Thesis (M. Phil.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 104-132).

The anterior visceral endoderm (AVE) is a specialised subpopulation of the visceral endoderm (VE), a single layer simple epithelium that surrounds the extra-embryonic ectoderm and epiblast of the egg cylinder stage embryo. Initially induced at the distal tip of the egg cylinder, AVE cells undergo a stereotypic migration towards the prospective anterior, stopping at the interface between the underlying epiblast and extra-embryonic ectoderm (ExE). Previous research has shown that membrane enrichment of Dvl2 is present in the VE overlying the epiblast (Epi-VE). In this thesis I confirm the presence of planar cell polarity (pep) signalling in this region by assaying the subcellular localisation of additional core pep proteins Vangl2 and Daaml. I show that null embryos of the Nodal antagonist Lefty1 exhibit ectopic membrane enrichment of Dvl2 and a previously unreported AVE over-migration phenotype. Furthermore, using pharmacological inhibition of Nodal signalling I show that the TGF~ protein Nodal modulates pep signalling in the YE. Utilising DIe and confocal microscopy I perform detailed time-lapse analyses of the VE to quantify the dynamic cell behaviour and topology. Using this assay I show that wild-type embryos exhibit dynamic cell movement, which is regionally restricted to the Epi-VE. Analysis of Leftyl-/- and ROSA26lyn-Celsr-l mutants, both of which exhibit disrupted pep signalling and AVE over-migration phenotypes, indicates that normal VE dynamics and topology are disrupted. The results of this quantitation indicate that these mutants exhibit increased cell migration and neighbour exchange across the YE. These data show that regional restriction of movement is lost and results in the AVE over-migration phenotypes observed. Together these results show that regionally restricted pep signalling in the VE acts to modulate cell behaviour and topology, which in turn determines the regional restriction and normal end-point of AVE migration.

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