published_or_final_version / Biochemistry / Master / Master of Philosophy

published_or_final_version / abstract / Surgery / Master / Master of Philosophy

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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.

A pancreatic stem cell could provide the tissue necessary for widespread β-cell transplantation therapy for diabetes. It is disputed whether pancreatic stem cells or β-cell replication are responsible for maintenance and regeneration of endocrine cells. Evidence presented here shows that pancreatic stem cells express insulin and produce multiple endocrine, exocrine and neural cells in vitro and in vivo. The human pancreas also contains stem cells that produce functional β-cells capable of reducing blood sugar levels in a diabetic mouse. Initial studies of pancreatic stem cells grown clonally in vitro indicated that they produced large numbers of neurons, suggesting they may be derived from the neural crest. Evidence shows that there are at least two distinct developmental origins for stem cells in the pancreas; one from the pancreatic lineage that produces endocrine and exocrine cells and one from the neural crest lineage that produces neurons and Schwann cells. Furthermore, pancreatic stem cells require the developmental transcription factor, Pax6, for endocrine cell formation suggesting they are using expected differentiation pathways. There is an interesting evolutionary connection between pancreatic β-cells and neurons which was applied to the derivation of pancreatic stem cells from human embryonic stem cells by using a clonal neural stem cell assay. These pancreatic stem cells express pancreatic and neural markers, self-renew and differentiate into insulin-expressing cells. The overexpression of SOX17 in these cells increases stem cell formation and self-renewal but inhibits differentiation. Overall I will show that there is a genuine stem cell in the adult mammalian pancreas capable of producing functional β-cells, that this stem cell is derived from the pancreatic developmental lineage but the pancreas also contains stem cells from the neural crest lineage, and that the neural stem cell assays that have identified these adult stem cells can be applied to the derivation of a pancreatic stem cell from hESCs.

A pancreatic stem cell could provide the tissue necessary for widespread β-cell transplantation therapy for diabetes. It is disputed whether pancreatic stem cells or β-cell replication are responsible for maintenance and regeneration of endocrine cells. Evidence presented here shows that pancreatic stem cells express insulin and produce multiple endocrine, exocrine and neural cells in vitro and in vivo. The human pancreas also contains stem cells that produce functional β-cells capable of reducing blood sugar levels in a diabetic mouse. Initial studies of pancreatic stem cells grown clonally in vitro indicated that they produced large numbers of neurons, suggesting they may be derived from the neural crest. Evidence shows that there are at least two distinct developmental origins for stem cells in the pancreas; one from the pancreatic lineage that produces endocrine and exocrine cells and one from the neural crest lineage that produces neurons and Schwann cells. Furthermore, pancreatic stem cells require the developmental transcription factor, Pax6, for endocrine cell formation suggesting they are using expected differentiation pathways. There is an interesting evolutionary connection between pancreatic β-cells and neurons which was applied to the derivation of pancreatic stem cells from human embryonic stem cells by using a clonal neural stem cell assay. These pancreatic stem cells express pancreatic and neural markers, self-renew and differentiate into insulin-expressing cells. The overexpression of SOX17 in these cells increases stem cell formation and self-renewal but inhibits differentiation. Overall I will show that there is a genuine stem cell in the adult mammalian pancreas capable of producing functional β-cells, that this stem cell is derived from the pancreatic developmental lineage but the pancreas also contains stem cells from the neural crest lineage, and that the neural stem cell assays that have identified these adult stem cells can be applied to the derivation of a pancreatic stem cell from hESCs.

Neural crest cells (NCC) arise from the neural tube (NT) and migrate through given regions of embryos, where they generate most of the peripheral nervous system (PNS), facial skeleton and pigment cells. Defective NCC development gives rises to malformations in multiple NCC-derived structures, collectively known as neurocristopathies. NCC from the NT vagal and trunk levels express Hoxb5 plus a number of other Hox proteins. Hoxb5 is a member of Hox transcription factors family that binds to specific target nucleotide sequences in the genome via their DNA-binding domain, where they regulate gene expressions. Vagal NCC migrate to the intestine and generate the enteric nervous system (ENS). To test the Hoxb5 function in vagal NCC, we made use a transgenic mouse line (enb5) and showed that perturbation of Hoxb5 signaling in NCC resulted in down-regulation of Ret and defective ENS, indicating that normal Hoxb5 function was required for the development of vagal NCC. Current project aims to investigate the function of Hoxb5 in trunk NCC development. Transgenic mouse enb5 can be induced by Cre recombinase to express a hybrid protein namely engrailed-Hoxb5 (enb5), in which the transactivation domain of the mouse Hoxb5 is replaced with a repressor domain of the Drosophila engrailed (en) protein. With the intact DNA-binding domain, enb5 binds to target genes of Hoxb5, repressing the expression of target genes instead of induction. Therefore, enb5 produces a dominant negative effect on the developmental pathways that normally require Hoxb5. In this study, enb5 mice were crossed to Wnt1-Cre mice to express enb5 in NCC that arose from the entire length of NT. Wnt1-Cre/enb5 mutants displayed apoptosis of NCC, skin hypopigmentation and PNS defects (hypoplastic dorsal root ganglion and defective ENS). Expression of Sox9, Foxd3 and Ret was down-regulated in Wnt1-Cre/enb5 embryos. Conditional deletion of Sox9 and Foxd3 by Wnt1-Cre, or conventional deletion of Ret in mice produced NCC phenoptypes similar to those of Wnt1-Cre/enb5. Taken all these prompted me to further investigate if Hoxb5 functioned in the same pathway as Sox9 and Foxd3 for NCC development using multiple experimental approaches. In ovo electroporation of enb5 in chick embryos induced apoptosis of NT, and co-electroporation of Hoxb5, Ret, Sox9 or Foxd3 rescued enb5-induced cell death. By bioinformatics analysis, Hoxb5 binding sites were identified in SOX9 and FOXD3 promoter sequences. Binding of Hoxb5 protein onto these binding sites of SOX9 and FOXD3 promoters was revealed by electro-mobility shift assay and further confirmed by chromatin immuno-precipitation assay. In addition, enb5 was also shown to bind to the same regions of SOX9 and FOXD3 promoters as Hoxb5. Using dual luciferase reporter assay, Hoxb5 was shown to induce transcription from SOX9 and FOXD3 promoters, and enb5 blocked the induction. Taken all these indicate that (i) Hoxb5 binds and induces transcriptions from SOX9 and FOXD3 promoters, (ii) enb5 blocks the induction. In summary, Hoxb5 regulates NCC development by controlling the expression of Sox9, Foxd3 and Ret, and perturbation of Hoxb5 signaling results in NCC death and neurocristopathies. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy