Thesis (Ph. D.)--University of Oregon, 1999. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 110-119). Also available for download via the World Wide Web; free to University of Oregon users. Address: http://wwwlib.umi.com/cr/uoregon/fullcit?p9955919.

In recent years, State Road Route 114 which is located in Montgomery County, Virginia, has gained a bad reputation because of numerous traffic accidents. Most of these accidents resulted in loss of lives and property. Although there are many suggestions and proposals designed to prevent these acidents, to date no actions is taken yet. The focus of this research is to explore a technology-based, low cost solution that will lower or eliminate the risk of accidents on this two-lane rural highway. / Master of Science

腸神經系統(enteric nervous system, ENS)是由大量神經元和神經膠質細胞聚集而成的最複雜的周圍神經系統。這些腸道的神經元和神經膠質細胞來源于迷走神經脊和骶神經脊細胞，在胚胎發育過程中，這些神經脊細胞沿著腸道移動最終占滿整個腸道。儘管神經脊細胞的遷移對於腸道神經系統的形成及功能的正常發揮起到很重要的作用，然而影響神經脊細胞遷移的分子機制的研究卻相對較少。因此找出參與調控神經脊細胞遷移的基因對於更好的瞭解腸道神經脊系統的發育起到非常重要的作用，並且為治療腸道神經系統紊亂所導致的相關疾病提供治療靶點。 / 本研究論文是由兩部份實驗課題所組成來研究影響腸和調控道神經脊細胞遷移及腸道神經系統發育的相關基因。第一部份課題主要研究的是Semaphorin3A (Sema3A)對於骶神經脊細胞遷移的影響。本論文的研究發現Sema3A不僅被腸道內的上皮細胞所表達，腸道兩側的盆神經節周圍的間質細胞也表達Sema3A。同時Sema3A的受體neuropilin-1被骶神經脊細胞所表達。體外培養的實驗表明Sema3A能夠抑制骶神經脊細胞的遷移。另外，當表達Sema3A的腸道末端與骶神經脊細胞共同培養時，骶神經脊細胞的遷移同樣也受到抑制。這些研究結果表明由腸道末端的上皮細胞和腸道外圍的間質細胞所表達的Sema3A共同作用來調控骶神經脊細胞在停滯時期的遷移活動。 / 第二部份的研究課題主要研究的是轉錄因子Sox10以及其靶基因對於迷走神經脊細胞遷移的影響。Dominant megacolon (Dom)是一種攜帶有Sox10突變的巨結腸癥小鼠模型。本研究利用這種小鼠模型來發現突變鼠中可能影響迷走神經脊細胞遷移的基因。從迷走神經脊細胞體外培養發現： 由於Sox10突變，迷走神經脊細胞在體外培養24小時后，細胞遷移延遲，細胞的分化能力被改變，並且細胞死亡增加。利用基因芯片的方法比較了純和變異鼠迷走神經脊細胞和正常鼠迷走神經脊細胞的基因表達的差異。螢光素酶報告基因分析顯示，Sox10可以結合Lama4, Itga4和Gfra2的啟動子并激活它們的表達。 Sox10能與Gfra2啟動子上-116bp到-58bp之間序列的結合誘導Gfra2的表達。在純和變異鼠迷走神經脊細胞中，通過上調Gfra2信使RNA的表達，細胞死亡的數目大大下降，表明Gfra2作為Sox10的靶基因，對迷走神經脊細胞的存亡有著重要作用。 / 綜上所述，我們發現在骶神經脊細胞未進入腸道末端的這段停滯期內，Sema3A對於骶神經脊細胞的遷移起到抑制作用，Sema3A通過其表達在這段停滯期內的時空改變來調控骶神經脊細胞進入腸道。另外我們發現由於Sox10的突變，迷走神經脊細胞表現出非正常的遷移和基因表達的變化。作為Sox10的靶基因，Gfra2對於迷走神經脊細胞的死亡有重要的作用。 / The enteric nervous system (ENS) is the most complex part of the peripheral nervous system which is composed of a vast number of neurons and glial cells. The enteric neurons and glial cells arise from vagal and sacral neural crest cells (NCCs) which migrate along the gastrointestinal tract to colonize the whole gut during the embryonic development. The molecular mechanisms regulating the NCC migration are poorly characterized despite the importance of this migration process in the ENS formation. Therefore, identification and characterization of molecules involved in the modulation of NCC migration are essential to understand the ENS development and could provide potential therapeutic targets for the treatment of human ENS disorders. / The present study was aimed to identify and characterize the molecules involved in modulating the NCC migration during the ENS development, and was divided into two parts. The first part focused on semaphorin3A (Sema3A) signaling, Sema3A was found to be expressed in the hindgut epithelium and also the adjacent regions of pelvic ganglia, while its receptor, neuropilin-1, was expressed by sacral NCCs before sacral NCCs entered the hindgut. Sacral NCC migration and neuronal fiber extension in vitro were retarded in the culture medium containing Sema3A. When a hindgut segment expressing Sema3A was co-cultured with sacral NCCs, sacral NCC migration and neuronal fiber extension were also suppressed by the hindgut segment. These findings provide evidence for the repulsive activity of Sema3A before the entry of sacral NCCs to the hindgut. / The second part focused on the potential target genes of the transcription factor Sox10 which is expressed by migrating NCCs. A naturally occurring mouse mutant Dominant megacolon (Sox10Dom) which expresses a mutant Sox10 was used to identify candidate molecules which may possibly affect the NCC migration. After 24 hours in culture, vagal NCCs from Sox10Dom/Dom embryos showed retarded migration, abnormal cell differentiation and excessive cell death in vitro when compared to Sox10⁺/⁺ vagal NCCs. Results of microarray analyses revealed differentially expressed genes in Sox10Dom/Dom as compared to Sox10⁺/⁺ vagal NCCs after 24 hours in culture. Among these genes, Sox10 was able to bind to the promoter of Itga4, Lama4, and Gfra2 to induce their expression. Sox10 activated Gfra2 promoter by direct binding to the critical region located between -116bp and -58bp upstream of the Gfra2 transcription start site. Finally, re-expression of Gfra2 in Sox10Dom/Dom vagal NCCs resulted in decreased cell death, suggesting that down-regulation of Gfra2 in the mutant mice played an important role in early cell death of vagal NCCs. / In conclusion, before sacral NCCs entered into the hindgut, Sema3A inhibited the sacral NCC migration, and the spatiotemporal change of the Sema3A distribution regulated the entry of sacral NCCs into hindgut. Furthermore, retarded cell migration, abnormal cell differentiation, increased cell death and differential gene expression were found in Sox10Dom/Dom vagal NCCs as compared with those from Sox10⁺/⁺ embryos in vitro. The expression of Gfra2, a potential target gene of Sox10, promoted the cell viability of vagal NCCs. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wang, Cuifang. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 180-196). / Abstracts also in Chinese. / Wang, Cuifang.

