Finally, as the first step to study the regulation of Kit system, we found that IGF-I was a potent regulatory factor that up-regulated the expression of kitlga in zebrafish follicle cells. The stimulation involved transcription but not translation, indicating that the kitlga gene is a direct downstream target of IGF-I. The effect of IGF-I on kitlga was exerted via PI3K-Akt but not MAPK pathway. In contrast, the MAPK pathway may play a negative role in controlling kitlga expression. / Kit ligand (also named stem cell factor, SCF) is a pleiotropic growth factor with diverse biological functions. It exerts effects on target cells by binding to its cognate tyrosine kinase receptor, Kit. In mammals, accumulated evidence has demonstrated important roles for Kit ligand and Kit in gametogenesis, melanogenesis and haematopoiesis. However, very little is known about Kit system in other vertebrates. In the present study, we used zebrafish as the model to investigate the expression, regulation and function of the Kit system in the ovary. / On the other hand, cAMP is involved in regulating the expression of kitlga in zebrafish follicle cells. Two cAMP-activated effectors, PKA and Epac, have reverse effects. PKA promotes but Epac inhibits the expression of kitlga, which was identified by the respective activator. The effect of forskolin and H89 on IGF-I-induced expression of kitlga suggests a cross-talk between the two signaling pathways. Both hCG and PACAP inhibited IGF-I-induced kitlga expression, indicating that they may have negative regulation through cAMP signaling pathways in the full-grown follicles. (Abstract shortened by UMI.) / The zebrafish has two homologues of Kit ligand (kitlga and kitlgb) and Kit (kita and kitb ) instead of one copy for each as in mammals. The present study proposed the origin of these homologues in the zebrafish by phylogenetic and chromosome synteny analyses, and provided further evidence for neo- or subfunctionalization for both Kit ligands and Kit receptors in the zebrafish ovary. All four Kit system members exhibited distinct and significant changes in mRNA expression during folliculogenesis, particularly in the periovulatory period before and after final oocyte maturation and ovulation. / Then we further studied the spatial localization of each member within the follicle. The present study demonstrated that kitlga and kitb are exclusively expressed in the follicle layer, while kitlgb and kita only in the oocyte. Using CHO cell line as a bioreactor, we produced recombinant zebrafish Kitlga and Kitlgb. Analysis in mammalian COS-1 cells and zebrafish primary follicle cells confirmed their biological activity and binding specifity. Two opposite paracrine pathways of Kit system in the zebrafish ovary have been shown. Kitlga from the follicle cells preferably activates Kita in the oocyte in spite of the weak response of Kitb to it. Kitlgb from the oocyte, however, exclusively activates Kitb in the follicle cells without any effects on Kita. / Yao, Kai. / Adviser: Ge Wei. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 136-150). / 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, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

In the present study using zebrafish as the model organism, we have investigated the function of UII/UII-receptor (UIIR) signaling pathway during early embryogenesis. Herein we presented five lines of evidence supporting the hypothesis that UII/ UIIR signaling pathway is required for normal determination of asymmetric axis during early embryogenesis. First, function-loss of UII results in a concordant randomization of viscus asymmetries in embryos, including abnormalities in cardiac looping and positioning of visceral organs. Second, knockdown of UII randomizes the left-sided expression of asymmetrical genes including lefty2, spaw and pitx2c in the lateral plate mesoderm (LPM) and bmp4 in the developing heart domain and the LPM. Third, reduced UII levels interfere with the normal organogenesis of Kupffer's vesicle (KV), an organ implicated in the early steps of left-right (L-R) patterning of embryos. Fourth, repression of UII function perturbs the asymmetrical distribution of free Ca2+ (intracellular Ca2+) at the region surrounding embryo KV during early somitogenesis, which is one of the signaling mechanisms that propagandize and amplify the early clue of left-right (L-R) asymmetry. Fifth, depressing UII levels alters the normal pattern of Bmp and Nodal signaling, which modulate the establishment of L-R axis of developmental embryo. Collectively, these observations support a model in which UII/UIIR signal system takes part in the early molecular events of L-R asymmetry patterning of embryo by modulating Bmp and Nodal signaling, regulating KV normal morphogenesis, so then, maintaining the asymmetrical distribution of free intracellular Ca2+ at the peripheral region surrounding embryo KV. This study documents a role of UII/UIIR signaling pathway in the establishment of L-R axis of embryos which promises to reveal the molecular mechanisms responsible for human congenital diseases with heterotaxy. / Urotensin II (UII) is the most potent vasoconstrictor identified so far. This cyclic peptide stimulates its G protein-coupled receptor (GPR) to modulate cardiovascular system function in humans and in other animal species. / Li, Jun. / Advisers: Christopher HK Cheng; Mingliang He. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 143-168). / 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, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Chow, Wing Shan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 118-129). / Abstract also in Chinese. / Abstract --- p.i / 摘要 --- p.iv / Acknowledgements --- p.viii / Table of Contents --- p.ix / List of Tables --- p.xiii / List of Figures --- p.xv / List of Abbreviations --- p.xviii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Pesticide contaminations in the environment --- p.1 / Chapter 1.2 --- Pesticides --- p.1 / Chapter 1.2.1 --- Usage of pesticide in the world --- p.1 / Chapter 1.2.2 --- Organochlorine (OC) pesticides --- p.3 / Chapter 1.2.3 --- Organophosphate (OP) pesticides --- p.4 / Chapter 1.2.4 --- Carbamate pesticides: --- p.6 / Chapter 1.2.5 --- Pyrethroid pesticides: --- p.6 / Chapter 1.3 --- Toxicological model: Zebrafish --- p.7 / Chapter 1.4 --- Biomarkers --- p.9 / Chapter 1.4.1 --- Cytochrome P450 1A (CYP1A) --- p.12 / Chapter 1.4.2 --- Cytochrome P450 3A65 (CYP3A65) --- p.14 / Chapter 1.4.3 --- Biomarker for estrogenicity - Vitellogenin (VTG1) --- p.15 / Chapter 1.4.4 --- Catalase (CAT) and Glutathione S-transferase (GST) --- p.18 / Chapter 1.4.4.1 --- Catalase (CAT) --- p.18 / Chapter 1.4.4.2 --- Glutathion S-transferase (GST) --- p.19 / Chapter 1.4.5 --- Multiple Drug Resistance (MDR1) --- p.20 / Chapter 1.4.6 --- Acetylcholinesterase (AChE) --- p.21 / Chapter 1.5 --- Objectives of this study --- p.26 / Chapter Chapter 2 --- "Toxicity assay and biomarker studies on zebrafish embryo-larvae exposed to organochlorine pesticides: endosulfan, heptachlor and methoxychlor" --- p.28 / Chapter 2.1 --- Introduction --- p.28 / Chapter 2.2 --- Materials and methods --- p.30 / Chapter 2.2.1 --- Chemicals tested --- p.30 / Chapter 2.2.2 --- Zebrafish cultivation and egg production --- p.30 / Chapter 2.2.3 --- Determination of 96h-EC50 and 96h-LC50 of organochlorine pesticides and bisphenol-A for zebrafish embryo-larvae --- p.31 / Chapter 2.2.4 --- Pesticide exposure for determination of mRNA levels of biomarkers --- p.31 / Chapter 2.2.5 --- Extraction of total RNA from the exposed embryo-larvae samples --- p.32 / Chapter 2.2.6 --- Reverse Transcription --- p.33 / Chapter 2.2.7 --- Quantifications of mRNA levels by qPCR --- p.35 / Chapter 2.2.7.1 --- Primer design --- p.35 / Chapter 2.2.7.2 --- Validation of qPCR conditions --- p.36 / Chapter 2.2.7.3 --- Quantification of biomarker gene expression levels in zebrafish embryo-larvae --- p.42 / Chapter 2.2.8 --- Statistical analysis --- p.43 / Chapter 2.3. --- Results --- p.44 / Chapter 2.3.1 --- Toxicities of OC pesticides and bisphenol-A --- p.44 / Chapter 2.3.2 --- Effects of OC pesticides and bisphenol-A on biomarker gene expression levels --- p.44 / Chapter 2.4. --- Discussions --- p.60 / Chapter 2.4.1 --- Toxicities of OC pesticides and bisphenol-A --- p.60 / Chapter 2.4.2 --- Effects of OC pesticides on CYP1A gene expression --- p.61 / Chapter 2.4.3 --- Effects of OC pesticides on CYP3A65 gene expression --- p.61 / Chapter 2.4.4 --- Effects of OC pesticides on VTG1 gene expression --- p.63 / Chapter 2.4.5 --- Effects of OC pesticides on MDR1 gene expression --- p.64 / Chapter 2.5 --- Conclusion --- p.65 / Chapter Chapter 3 --- "Toxicity assay and biomarker studies on zebrafish embryo-larvae exposed to a organochlorine pesticide, chlorpyrifos" --- p.66 / Chapter 3.1 --- Introduction --- p.66 / Chapter 3.2 --- Materials and methods --- p.68 / Chapter 3.2.1 --- Chemicals tested --- p.68 / Chapter 3.2.2 --- Zebrafish cultivation and egg production --- p.68 / Chapter 3.2.3 --- Determination of 96h-EC50 and 96h-LC50 of chlorpyrifos for zebrafish embryo-larvae --- p.68 / Chapter 3.2.4 --- Pesticide exposure for determination of mRNA levels of biomarkers --- p.68 / Chapter 3.2.5 --- Extraction of total RNA from the exposed embryo-larvae samples --- p.69 / Chapter 3.2.6 --- Reverse Transcription --- p.69 / Chapter 3.2.7 --- Quantifications of mRNA levels by qPCR --- p.70 / Chapter 3.2.7.1 --- Primer design --- p.70 / Chapter 3.2.7.2 --- Validation of qPCR conditions --- p.70 / Chapter 3.2.7.3 --- Quantification of biomarker gene expression levels in zebrafish embryo-larvae --- p.75 / Chapter 3.2.8 --- Determination of acetylcholinesterase (AChE) activities --- p.76 / Chapter 3.2.9 --- Statistical analysis --- p.77 / Chapter 3.3 --- Results --- p.78 / Chapter 3.3.1 --- Toxicities of chlorpyrifos --- p.78 / Chapter 3.3.2 --- Effects of chlorpyrifos on CAT and GST gene expression levels --- p.81 / Chapter 3.3.3 --- Effects of chlorpyrifos on acetylcholinesterase (AChE) activity --- p.83 / Chapter 3.4 --- Discussions --- p.86 / Chapter 3.4.1 --- Toxicity of chlorpyrifos --- p.86 / Chapter 3.4.2 --- Effect of chlorpyrifos on CAT and GST gene expressions --- p.86 / Chapter 3.4.3 --- Effect of chlorpyrifos on AChE activity --- p.88 / Chapter 3.5 --- Conclusions --- p.89 / Chapter Chapter 4 --- Toxicity assay and biomarker studies on zebrafish embryo-larvae exposed to carbamate and pyrethroid pesticides --- p.90 / Chapter 4.1 --- Introduction --- p.90 / Chapter 4.2 --- Materials and methods --- p.92 / Chapter 4.2.1 --- Chemicals tested --- p.92 / Chapter 4.2.2 --- Zebrafish cultivation and egg production --- p.92 / Chapter 4.2.3 --- Determination of 96h-EC50 and 96h-LC50 of aldicarb and cypermethrin for zebrafish embryo-larvae --- p.92 / Chapter 4.2.4 --- Pesticide exposure for determination of mRNA levels of biomarkers --- p.92 / Chapter 4.2.5 --- Quantification of biomarker gene expression levels in zebrafish embryo- larvae and Determination of acetylcholinesterase (AChE) activity --- p.94 / Chapter 4.2.6 --- Statistical analysis --- p.94 / Chapter 4.3 --- Results --- p.95 / Chapter 4.3.1 --- Toxicities of aldicarb and cypermethrin --- p.95 / Chapter 4.3.2 --- Effects of aldicarb and cypermethrin on CAT and GST gene expression levels.. --- p.99 / Chapter 4.3.3 --- Effects of aldicarb on acetylcholinesterase (AChE) activity --- p.102 / Chapter 4.4 --- Discussion --- p.105 / Chapter 4.4.1 --- Toxicity of aldicarb of cypermethrin --- p.105 / Chapter 4.4.2 --- Effect of aldicarb and cypermethrin on CAT and GST gene expressions --- p.105 / Chapter 4.4.3 --- Effect of aldicarb on AChE activity --- p.107 / Chapter 4.5 --- Conclusion --- p.108 / Chapter Chapter 5 --- General Conclusion --- p.109 / Chapter 5.1 --- Toxicities of pesticides --- p.109 / