by Cheung Chui Shan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 96-107 (2nd gp.)). / Abstracts in English and Chinese. / ABSTRACT (ENGLISH) --- p.i / ABSTRACT (CHINESE) --- p.iii / ACKNOWLEDGEMENTS --- p.v / TABLE OF CONTENT --- p.vii / Chapter CHAPTER ONE --- GENERAL INTRODUCTION / Chapter 1.1 --- Early development of the central nervous system --- p.1 / Chapter 1.2 --- Neural crest cells and Cardiac neural crest cells --- p.1 / Chapter 1.3 --- Role of neural crest cells in cardiovascular development --- p.4 / Chapter 1.4 --- Methods in tracing neural crest cells --- p.7 / Chapter 1.5 --- Neural crest-related defects --- p.14 / Chapter 1.6 --- Animal models for studying neural crest defects --- p.16 / Chapter 1.7 --- Recent studies on the migration of cardiac neural crest cells in mammals --- p.19 / Chapter 1.8 --- Objectives of the present study --- p.22 / Chapter CHAPTER TWO --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.26 / Pregnant mice --- p.26 / Pregnant Splotch mice (Sp2H) --- p.26 / Preparation of the handling medium --- p.27 / Preparation of the culture medium --- p.27 / Gas mixtures for embryo culture --- p.30 / Preparation of wheat germ agglutinin-gold conjugates (WGA-Au) --- p.30 / Preparation of the fixative --- p.30 / DNA solution for genotyping of Splotch embryos --- p.31 / Primers used in PCR for genotyping of Splotch embryos --- p.31 / PCR reagent system --- p.32 / 10XTBE --- p.32 / Chapter 2.2 --- Methods --- p.33 / Isolation of embryos from pregnant mice --- p.33 / In situ labelling of exogenous dye --- p.34 / Orthotopical grafting of neural crest fragment --- p.36 / Whole embryo culture --- p.37 / Morphological examination of cultured embryos --- p.38 / Histological examination of cultured embryos --- p.38 / Examination of labelled cells in sectioned embryos --- p.39 / Genotyping of Splotch embryos by PCR --- p.40 / Gel electrophoresis --- p.41 / Chapter CHAPTER THREE --- RESULTS / Chapter 3. 1 --- Initial migration of cardiac neural crest cells in normal ICR mouse embryos --- p.43 / Gross morphological examination of cultured embryos --- p.43 / Distribution of WGA-Au labelled cells in ICR normal mouse embryos --- p.45 / Chapter 3.2 --- Initial migration of cardiac neural crest cells in Splotch embryos --- p.50 / Genotyping --- p.50 / Morphological examination of Splotch mutant embryos --- p.50 / Morphological examination of in vivo Splotch embryos --- p.53 / Distribution of WGA-Au labelled cells in Splotch Embryos --- p.54 / Chapter 3.3 --- Transplantation of neural crest fragments in Splotch embryos --- p.60 / Morphological features of Splotch embryos after orthotopic grafting --- p.60 / Histological examination of Splotch embryos after grafting --- p.61 / Distribution of WGA-Au labelled cells in Splotch embryos after grafting --- p.62 / Chapter CHPATER FOUR --- DISCUSSION / Chapter 4.1 --- Development of embryos in vitro --- p.65 / Chapter 4.2 --- Methodology --- p.70 / In situ labelling of WGA-Au in embryos --- p.70 / Counting of labelled cells in Sploch embryos --- p.72 / Transplantation of neural crest fragments --- p.72 / Chapter 4.3 --- Initial migration of cardiac neural crest cells --- p.74 / Distribution of cardiac neural crest cellsin normal mouse embryos --- p.74 / Differences in the distribution of labelled neural crest cells In different genotypes of Splotch embryos --- p.78 / Distribution of cardiac neural crest cells in Splotch embryos After transplanting of neural crest fragments --- p.83 / Chapter 4.4 --- Factors in extracellular matrix affecting the migration of neural crest cells --- p.88 / Chapter CHAPTER FIVE --- CONCLUSION --- p.91 / REFERENCES --- p.96 / FIGURES AND LEGEND / TABLES / GRAPHS

