臺灣博碩士論文加值系統

父系印記基因10 (Peg10)，是一個表現在老鼠胚胎早期的基因。但是對於Peg10在老鼠前腦發育的表現模式以及功能仍是未知。因此我的論文主要針對Peg10在老鼠發育時期前腦的表現模式以及功能進行研究。在論文的第一部分先主要研究Peg10在老鼠發育時期前腦的表現模式，我們利用西方墨點法發現在胚胎時期13.5天老鼠的前腦有表現RF-1 的蛋白質，但不表現RF-1/2 的蛋白質，接著針對Peg10做原位雜交技術以及免疫染色發現Peg10表現，在發育早期，胚胎時期第12.5天，Peg10主要表現在外側神經節隆起（LGE，紋狀體前驅結構），內側神經節隆起，尾神經節隆起，視前區，腹丘腦和下丘腦，並且與紋狀體標記基因，Nolz-1以及Islet-1共同表現在同一顆細胞的情形，而在胚胎發育時期第15.5天，Peg10大量表現在腹側紋狀體的伏隔核前驅結構，相反的，Peg10在背側紋狀體的尾狀核與殼核前驅結構則表現量少。在出生後Peg10持續表現，且大多表現在伏隔核。在出生後的第7天伏隔核可分成核心跟外殼兩部分，Peg10主要表現在核心，少數Peg10 細胞表現在外殼。在出生後第7天以及第14天的的伏隔核中Peg10與subtanceP同時表現在紋狀體投射到黑質的細胞中，Peg10亦與enkephalin同時表現在紋狀體投射到蒼白球的細胞中。就伏隔核神經元新生的時間點分析而言，Peg10細胞較在胚胎時期帶15.5出生的數量多於胚胎時期12.5天出生的數量，這表示在伏隔核Peg10表現的神經元大多是在發育時期晚期出生的。這些表現模式的研究結果推測Peg10的表現可能跟伏隔核核心神經元的形成有關聯。在論文的第二部分先主要想探討Peg10的下游基因以及Peg10在發育中的伏隔核的神經生物性功能。利用在胚胎時期第13.5天利用子宮內電穿孔技術將Peg10干擾核糖核酸的建構質體表現在前腦腹側外神經隆突降低Peg10的表現，在胚胎時期第15.5天的紋狀體分析的結果發現表現Tuj1的分化中的細胞數量顯著增加，而表現Ki67的還在進行細胞增生的細胞數量有減少的趨勢，這表示Peg10在外側神經節隆起的神經祖源細胞持續進行細胞增生是需要的。而我們也發現Astn2 (星形膠質細胞2)為潛在的目標基因被Peg10所抑制，因為降低Peg10的表現則會使Astn2表現量在尾狀核與殼核以及伏隔核中表現量上升，之前的文獻闡釋在小腦Astn2參與細胞的遷移。我們在胚胎時期13.5天利用子宮內電穿孔技術將Peg10干擾核糖核酸的建構質體表現在前腦腹側外神經隆突降低Peg10的表現以及同時表現GFP的報告基因並在胚胎時期第15.5天與第18.5天進行分析，結果發現GFP的報告基因在背側到腹側的分布是沒有受到顯著影響。最後，我們利用Dlx6-Cre使Nolz-1基因過度表現的轉殖老鼠腦中發現在伏隔核Peg10的表現量變少，這樣的結果與我們實驗室之前看到在剔除Nolz-1表現的基因轉殖老鼠的尾狀核與殼核以及伏隔核中，Peg10的表現量上升結果是一致的。總結本論文研究，我們發現在發育的過程中，Peg10為伏隔核高量表現的基因。一個重要的問題是，在紋狀體發育的過程中是如何區分成背部及腹部的紋狀體。在尾狀核與殼核以及伏隔核發育的過程中，Nolz-1調節Peg10以及Peg10調節Astn2，這些生物訊息路徑如何對於背部及腹部的紋狀體區分等待更進一步的深入研究。

Peg10 (Paternally Expressed Gene 10) is expressed in early stages of mouse embryos. Peg10 is expressed in the mouse brain, but its detailed expression pattern and function in developing mouse brain are yet unkown. The main focus of my thesis was to study the expression pattern and function of Peg10 in developing mouse forebrain. In the first part of my thesis study, we investigated the expression pattern of Peg10 in developing mouse forebrain. Western blotting showed that Peg10-RF1 isoform, but not Peg10-RF1/2 isoform, was expressed in E13.5 developing mouse forebrain. In situ hybiridzation and immunostaining of Peg10 showed that Peg10 was expressed in the lateral ganglionic eminence (LGE, striatal primordia), medial ganglionic eminence, caudal ganglionic eminence, preoptic area, ventral thalamus and hypothalamus as early as E12.5. Peg10 was co-expressed with striatal markers of Nolz-1 and Islet-1 in E13.5. By E15.5, Peg10 was preferentially expressed at high levels in the primordium of nucleus accumbens (NAc) of ventral striatum. In contrast, Peg10 was expressed at low levels in the primordium of caudate-putamn (CP) of dorsal striatum. The NAc-seletive expression pattern of Peg10 in NAc continues postnatally. By postnatal day (P) 7 when the core and shell regions of NAc were distinguisable, Peg10 was predominatly expressed in the core with some Peg10-positive cells scattered in the shell. Moreover, Peg10 was co-expressed in subtance P-positive striatonigral and enkephalin-positive striatopallidal neurons in P7 and P14 NAc. In terms of neurogenesis, the birthdating analysis indicated that the number of Peg10;BrdUE15.5 cells were higher than Peg10;BrdUE12.5 cells in NAc, suggesting that Peg10-positive cells are born at later stages. The results of the expression pattern analyses suggest that Peg10-positive cell lineages contribute to the formation of the core region of NAc. For the second part of my thesis study, we attempted