published_or_final_version / Biochemistry / Master / Master of Philosophy

published_or_final_version / abstract / Surgery / Master / Master of Philosophy

No description available.

No description available.

The neural crest (NC) is a multipotent embryonic cell type derived from the ectoderm during neurulation giving rise to a variety of cell lineages such as neurons, glia and pigment cells. Most genes associated with the correct initiation, differentiation and migration of the neural crest have been found through reverse genetics. Similarities between neural crest development and some features of cancer progression are remarkable. For instance, it has been suggested that some cancer types recapitulate NC processes in an unregulated manner such as epithelial-mesenchymal transition or active cell migration throughout the body to form distant metastases. However, to date very little is known about initiators and drivers that direct neural crest cell migration to specific target sites. The Medaka mutant hirame represents an interesting melanocyte specific migration defect on the yolk sac caused by a loss of functional Yes-associated protein (YAP). Medaka hirame mutants were initially studied for their profound changes in body morphology. Genomic mapping identified the causal mutation as a nonsense point mutation within the first WW domain in the Yes-associated protein 1 (YAP1), causing translation of a dysfunctional YAP protein. YAP is a downstream transcriptional co-activator of the recently discovered and evolutionarily conserved Hippo pathway. Alterations within Hippo signalling are linked to cell survival, proliferation and abnormal tissue overgrowth. We demonstrate that hirame melanocyte precursors (melanoblasts) are initially present in normal abundance, but show an early migration defect with a lack of melanoblasts on the yolk sac, and corresponding accumulation in the lateral parts of the body. Subsequently, we observe an overall decline in differentiated melanocyte numbers during late stage embryogenesis. We designed an overexpression cassette linking enhanced GFP to either wild type or a mutated activated version of YAP and present evidence that it can efficiently rescue the melanocyte defect after injection of mRNA into one-cell stage embryos. Furthermore, analysis of the yolk sac anatomy via transmission electron microscopy indicates that a fraction of yolk membrane cells undergo apoptosis and we propose that this may contribute to the establishment of altered environmental cues leading to abnormal melanoblast migration onto the yolk sac. Injection of yap mRNA directly into the yolk sac however, failed to rescue melanoblast patterning. To advance our study, we isolated and characterised a 3.6 kb Medaka dopachrome tautomerase (Dct) promoter fragment, and used it to drive expression of enhanced green fluorescent protein (eGFP) in vivo. We generated germline transgenics with this construct that showed lineage-specific expression of eGFP within early migrating melanoblasts, a phenotype that is maintained in differentiated melanocytes throughout embryogenesis. In addition, using this promoter we overexpressed our egfp-yap fusion cassette and established transgenic lines to assess the cell autonomy of YAP within the melanocyte lineage. However, no fluorescent signal could be detected in the latter transgenics, necessitating future experimentation to properly characterise these lines. Finally, we analysed a range of neural crest markers to examine the extent of the neural crest defects in hirame mutants. In addition to the melanocyte phenotype, we identified a dramatic reduction in xanthophore numbers, although early leucophore development appears unaffected. We also observed a decreased number of dorsal root ganglia in the peripheral nervous system as well as smaller and partly ectopic cranial neural crest ganglia populations within the epibranchial arches. The characterisation of a novel Medaka melanocyte specific promoter as well as additional novel NC markers will be widely applicable and useful to the wider Medaka research community as a tool for the study of neural crest related mechanisms during development.

by Yung, Kim Ming. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 135-153). / Abstract --- p.i / Acknowledgments --- p.iv / List of content --- p.v / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Neural crest cells and cardiac neural crest cells --- p.1 / Chapter 1.2 --- The role of cardiac neural crest cells in the septation of the outflow tract --- p.5 / Chapter 1.3 --- Neural crest-related malformations --- p.8 / Chapter 1.4 --- Early changes in cardiovascular development induced by neural crest ablation --- p.11 / Chapter 1.5 --- Experimental strategies commonly employed in tracing the premigratory neural crest cells --- p.14 / Chapter 1.6 --- Objectives of the present study --- p.21 / Chapter Chapter 2 --- Location of the cardiac neural crest along the neural axis in the mouse embryo --- p.24 / Chapter 2.1 --- Introduction --- p.24 / Chapter 2.2 --- Materials and Methods --- p.29 / Chapter 2.2.1 --- Preparation of DiI --- p.29 / Chapter 2.2.2 --- Embryo collection --- p.29 / Chapter 2.2.3 --- Microinjection of DiI --- p.30 / Chapter 2.2.4 --- Isolation of tissue fragments from the lateral neural epithelium --- p.31 / Chapter 2.2.5 --- Dil labelling of the donor fragment isolated from the lateral neural epithelium --- p.32 / Chapter 2.2.6 --- Grafting of DiI labelled fragments from the lateral neural epithelium --- p.32 / Chapter 2.2.7 --- Embryo culture --- p.33 / Chapter 2.2.8 --- Examination of cultured embryos --- p.34 / Chapter 2.2.9 --- Cryosection --- p.35 / Chapter 2.3 --- Results --- p.36 / Chapter 2.3.1 --- Development of the cultured embryos in control and experimental groups --- p.36 / Chapter 2.3.2 --- Location of the cardiac neural crest region along the neural axis --- p.38 / Chapter 2.4 --- Discussion --- p.44 / Chapter 2.4.1 --- Development of embryos in vitro --- p.44 / Chapter 2.4.2 --- Comparison of the two methods for tracing cell migration: focal labelling and orthotopic grafting --- p.49 / Chapter 2.4.3 --- Location of the cardiac neural crest region along the neural tube --- p.53 / Chapter Chapter 3 --- Initial and terminal stages of cardiac neural crest cell migration --- p.56 / Chapter 3.1 --- Introduction --- p.56 / Chapter 3.2 --- Materials and Methods --- p.62 / Chapter 3.2.1 --- Examination of the initial and terminal stages of migration of cardiac neural crest cells by haematoxylin and eosin (H&E) staining --- p.62 / Chapter 3.2.2 --- Preparation of WGA-Au --- p.62 / Chapter 3.2.3 --- Collection of embryos for microinjection of WGA-Au --- p.63 / Chapter 3.2.4 --- WGA-Au labelling of the presumptive cardiac neural crest region --- p.64 / Chapter 3.2.5 --- Embryo culture --- p.65 / Chapter 3.2.6 --- Examination of cultured embryos --- p.66 / Chapter 3.2.7 --- Silver enhancement staining --- p.66 / Chapter 3.3 --- Results --- p.67 / Chapter 3.3.1 --- Initial stage of cardiac neural crest migration studied by haematoxylin and eosin staining and silver enhancement staining --- p.67 / Chapter 3.3.2 --- Terminal stage of cardiac neural crest migration studied by haematoxylin and eosin staining and silver enhancement staining --- p.69 / Chapter 3.4 --- Discussion --- p.71 / Chapter 3.4.1 --- Wheat germ agglutinin-gold conjugate (WGA-Au) as a cell marker --- p.71 / Chapter 3.4.2 --- Initial stage for cardiac neural crest cell migration --- p.72 / Chapter 