Siu-wai Michael Sung. / Thesis submitted in: December 2000. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 97-111). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Abbreviations --- p.v / Table of Contents --- p.vii / Chapter Chapter 1 --- General Introduction / Chapter l. 1 --- Drosophila as a model for studying development --- p.1 / Chapter l .2 --- The formation of the body axis in Drosophila --- p.2 / Chapter l .3 --- Maternal genes are essential for development --- p.9 / Chapter 1.4 --- Maternal gene bicoid is essential for formation of the anterior structures in the embryo --- p.13 / Chapter 1.5 --- Establishment of an anterior to posterior bicoid protein gradient --- p.13 / Chapter 1.6 --- The bicoid protein gradient controls the downstream zygotic target genes in a concentration-dependent manner --- p.17 / Chapter 1.7 --- Bicoid protein acts as transcriptional regulators \9 --- p.19 / Chapter 1.8 --- Bicoid protein acts as transcriptional regulators --- p.21 / Chapter 1.9 --- The anterior localization of bcd mRNA --- p.21 / Chapter 1.10 --- Components required for bcd mRNA localization at anterior pole of oocyte / Chapter 1.10.1 --- Cis-acting elements --- p.22 / Chapter 1.10.1.1 --- BLE1 at 3' UTR directs localization of bcd mRNA --- p.23 / Chapter 1.10.2 --- Trans-acting elements / Chapter 1.10.2.1 --- "Exuperantia, swallow, and staufen are necessary for localization for bcd mKNA" --- p.27 / Chapter 1.10.2.2 --- exu protein is an absolute requirement for localization for bcd mRNA --- p.30 / Chapter 1.10.2.3 --- Microtubules dependence of localization --- p.31 / Chapter 1.11 --- Functions of exu in localization of bcd mRNA --- p.32 / Chapter 1.12 --- Characteristics of Bicoid protein and Bic-D gene --- p.33 / Chapter 1.13 --- Aim of Project --- p.36 / Chapter CHAPTER 2 --- Materials and Methods / Chapter 2.1 --- Fly Food --- p.37 / Chapter 2.2 --- Conditions in maintaining the fly stocks and working stocks --- p.37 / Chapter 2.3 --- Localization of exu protein and other intracellular elements by indirect immunofluorescence detection / Chapter 2.3.1 --- Immunohistrochemical distribution of exu and Bic-D protein --- p.38 / Chapter 2.3.2 --- Immunohistrochemical distribution of β-tubulin --- p.39 / Chapter 2.4 --- Preparation of total protein from the female and male flies --- p.41 / Chapter 2.5 --- Analysis of interactions between exu and trans-acting elements / Chapter 2.5.1 --- 35S-methionine metabolic labelling and immunoprecipitation by RIPA buffer --- p.41 / Chapter 2.5.2 --- 35S-methionine metabolic labelling and immunoprecipitation by Mach and Lehmann buffer system --- p.43 / Chapter 2.6 --- Co-immunoprecipitation of exu and Bic-D protein / Chapter 2.6.1 --- Co-immunoprecipitation of exu and Bic-D protein synthesized by in vitro coupled transcription and translation system with modified Mach and Lechmann buffer system --- p.44 / Chapter 2.7 --- in vivo ovary extract co-immunoprecipitation / Chapter 2.7.1 --- in vivo ovary extraction co-immunoprecipitation of exu and Bic-D protein with modified Mach and Lehmann buffer system supplemented with recombinant exu protein --- p.45 / Chapter CHAPTER 3 --- Results / Chapter 3.1 --- Analysis of co-localization of exu and Bic-D protein by double immuno-fluorescence staining on w1118 flies --- p.47 / Chapter 3.2 --- Analysis of co-localization of exu protein and β-tubulin protein by double immuno-fluorescence staining on w1118 flies --- p.51 / Chapter 3.3 --- Analysis of co-localization of exu and Bic-D protein by double immuno-fluorescence staining on Bic-D mutants --- p.55 / Chapter 3.4 --- Co-immunoprecipitation of exu and Bic-D protein synthesized by in vitro coupled transcription and translation system --- p.61 / Chapter 3.5 --- 35S-Methionine metabolic labelling and co-immunoprecipitation of exu and Bic-D protein with RIP A buffer system --- p.65 / Chapter 3.6 --- 35S-Methionine metabolic labelling and co-immunoprecipitation of exu and Bic-D protein with Mach and Lehmann buffer system --- p.68 / Chapter 3.7 --- in vivo ovary extract co-immunoprecipitation of exu and Bic-D protein with modified Mach and Lehmann buffer system supplemented with recombinant exu protein --- p.71 / Chapter CHAPTER 4 --- Discussion / Chapter 4.1 --- Analysis of co-localization of exu protein and other intracellular elements by indirect double immunofluorescence staining detection --- p.74 / Chapter 4.2 --- Analysis of co-localization of exu and BicD protein by double immuno- fluorescence staining on Bic-D mutants --- p.78 / Chapter 4.3 --- Co-immunoprecipitation of exu and BicD protein synthesized by in vitro coupled transcription and translation system --- p.79 / Chapter 4.4 --- Analysis of interactions between exu and trans-acting elements by 35S- Methionine metabolic labelling and immunoprecipitation --- p.82 / Chapter 4.5 --- "in vivo ovary extract coimmunoprecipitation of exu and Bic-D protein with modified Mech and Lehmann buffer system, supplemented with recombinant exu protein" --- p.84 / Chapter 4.6 --- Recent developments on the concept of ribonucleoprotein --- p.86 / Appendix A Supplementary protocols --- p.91 / Appendix B Reagents --- p.95 / Reference --- p.97

Objetivo: Contribuir para a ampliação do conhecimento das fissuras orais, descrevendo, sob o aspecto genético-clínico, uma amostra de indivíduos com fissura pré-forame incisivo uni/bilateral incompleta e fissura pós-forame incisivo associadas. Casuística e metodologia: Foram selecionados 356 indivíduos com fissura pré-forame incisivo uni/bilateral incompleta, sem acometimento do arco alveolar, associada à fissura pós-forame incisivo, cadastrados e em tratamento no Hospital de Reabilitação de Anomalias Craniofaciais da Universidade de São Paulo, Bauru, SP. Dados de razão sexual, idade parental na época da concepção, consanguinidade parental, recorrência familial, lateralidade da fissura e presença de anomalias associadas à fissura foram investigados. Para análise dos resultados foram destacados dois grupos (Grupos I e Grupo II) da amostra total. No Grupo I foram incluídos os indivíduos que apresentaram fissura pré-forame incisivo cicatricial, independentemente do tipo de acometimento pós-forame. Indivíduos do Grupo I, que, além de apresentarem fissura pré-forame incisivo cicatricial apresentaram também algum tipo de microforma de fissura pós-forame incisivo, foram destacados para formarem também o Grupo II. Testes estatísticos de comparação foram realizados entre os Grupos e o restante da amostra e entre a amostra total e dados da literatura pertinente. Resultados e Discussão: Observou-se diferença estatisticamente significativa entre a amostra total e os dados da literatura em relação à lateralidade da fissura, razão sexual, consanguinidade, recorrência familial e presença de anomalias associadas. Observou-se, ainda, diferença estatisticamente significativa entre o Grupo II e o restante da amostra total quanto à idade paterna e, entre os Grupos I e II e a amostra total, em relação à ocorrência de múltiplas anomalias associadas à fissura. A amostra estudada apresentou, em geral, as mesmas características genético-clínicas do grupo das fissuras pré e transforame incisivo (FL/P). As diferenças encontradas não permitiram afirmar a distinção da fissura pré-forame associada à fissura pós-forame incisivo, sem acometimento do arco alveolar (FL+FP) das FL/P. Da mesma forma não foi possível afirmar, pelos resultados obtidos, que os Grupos I e II eram distintos da amostra total. Conclusão: Embora não se possa afirmar que FL+FP seja distinta das FL/P, suas características peculiares apontam para essa diferenciação. Os indivíduos com quadros de microformas de fissura constituem um grupo alvo de investigações sobre possíveis mecanismos genéticos que levam à gravidade variável dessas malformações. / Purpose: To contribute to the expansion of knowledge about oral clefts, describing the clinical and genetic aspect of a sample of individuals with cleft lip associated with cleft palate, without alveolar arch involvement, showing or not other abnormalities. Patients and methods: We selected 356 patients with incomplete cleft lip uni/bilateral associated with cleft palate, without alveolar arch involvement, registered and in treatment at the Hospital de Reabilitação de Anomalias Craniofaciais da Universidade de São Paulo, Bauru, SP. Data for sexual ratio, parental age at the time of conception, parental consanguinity, familial recurrence, laterality of cleft and presence of associated anomalies were investigated. Regarding the analysis of the results two groups were detached (Group I and Group II) from the total sample. In Group I it was included individuals who had healed cleft lip, regardless of the type of palate involvement. Individuals in Group I, which, besides having had healed cleft lip also had some type of microform cleft palate were also detached to form Group II. Statistical tests were performed for comparison between groups and remainder of the sample, and between the total sample and literature data. Results and Discussion: There was a statistically significant difference between the total sample and literature data regarding laterality of the cleft, sexual ratio, consanguinity, familial recurrence and presence of associated anomalies. There was also a statistically significant difference between Group II and the remainder of the sample regarding paternal age, and between Groups I and II and the total sample in relation to the occurrence of multiple anomalies associated with cleft. The sample has, in general, the same genetic and clinical characteristics of the group of cleft lip with or without cleft palate (CL/P). The differences did not allow distinction between cleft lip associated cleft palate without involvement of the alveolar arch (CL+CP) and CL/P. Likewise it is not possible to affirm, from the results obtained, that Groups I and II are distinct from the total sample. Conclusion: Although we can not say that CL+CP is distinct from the CL/P, its peculiar features indicate to this differentiation. Individuals with microforms of cleft constitute a target group for research on possible genetic mechanisms that lead to varying severity of these malformations.

The CCAAT/enhancer-binding protein delta (C/EBPδ) is a highly conserved transcription factor capable of regulating numerous cell fate processes, such as cell growth, differentiation, proliferation and apoptosis. C/EBPδ is inducible during cellular stress responses, including inflammation and responses to growth factor deprivation or thermal stress. C/EBPδ is stress-inducible in a diversity of fishes, including the zebrafish Danio rerio; however, little is known about its role in fish development. Here I show that overexpression of C/EBPδ leads to severe developmental defects, including reduced body length, edema, liver malformation and retinal abnormalities. The proportion of individuals that display developmental abnormalities is significantly greater in C/EBPδ-overexpressing embryos compared to control embryos and overexpression significantly reduces survival of larvae over time. TUNEL analysis suggests C/EBPδ-overexpressing embryos exhibit a pattern of apoptotic cell death which is spatially distinct from control embryos. These data support a critical role for C/EBPδ in numerous developmental processes, including promoting programmed cell death during development. Mutations in C/EBPδ have been implicated in the progression of human tumors, including those of myeloid, hepatocellular and breast cancers. Therefore, the C/EBPδ-overexpressing zebrafish will serve as a valuable model for examining the role of this gene during development, as a part of the cellular response to stress and in pathological states such as tumor progression.

Abstract not available

Abstract not available

Abstract not available

A genetic selection study to determine the effects on egg hatchability and subsequent chick performance of Japanese quail (Coturnix japonica) eggs incubated at 100 F dry bulb temperature (Control, Line C) when compared to other eggs incubated at 102 F (Selected, Line S) was conducted over 10 consecutive generations. Eggs from a randomly mated population (designated as Generation 0) of Japanese quail maintained at the Oregon Agricultural Experiment Station were randomly allocated to two treatment groups (Lines C and S) and incubated at the different temperatures in separate but identical Jamesway 252 machines. On day 14 of incubation all eggs were transferred to a common hatcher (98.5 F). Using family-based selection, the chicks that hatched from the two lines were subsequently used as breeders (25 paired matings per line) and the resulting eggs from each line incubated at their respective temperatures for 10 consecutive generations. Following the 10th generation percent egg fertility and percent hatch of fertile eggs were greater in Line C vs. Line S (p<O.O3 and p<O.0001, respectively). Embryo development time was shortened in Line S by 24 hours and mean 4- or 5- week body weights were greater (p<0.001) in Line S. Ten-day post-hatch mortality increased greatly in Line S vs. Line C after generation 6 (p<0.001) and hen-day egg production decreased after generation 4 in Line S vs. Line C (p<0.0001). The results indicate that embryo development time can be reduced by high temperature incubation, but at the expense of reproductive traits such as egg production, fertility, and hatchability of fertile eggs. / Graduation date: 2005