to indeifity the downstream traget genes of Peg10 and the neuobiological function of Peg10 in developing NAc. In utero electroporation of SH-Peg10 knockdon construct into E13.5 LGE resulted in an increase of Tuj1-positive differentaing neurons and a trend of decrease in proliferating Ki67-positive cells in E15.5 striatum, which suggests that Peg10 is required to maintain proliferation of neural progenitors in the LGE. We also identified Astn2 (astrotactin 2) as a potential target gene that was repressed by Peg10, because Peg10 knockdown up-regulated Astn2 expression in CP and NAc. Although previous study has shown involvement of Astn2 in neuronal migration in developing cerebellum, electroporation of SH-Peg10 knockdown and GFP reporter gene constructs into E13.5 LGE did not affect the distribution pattern of GFP-positive cells along the dorsal-ventral axis of E18.5 striatum, Nor did it affect the distribution pattern of GFP+ cells at E15.5 striatum, except that GFP+ cells were increased in the region of bin 4 in SH-Peg10 knockdown group compared to control group at level 2. Finally, based on transgenic over-expression of Nolz-1, we found reduction of Peg10 expression in NAc of Dlx6-cCre;Nolz-1 Tg brains, which was consistent with our previous finding of up-regulation of Nolz-1 in CP and NAc of Nolz-1 knockout brains. In summary, we have identified Peg10 as a NAc-enriched gene during development. An important question in striatal development is how dorsal and ventral striatum are parcellated during development. The biological signifcance of the Nolz-1-Peg10 and Peg10-Astn2 signaling pathways in developing CP and NAc with relevance to partition of dorsal and ventral striautm awaits furture studies.

CONTENTS
Acknowledgement I
中文摘要 III
ABSTRACT V
CONTENTS VII
FIGURE CONTENT XI
TABLE CONTENT XIII
CHAPTER 1 Introduction 1
1.1 Stiatum 1
1.2 Nucleus accumbens 2
1.3 Paternally expressed gene 10, Peg10 4
1.4 Goals and signifcance of the thesis 5
CHAPTER 2 Materials and Methods 6
2.1 Animals 6
2.2 Brain harvesting 6
2.3 PCR genotying of transgenic mice 7
2.4 Prepareration of brain sections 7
2.5 Administration of 5-bromo-2’-deoxyuridine 7
2.6 Immunohistochemistry 7
2.7 In situ hybridization with digoxigenin-labeled probes 8
2.8 SH-Peg10 knockdown plasmid construction 9
2.10 Validation of SH-Peg10 knockdown efficiency in N2a cell line 10
2.11 Quantitative RT-PCR 10
2.12 Western blotting 11
2.13 In utero electroporation 12
2.14 Microscopic image analyses, quantification and statistics 12
CHAPTER 3 Results 14
3.1 Ontogeny of Peg10 mRNA expression in developing mouse brains 14
3.2 Developmental origins of Peg10 cell lineages in the mouse forebrain 17
Peg10 is selectively expressed in the ventral LGE: Complementary expression of Pax6 and Peg10 in E12.5 and E15.5 telencephalon 17
3.3 Peg10 is expressed in Ki67-positive progenitors and Tuj1-positive differentiating neurons of E12.5 LGE and E15.5 ventral striatum (vST) 18
3.4 Birthdating analysis of Peg10-positive cells in developing NAc 18
3.5 Analysis of Peg10 cell populations in NAc 19
3.6 Functional studies of Peg10 in developing NAc 21
CHAPTER 4 Discussion 28
4-1 Peg10-positive progentiors in the vLGE 28
4-2 Peg10 expression in the developing nucleus accounbens 29
4-3 Knowndown Peg10 promotes neuronal differentiation 30
4-4 Astn2 is a potential downstream effector of Peg10 in developing striatum 31
4-5 Nolz-1 acts upstream of Peg10 gene to repress Peg10 expression in developing striatum 32
4-6 The potential Nolz-1-Peg10-Astn2 signaling pathway in regulation of CP and NAc development 32
Figure and Figure ledgens 34
Tables 72
References 76
Appendixes 80

FIGURE CONTENT
Figure 1. Expression of Peg10 protein in E13.5 and adult mouse forebrain 36
Figure 2. Peg10 mRNA expression pattern in E12.5 mouse forebrain 37
Figure 3. Peg10 mRNA expression pattern in E13.5 mouse forebrain 38
Figure 4. Peg10 mRNA expression pattern in E15.5 and E17.5 mouse forebrain 39
Figure 5. Peg10 mRNA expression pattern in P0 mouse forebrain 40
Figure 6. Peg10 mRNA expression pattern in P7 mouse forebrain 42
Figure 7. Peg10 mRNA expression pattern in P14 mouse forebrain 43
Figure 8. Peg10 expression is absent in adult mouse forebrain 44
Figure 9. Complementary expression of Pax6 and Peg10 in E12.5 and E15.5 telencephalon 45
Figure 10. Peg10 is expressed in Ki67-positive progenitors and Tuj1-positive differentiating neurons of E12.5 LGE and E15.5 vST 46
Figure 11. Birthdating analysis of Peg10-positive cells in NAc 49
Figure 12. Peg10 is co-localized with Islet-1 and Nolz-1 of striatal differentiating markers, in E12.5 LGE and E15.5 vST 51
Figure 13. Double staining of Peg10 and MGE marker, Nkx2.1 to trace MGE-derived Peg10-positive cells in E12.5 and E15.5 forebrain 53
Figure 14. Peg10 is expressed in striatonnigral and striatopallidal neurons in P7 NAc 55
Figure 15. Validation of Peg10 knockdown in N2a cells 57
Figure 16. Validation of Peg10 knockdown in the electroporated brains 59
Figure 17 Peg10 knockdown promotes neuronal differentiation in E15.5 LGE 62
Figure 18. Knockdown Peg10 increases Astn2 expression in E18.5 65
Figure 19. Peg10 knockdown does not affect migration pattern of GFP+ cells in developing CP and NAc 68
Figure 20. Transgenic over-expression of Nolz-1 suppresses Peg10 expression in the striatum at P7 70
Figure 21. Peg10 knockdown does not affect Nolz-1 expression in E18.5 CP and NAc 71

TABLE CONTENT
Table 1. Genotyping primer sequences and related conditions 73
Table 2. Antibody conditions 74
Table 3. Summary of the cRNA probes for in situ hybridization 75

Alexander B, Warner-Schmidt J, Eriksson TM, Tamminga C, Arango-Lievano M, Ghose S, Vernov M, Stavarache M, Musatov S, Flajolet M (2010) Reversal of depressed behaviors in mice by p11 gene therapy in the nucleus accumbens. Science translational medicine 2:54ra76-54ra76.
Al-Hasani R, McCall JG, Shin G, Gomez AM, Schmitz GP, Bernardi JM, Pyo C-O, Park SI, Marcinkiewcz CM, Crowley NA (2015) Distinct subpopulations of nucleus accumbens dynorphin neurons drive aversion and reward. Neuron 87:1063-1077.
Bertran-Gonzalez J, Bosch C, Maroteaux M, Matamales M, Hervé D, Valjent E, Girault J-A (2008) Opposing patterns of signaling activation in dopamine D1 and D2 receptor-expressing striatal neurons in response to cocaine and haloperidol. Journal of Neuroscience 28:5671-5685.
Blood AJ, Zatorre RJ (2001) Intensely pleasurable responses to music correlate with activity in brain regions implicated in reward and emotion. Proceedings of the National Academy of Sciences 98:11818-11823. Bertran-Gonzalez J,
Carelli RM, Ijames SG, Crumling AJ (2000) Evidence that separate neural circuits in the nucleus accumbens encode cocaine versus “natural”(water and food) reward. Journal of Neuroscience 20:4255-4266.
Chang C-W, Tsai C-W, Wang H-F, Tsai H-C, Chen H-Y, Tsai T-F, Takahashi H, Li H-Y, Fann M-J, Yang C-W (2004) Identification of a developmentally regulated striatum-enriched zinc-finger gene, Nolz-1, in the mammalian brain. Proceedings of the National Academy of Sciences of the United States of America 101:2613-2618.
Chang SL-Y, Chen S-Y, Huang H-H, Ko H-A, Liu P-T, Liu Y-C, Chen P-H, Liu F-C (2013) Ectopic expression of nolz-1 in neural progenitors promotes cell cycle exit/premature neuronal differentiation accompanying with abnormal apoptosis in the developing mouse telencephalon. PloS one 8:e74975.
Deacon T, Pakzaban P, Isacson O (1994) The lateral ganglionic eminence is the origin of cells committed to striatal phenotypes: neural transplantation and developmental evidence. Brain research 668:211-219.
Di Martino A, Scheres A, Margulies DS, Kelly A, Uddin LQ, Shehzad Z, Biswal B, Walters JR, Castellanos FX, Milham MP (2008) Functional connectivity of human striatum: a resting state FMRI study. Cerebral cortex 18:2735-2747.
Gerfen CR, Surmeier DJ (2011) Modulation of striatal projection systems by dopamine. Annual review of neuroscience 34:441-466. Bertran-Gonzalez J, Bosch C, Maroteaux M, Matamales M, Hervé D, Valjent E, Girault J-A (2008) Opposing patterns of signaling activation in dopamine D1 and D2 receptor-expressing striatal neurons in response to cocaine and haloperidol. Journal of Neuroscience 28:5671-5685.
Hasbi A, O'Dowd BF, George SR (2011) Dopamine D1-D2 receptor heteromer signaling pathway in the brain: emerging physiological relevance. Molecular brain 4:26.
Kawaguchi Y, Wilson CJ, Augood SJ, Emson PC (1995) Striatal interneurones: chemical, physiological and morphological characterization. Trends in neurosciences 18:527-535.
Lu K-M, Evans SM, Hirano S, Liu F-C (2014) Dual role for Islet-1 in promoting striatonigral and repressing striatopallidal genetic programs to specify striatonigral cell identity. Proceedings of the National Academy of Sciences 111:E168-E177.
Lux A, Beil C, Majety M, Barron S, Gallione CJ, Kuhn H-M, Berg JN, Kioschis P, Marchuk DA, Hafner M (2005) Human retroviral gag-and gag-pol-like proteins interact with the transforming growth factor-β receptor activin receptor-like kinase 1. Journal of Biological Chemistry 280:8482-8493.
Lux H, Flammann H, Hafner M, Lux A (2010) Genetic and molecular analyses of PEG10 reveal new aspects of genomic organization, transcription and translation. PloS one 5:e8686.
Marı́n O, Anderson SA, Rubenstein JL (2000) Origin and molecular specification of striatal interneurons. Journal of Neuroscience 20:6063-6076.
Miyamoto Y, Iida A, Sato K, Muramatsu S-i, Nitta A (2015) Knockdown of dopamine D2 receptors in the nucleus accumbens core suppresses methamphetamine-induced behaviors and signal transduction in mice. International Journal of Neuropsychopharmacology 18:pyu038.
Muñoz-Manchado A, Foldi C, Szydlowski S, Sjulson L, Farries M, Wilson C, Silberberg G, Hjerling-Leffler J (2016) Novel striatal GABAergic interneuron populations labeled in the 5HT3aEGFP mouse. Cerebral Cortex 26:96-105.
Nirenberg MJ, Chan J, Pohorille A, Vaughan RA, Uhl GR, Kuhar MJ, Pickel VM (1997) The dopamine transporter: comparative ultrastructure of dopaminergic axons in limbic and motor compartments of the nucleus accumbens. The Journal of neuroscience 17:6899-6907.
Okabe H, Satoh S, Furukawa Y, Kato T, Hasegawa S, Nakajima Y, Yamaoka Y, Nakamura Y (2003) Involvement of PEG10 in human hepatocellular carcinogenesis through interaction with SIAH1. Cancer research 63:3043-3048.
Ono R, Kobayashi S, Wagatsuma H, Aisaka K, Kohda T, Kaneko-Ishino T, Ishino F (2001) A retrotransposon-derived gene, PEG10, is a novel imprinted gene located on human chromosome 7q21. Genomics 73:232-237.
Ono R, Nakamura K, Inoue K, Naruse M, Usami T, Wakisaka-Saito N, Hino T, Suzuki-Migishima R, Ogonuki N, Miki H (2006) Deletion of Peg10, an imprinted gene acquired from a retrotransposon, causes early embryonic lethality. Nature genetics 38:101-106.
Paxinos G (2014) The rat nervous system: Academic press.
Perreault ML, Hasbi A, Alijaniaram M, Fan T, Varghese G, Fletcher PJ, Seeman P, O'Dowd BF, George SR (2010) The dopamine D1-D2 receptor heteromer localizes in dynorphin/enkephalin neurons increased high affinity state following amphetamine and in schizophrenia. Journal of Biological Chemistry 285:36625-36634.
Pfaus J, Damsma G, Wenkstern D, Fibiger H (1995) Sexual activity increases dopamine transmission in the nucleus accumbens and striatum of female rats. Brain research 693:21-30.
Reiner O, Karzbrun E, Kshirsagar A, Kaibuchi K (2016) Regulation of neuronal migration, an emerging topic in autism spectrum disorders. Journal of Neurochemistry 136:440-456.
Robison AJ, Vialou V, Mazei-Robison M, Feng J, Kourrich S, Collins M, Wee S, Koob G, Turecki G, Neve R (2013) Behavioral and structural responses to chronic cocaine require a feedforward loop involving ΔFosB and calcium/calmodulin-dependent protein kinase II in the nucleus accumbens shell. Journal of Neuroscience 33:4295-4307.
Sadikot AF, Sasseville R (1997) Neurogenesis in the mammalian neostriatum and nucleus accumbens: Parvalbumin‐immunoreactive GABAergic interneurons. Journal of Comparative Neurology 389:193-211.
Salgado S, Kaplitt MG (2015) The nucleus accumbens: a comprehensive review. Stereotactic and Functional Neurosurgery 93:75-93.
Schiffmann SN, Vanderhaeghen J-J (1993) Adenosine A2 receptors regulate the gene expression of striatopallidal and striatonigral neurons. Journal of Neuroscience 13:1080-1087.
Schilström B, Svensson H, Svensson T, Nomikos G (1998) Nicotine and food induced dopamine release in the nucleus accumbens of the rat: putative role of α7 nicotinic receptors in the ventral tegmental area. Neuroscience 85:1005-1009.
Stenman J, Toresson H, Campbell K (2003) Identification of two distinct progenitor populations in the lateral ganglionic eminence: implications for striatal and olfactory bulb neurogenesis. Journal of Neuroscience 23:167-174.
Sussel L, Marin O, Kimura S, Rubenstein J (1999) Loss of Nkx2. 1 homeobox gene function results in a ventral to dorsal molecular respecification within the basal telencephalon: evidence for a transformation of the pallidum into the striatum. Development 126:3359-3370.
Taupin P (2007) BrdU immunohistochemistry for studying adult neurogenesis: paradigms, pitfalls, limitations, and validation. Brain Research Reviews 53:198-214.
Voorn P, Vanderschuren LJ, Groenewegen HJ, Robbins TW, Pennartz CM (2004) Putting a spin on the dorsal–ventral divide of the striatum. Trends in neurosciences 27:468-474.
Wall NR, De La Parra M, Callaway EM, Kreitzer AC (2013) Differential innervation of direct-and indirect-pathway striatal projection neurons. Neuron 79:347-360.
Watson C (2012) Chapter 7, page 173-193. The mouse nervous system: Academic Press.
Wilson PM, Fryer RH, Fang Y, Hatten ME (2010) Astn2, a novel member of the astrotactin gene family, regulates the trafficking of ASTN1 during glial-guided neuronal migration. The Journal of Neuroscience 30:8529-8540.
Wichterle H, Turnbull DH, Nery S, Fishell G, Alvarez-Buylla A (2001) In utero fate mapping reveals distinct migratory pathways and fates of neurons born in the mammalian basal forebrain. Development 128:3759-3771.
Wichterle H, Garcia-Verdugo JM, Herrera DG, Alvarez-Buylla A (1999) Young neurons from medial ganglionic eminence disperse in adult and embryonic brain. Nature neuroscience 2:461-466.
Yun K, Potter S, Rubenstein J (2001) Gsh2 and Pax6 play complementary roles in dorsoventral patterning of the mammalian telencephalon. Development 128:193-205.
Zalocusky KA, Ramakrishnan C, Lerner TN, Davidson TJ, Knutson B, Deisseroth K (2016) Nucleus accumbens D2R cells signal prior outcomes and control risky decision-making. Nature 531:642-646.