3.4.3 --- Terminal stage for cardiac neural crest cell migration --- p.74 / Chapter Chapter 4 --- Migration pathways of cardiac neural crest cells… --- p.77 / Chapter 4.1 --- Introduction --- p.77 / Chapter 4.2 --- Materials and Methods --- p.82 / Chapter 4.2.1 --- Preparation of DiI --- p.82 / Chapter 4.2.2 --- Preparation of WGA-Au --- p.82 / Chapter 4.2.3 --- Embryo collection --- p.82 / Chapter 4.2.4 --- Microinjection of WGA-Au and DiI --- p.82 / Chapter 4.2.5 --- Isolation of tissue fragments from the lateral neural epithelium --- p.83 / Chapter 4.2.6 --- WGA-Au labelling of the donor fragments from the lateral neural epithelium --- p.83 / Chapter 4.2.7 --- DiI labelling of the donor neural epithelium --- p.83 / Chapter 4.2.8 --- Grafting of WGA-Au or DiI-labelled donor tissues from the lateral neural epithelium --- p.83 / Chapter 4.2.9 --- Coating of latex beads by WGA-Au --- p.83 / Chapter 4.2.10 --- Microinjection of WGA-Au-coated latex beads --- p.84 / Chapter 4.2.11 --- Embryo culture --- p.84 / Chapter 4.2.12 --- Examination of cultured embryos --- p.85 / Chapter 4.2.13 --- Silver enhancement staining of the WGA-Au labelled sections --- p.85 / Chapter 4.2.14 --- Cryosection --- p.85 / Chapter 4.3 --- Results --- p.86 / Chapter 4.3.1 --- Distribution of labelled cells after WGA-Au labelling or orthotopic grafting --- p.86 / Chapter 4.3.2 --- Distribution of labelled cells after DiI labelling or orthotopic grafting --- p.88 / Chapter 4.3.3 --- Distribution of latex beads --- p.90 / Chapter 4.4 --- Discussion --- p.92 / Chapter 4.4.1 --- Methodology --- p.92 / Chapter 4.4.2 --- Migration pathways of the cardiac neural crest cells --- p.94 / Chapter 4.4.3 --- Migration of latex beads --- p.98 / Chapter Chapter 5 --- Derivatives of cardiac neural crest cells in the developing mouse heart --- p.101 / Chapter 5.1 --- Introduction --- p.101 / Chapter 5.2 --- Materials and Methods --- p.110 / Chapter 5.2.1 --- DiI labelling of the cardiac neural crest region of the mouse embryo --- p.110 / Chapter 5.2.2 --- Collection of the embryonic hearts --- p.111 / Chapter 5.2.3 --- Heart organ culture --- p.111 / Chapter 5.2.4 --- Cryosectioning --- p.112 / Chapter 5.2.5 --- Paraffin wax sectioning --- p.113 / Chapter 5.2.6 --- Immunohistochemical staining --- p.113 / Chapter 5.3 --- Results --- p.118 / Chapter 5.3.1 --- Distribution of 2H3 positive cells in the heart developedin vivo --- p.118 / Chapter 5.3.2 --- Development of the heart at 10.5 d.p.c. in organ culture --- p.119 / Chapter 5.3.3 --- Distribution of DiI labelled cells in the heart one day after organ culture --- p.119 / Chapter 5.3.4 --- Distribution of 2H3 positive cells in the hearts one day after organ culture --- p.120 / Chapter 5.4 --- Discussion --- p.121 / Chapter 5.4.1 --- Relationship between 2H3 positive cells and cardiac conduction system --- p.121 / Chapter 5.4.2 --- Development of the mouse embryonic hearts in vitro --- p.123 / Chapter 5.4.3 --- Distribution patterns of the 2H3 immunopositive cellsin the hearts developed in vitro and in vivo --- p.125 / Chapter 5.4.4 --- Relationship between the DiI labelled cells and2H3 immunopositive cells --- p.125 / Chapter 5.4.5 --- Genes that express in the cardiac neural crest cells --- p.127 / Chapter Chapter 6 --- Conclusion --- p.129 / References --- p.135 / Appendix --- p.154

The neural crest is a transient, multipotent, cell population that gives rise to several important tissues during embryonic development, including the craniofacial skeleton, peripheral nervous system, and melanocytes. The neural crest arises from the ectoderm, along with the skin and central nervous system. This process of specification is dependent on a gene regulatory network (GRN) which is made up of transcription factors that regulate each other. While we know many of the members of this GRN, the direct connections among the members are largely unsolved. Breakdown of this GRN can lead to birth defects, such as cleft lip and palate, and cancer of neural crest derivatives, such as melanoma, thus understanding the intricate details of this network is important. The transcription factor Tfap2a is an important member of the GRN, as loss of tfap2a and its paralog tfap2c leads to loss of pre-migratory neural crest and all neural crest derivatives. Despite its importance in this network little is known about how its expression is regulated. We hypothesized that, due to its importance in this network, it will have multiple enhancers that drive its expression in the neural crest. We have identified two neural crest enhancers of tfap2a. We found that one of these enhancers is responsive to WNT signals and is maintained by forming a positive feedback loop with Sox10. Our results suggest that this enhancer is important for both induction and maintenance of tfap2a expression in the neural crest. Tfap2 paralogs are important at several different stages throughout neural crest lineage specification. However, the only direct target of Tfap2a that has been identified is sox10. Thus, we wanted to determine the direct targets of Tfap2 in this network. Through the integration of several data sets, including ATAC-seq and expression profiling of tfap2a/c double mutants, we have identified several direct targets including sox9b and alx1. Melanoma is cancer of the melanocytes, a neural crest derivative. Recent studies have shown that melanoma and the neural crest share genetic similarities. TFAP2A expression is decreased in metastatic melanoma compared to primary tumors, thus we wanted to investigate the mechanism of TFAP2A in metastatic melanoma. We found that the promoter of TFAP2A is hypermethylated in some metastatic melanoma tumors. This was confirmed by samples in the TCGA database. Hypermethylation of the promoter contributes to the downregulation of TFAP2A in metastatic melanoma. In conclusion, we have further illuminated the connections among transcription factors in the GRN important for neural crest lineage specification. Further, we have identified a new mechanism regulating TFAP2A expression in metastatic melanoma. Together, these studies reveal regulatory mechanisms of TFAP2A gene expression.