependent autoregulatory enhancer controls the level of Nodal signals in the mouse embryo. Development 129 (14), 3455-3468 (2002).
30 Zakin, L., Reversade, B., Kuroda, H., Lyons, K.M., & De Robertis, E.M., Sirenomelia in Bmp7 and Tsg compound mutant mice: requirement for Bmp signaling in the development of ventral posterior mesoderm. Development 132 (10), 2489-2499 (2005).
31 Levine, A.J. & Brivanlou, A.H., Proposal of a model of mammalian neural induction. Dev Biol 308 (2), 247-256 (2007).
32 Theil, T., Alvarez-Bolado, G., Walter, A., & Ruther, U., Gli3 is required for Emx gene expression during dorsal telencephalon development. Development 126 (16), 3561-3571 (1999).
33 Dziadek, M. & Adamson, E., Localization and synthesis of alphafoetoprotein in post-implantation mouse embryos. J Embryol Exp Morphol 43, 289-313 (1978).
34 Hoodless, P.A. et al., FoxH1 (Fast) functions to specify the anterior primitive streak in the mouse. Genes Dev 15 (10), 1257-1271 (2001).
35 Shawlot, W. & Behringer, R.R., Requirement for Lim1 in head-organizer function. Nature 374 (6521), 425-430 (1995).
36 Capdevila, J., Vogan, K.J., Tabin, C.J., & Izpisua Belmonte, J.C., Mechanisms of left-right determination in vertebrates. Cell 101 (1), 9-21 (2000).
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