Une caractéristique fondamentale des vertébrés est leur organisation métamerique, visible au niveau de la colonne vertébrale. Cette organisation se met en place au cours du développement embryonnaire et l'émergence des précurseurs des vertèbres, les somites, formés à partir du mésoderm paraxial présomitique (PSM). Depuis la découverte d'une activité transcriptionelle oscillatoire de la voie de signalisation Notch dans le PSM, on propose que cette activité oscillatoire représente l'action d'une horloge embryonnaire, l'horloge de segmentation, responsable de contrôler la formation des somites de façon périodique. Dans cette étude, je présente la découverte d'une nouvelle association de la voie Wnt avec l'horloge de segmentation en décrivant l'activité oscillatoire de Axin2, une cible de la voie Wnt, dans le PSM. Ensuite, j'ai mise en place un system d'imagerie biphoton qui nous permet d'observer l'action de l'horloge de segmentation en temps réel et in vivo dans les embryons de souris. Ce système permet de mesurer directement les paramètres des oscillations. Ultérieurement je décris la découverte d'un gradient d'expression de la protéine -caténine dans le PSM. A l'aide d'expériences de recombinaisons homologues conditionnelles nous avons déterminé que ce gradient de - caténine contrôle la maturation et différentiation des cellules dans le PSM. De plus, ce gradient constitue un signal essentiel et permissif pour les oscillations de l'horloge de segmentation. En conclusion, je propose un nouveau modèle de mise en place des somites incorporant les résultats présentés.

The objective of this work is to investigate the elastic modulus of gastrula-stage avian embryos and the effect of substrate stiffness on presumptive precardiac cell fate. Our overall hypothesis is that the mechanical microenvironment, specifically, tissue modulus and substrate stiffness, influences gastrulation and cardiac induction. Large-scale morphogenetic movements during early embryo development are driven by complex changes in biochemical and biophysical factors. Current models for amniote primitive streak morphogenesis and gastrulation take into account numerous genetic pathways but largely ignore the role of mechanical forces. Here, we used atomic force microscopy (AFM) to obtain for the first time precise biomechanical properties of the early avian embryo. Our data reveal that the primitive streak is significantly stiffer than neighboring regions of the epiblast, and that it is stiffer than the pre-primitive streak epiblast. To test our hypothesis that these changes in mechanical properties are due to a localized increase of actomyosin contractility, we inhibited actomyosin contractility via the Rho kinase (ROCK) pathway using the small-molecule inhibitor Y-27632. Our results using several different assays show the following: 1) primitive streak formation was blocked; 2) the time-dependent increase in primitive streak stiffness was abolished; and 3) convergence of epiblast cells to the midline was inhibited. Taken together, our data suggest that actomyosin contractility is necessary for primitive streak morphogenesis, and specifically, ROCK plays a critical role. To better understand the underlying mechanisms of this fundamental process, future models should account for the findings presented in this study. As presumptive cardiac cells traverse the course of differentiation into cardiac myocytes during cardiogenesis, the sequence, magnitude, and spatiotemporal map of biomechanical and biochemical signals has not been fully explored. There have been many studies detailing the induction of cardiogenesis on a variety of substrates and extracellular matrix (ECM) proteins, but none have completed a rigorous study of the effects of substrate stiffness on the induction of precardiac cells prior to the onset of cardiac gene expression (smooth muscle alpha actin [SMAA] at stage 5.) We investigate the effects of the mechanical environment on precardiac cell behaviors in an in vitro setting to elucidate the effect of substrate stiffness and inducing factors on precardiac tissue and the potential connection between them. The cells in the anterior portion of the primitive streak are fated to form the heart, and we show differing levels of SMAA expression on substrates of differing moduli, which suggests that substrate stiffness may play a role in cardiac differentiation. We cannot determine the physical mechanisms during morphogenesis without understanding the response of precardiac cells to changes in their mechanical environment.