published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Recently, the timing of oocyte loss during murine development has been brought into question as authors using mouse vasa homologue (MVH) as a germ cell marker did not observe a loss of oocytes during fetal life. Instead the major loss was observed in the days following birth, after chromosome pairing has occurred. / In this study the controversy was addressed by establishing a new and reliable method to quantify murine oocytes in meiotic prophase, as well as to determine the gestation age and meiotic prophase stage of oocyte loss. Earlier limitations were overcome through the use of Germ Cell Nuclear Antigen-1 (GCNA-1) antibody as a germ cell specific marker, and the novel addition of a cytospin centrifugation step to the method. Progress through meiotic prophase was examined in chromosome spread preparations where meiotic stages were assessed using an antiserum against synaptonemal complex (SC) proteins. Quantification was accomplished by counting the number of GCNA-1 immunoreactive cells in chromosome spread preparations and estimated in histological sections using the ratio estimation model. (Abstract shortened by UMI.)

H1 histones are potentially significant to nuclear reprogramming during the oocyte-to-embryo transition. One characteristic distinguishing the H1 subtypes is that the somatic H1 histones are found primarily in dividing cells, whereas the H10 subtype is predominantly found in differentiated cells. The H1 complement in mouse oocytes and preimplantation embryos from wild-type and H10-/- animals was investigated. / Immunocytochemistry of wild-type cells demonstrated that H10 was predominant in oocytes while somatic H1 began accumulating in the 2-cell embryo. In H10-/- cells H10 was not detected, but, surprisingly, somatic H1 was detected beginning at the 1-cell stage. Radiolabeling of wild-type and H10-/- cells revealed that somatic H1 synthesis intensified after meiotic maturation, and therefore prior to its detection in embryos. The functional study found that loss of H10 impaired oogenesis but enhanced embryogenesis. The patterns of H1 immunodetection and synthesis are integrated, and the significance of H1 composition in development is discussed.

At fertilization, the remodelling of the sperm nucleus into the male pronucleus is critical for normal development. Morphological and functional changes to the nucleus are underpinned by biochemical changes in the chromatin composition, most notably the removal of sperm specific protamines and assembly of histones onto the paternal DNA. This exchange is controlled by oocyte factors, as exemplified in Xenopus by nucleoplasmin. Though mammalian factors remain unidentified, a functional assay based on antibodies recognizing core histones has been developed to test the activity in oocytes that transfers histones onto sperm DNA, named histone transfer activity (HTA). The assay was applied to growing and maturing murine oocytes to determine when during oogenesis HTA develops, and to probe potential regulatory mechanisms. Fully-grown oocytes develop HTA upon maturation, in a protein-synthesis dependent manner. Large, growing oocytes also develop HTA upon entry into M-phase. Small growing meiotically incompetent oocytes, ones that do not spontaneously enter M-phase, do not develop HTA, though this can be overcome by culture of oocytes to meiotic competence, or by treatment with strontium to induce intracellular calcium oscillations. Taken together these findings form a model of how HTA develops throughout oogenesis. Finally, an attempt is made to identify a potential mammalian HTA factor. Transcripts for two remodelling factors, mNAP and Npm3, are identified in the murine oocyte, and injection of anti-sense oligonucleotides reveals that Npm3 plays a significant role in the deposition of histories and the remodelling of sperm chromatin at fertilization. Combined with the findings of the HTA assay, the data forms a testable model of how Npm3 may be regulated throughout oogenesis.

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Next, the influences on the migration of neural crest cell from the microenvironment of the hindgut through which the neural crest cells migrate were studied. An organ culture system was established to recombine different gut segments together at E11.5 for gut culture in order to trace the migration of neural crest cells from the midgut of the +/+ or Dom/+ embryo to the hindgut of the same or different genotypes. At E11.5, the midgut of both +/+ and Dom/+ embryos had already been fully colonized by neural crest cells, thus an explanted midgut segment (donor midgut) could serve as the source of the neural crest cells, while the caudal half of the hindgut (recipient hindgut) acted as the recipient of the neural crest cells from the donor midgut segment because at this stage, the caudal half of the hindgut was completely devoid of neural crest cells. After three days of culture, when a segment of midgut from the +/+ embryo was used as the donor of migratory vagal neural crest-derived cells and combined with an aneural segment of the hindgut (segment without neural crest-derived cells) from Dom/+ or Dom/Dom embryos, neural crest-derived cells from the midgut segment successfully crossed the combination junction and migrated normally along the hindgut segment to reach its caudal end within a normal developmental time frame. However, the migration of neural crest-derived donor cells from the Dom/+ midgut segment was abnormal in the recipient hindgut with a genotype of +/+, Dom/+ or Dom/Dom as evidenced by the retarded rostrocaudal progression of the vagal neural crest-derived cells and the reduced number of migratory cells in the recipient hindgut segment. These results thus indicate that the migration of the vagal neural crest-derived cells is minimally influenced by the migratory environment of the hindgut of the Dom embryo, and that the neural crest cells themselves may be defective in migration leading to the retarded migration in the hindgut of Dom mouse embryos. / The vagal neural crest cells originating from the region of the neural tube adjacent to somites 1 to 7 migrate along defined pathways to the gastrointestinal tract and then colonize the gut to give rise to the majority of neurons and glia of the enteric nervous system. Mutation of Sox10 in the Dominant megacolon (Dom) mouse, which is an animal model of Hirschsprung's disease, leads to aganglionosis (absence of ganglia) in varying lengths of the hindgut. To investigate the underlying cellular mechanism of aganglionosis, the migration of vagal neural crest cells from the neural tube to the gut (pre-enteric migration) in Dom mouse embryos at E8.5 was firstly traced with extrinsic cell markers, such as wheat germ agglutinin gold conjugates (WGA-Au) or fluorescent dye DiI. After the vagal neural crest cells entered the gut at E9.5, their migration was then followed by the examination of the expression of specific markers for undifferentiated neural crest cells with immunohistochemical staining. It was found that, although vagal neural crest cells in embryos of the three genotypes examined migrated along similar pre-enteric pathways at a similar migratory rate, the numbers of neural crest cells in embryos heterozygous (Dom/+) and homozygous (Dom/Dom) for the Sox10 mutation were significantly reduced when compared with the number of neural crest cells in wild-type (+/+) embryos. After vagal neural crest had entered the gut and from E10.5 onwards, no neural crest-derived cells were found in the gut of Dom/Dom embryos, and the migration of neural crest cells along the Dom/+ gut was significantly retarded from E12.5 onwards as compared with the migration in stage-matched +/+ embryos. / To further trace the cause of defective migration of neural crest cells in the Dom embryo, the proliferation and survival of neural crest cells were investigated with BrdU labeling and TUNEL assay. It was found that, although there was no obvious difference in the proliferating ability of vagal neural crest cells in embryos of all the three Dom genotypes studied during the pre-enteric migration and the migration in the gut, more apoptotic neural crest cells were found along the pre-enteric migratory pathway of Dom/Dom embryos than Dom/+ and +/+ embryos. Therefore, the decreased surviving ability, but possibly not the reduced proliferating ability, of neural crest cells during their pre-enteric migration may be partly responsible for aganglionosis in the hindgut of the Dom mouse. / Wang Liang. / "June 2006." / Adviser: W. Y. Chan. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1380. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 287-307). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Shan Sze Wan. / "July 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 125-139) / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

by Lam, Sau Hing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 141-158). / Abstract also in Chinese. / Content / Acknowledgements / Abbreviation / Project Objectives / Abstract / Chapter Chapter One --- Introduction / Chapter 1.1 --- The definition of homeobox genes / Chapter 1.2 --- Homeobox genes as a transcription factor / Chapter 1.3 --- Homeobox in Drosophila / Chapter 1.3.1 --- The development of Drosophila / Chapter 1.3.2 --- Maternal genes / Chapter 1.3.3 --- Segmentation genes / Chapter 1.3.4 --- Homeobox genes / Chapter 1.4 --- Family of Hox genes in the mammalian system / Chapter 1.5 --- Some possible chemical mechanism in the cascade system of the Hox genes in the vertebrate / Chapter 1.6 --- Hox (Antp-Class homeobox gene) in mammal / Chapter 1.6.1 --- Labial Like homeobox genes / Chapter 1.6.2 --- Proboscipedia Like homeobox genes / Chapter 1.7 --- Divergent homeobox genes / Chapter 1.7.1 --- Paired (prd) Class / Chapter 1.7.2 --- Even-Skipped (Eve) Class / Chapter 1.7.3 --- Distal-less (Dll) Class / Chapter 1.7.4 --- Muscle-Specific Homeobox (Msx) Class / Chapter 1.8 --- Orphan homeobox gene / Chapter 1.8.1 --- The characteristic of Hox 11 sequence in human and mouse / Chapter 1.8.2 --- Novel homeobox genes related to hox 11 gene family / Chapter 1.8.3 --- The mechanism of HOX 11 inducing gene regulation and signal transduction pathways / Chapter 1.8.4 --- HOX 11 in human / Chapter 1.8.5 --- Hox11 in mouse / Chapter 1.8.6 --- Hox11 L1 in mouse / Chapter 1.9 --- Homeobox gene involved in haematopoiesis / Chapter 1.10 --- Some translocations of homeobox genes in blood lineage / Chapter 1.11 --- The development of mouse / Chapter 1.11.1 --- Early organogenesis / Chapter 1.11.2 --- Nervous system development / Chapter 1.11.3 --- Somite development / Chapter 1.11.4 --- Eye development / Chapter 1.11.5 --- Neural crest cell migration / Chapter 1.11.6 --- Branchial arches development / Chapter Chapter Two --- Materials and methods / Chapter 2.1 --- Mouse Embryos / Chapter 2.2 --- RNA extraction / Chapter 2.3 --- Large plasmid preparation / Chapter 2.4 --- The synthesis of cDNAs using Reverse Transcription / Polymerase Chain Reaction (RT-PCR) and ligation into Bluescript® II KS / Chapter 2.4.1 --- The synthesis of RT-PCR products / Chapter 2.4.2 --- The formation of blunt ends of cDNA / Chapter 2.4.3 --- The ligation of cDNA with plasmid vectors / Chapter 2.4.4 --- Transformation / Chapter 2.4.5 --- The miniprep plasmid purification / Chapter 2.5 --- T7 sequencing / Chapter 2.6 --- Double stranded DNA cycle sequencing of plasmid / Chapter 2.6.1 --- Gel electrophoresis / Chapter 2.7 --- Northern blot / Chapter 2.7.1 --- Preparation of Northern blot / Chapter 2.7.2 --- Hybridization of Northern blot / Chapter 2.8 --- DIG labeled probes in whole mount in situ hybridization / Chapter 2.8.1. --- Preparation of linear DNA to generate riboprobes / Chapter 2.8.2 --- Preparation of DIG labeled riboprobe / Chapter 2.8.3. --- Preparation of embryo powder / Chapter 2.8.4 --- Absorption of antibody / Chapter 2.8.5 --- Embryo preparation / Chapter 2.8.6 --- Embryos staining / Chapter 2.9 --- Sections from whole mount in situ hybridization / Chapter 2.10 --- The radiolabeled section in situ hybridization / Chapter 2.10.1 --- The preparation of paraffin wax block and sample sections / Chapter 2.10.2 --- Slide pretreatment / Chapter 2.10.3 --- Preparation of probe / Chapter 2.10.4 --- Hybridization / Chapter 2.10.5 --- Washing / Chapter 2.10.6 --- Emulsification and development / Chapter 2.11 --- DIG-label in situ hybridization of frozen / Chapter 2.12 --- H&E staining / Chapter Chapter Three --- Results and Discussion / Chapter 3.1 --- Synthesis of Tlx3 probes for use in the section and whole mount in situ hybridization / Chapter 3.1.1 --- Synthesis of the Tlx cDNA by RT-PCR method / Chapter 3.1.2 --- The Tlx3 genomic clone for detecting the developmental expression of Tlx3 by Northern Blot / Chapter 3.1.3 --- The characterization of Tlx3 cDNAs and the sonic hedgehog cDNA / Chapter 3.2 --- Section in situ hybridization using different probes / Chapter 3.2.1 --- Section in situ hybridization using the transcribed riboprobes of Pax2 cDNA / Chapter 3.2.2 --- The transcribed riboprobes of Tlx genomic clones were used to hybridize the section in situ hybridization / Chapter 3.2.3 --- The in situ hybridization of frozen sections of mouse embryos using the transcribed riboprobes from Tlx3 cDNA subclone / Chapter 3.3 --- Expression pattern of Tlx3 on whole mount embryos using both cDNA and genomic probes / Chapter 3.3.1 --- The expression of Tlx3 on whole mount in situ hybridization of the mouse embryos using the antisense probes from the Tlx3 genomic clone / Chapter 3.3.2 --- Whole mount in situ hybridization of mouse embryo using the transcribed riboprobes of Tlx3 cDNA / Chapter 3.3.3 --- The expression pattern of sonic hedgehog on embryos at 8.5 to9.5 dpc / Conclusion / Future prospective / Appendix / Reference / Acknowledgement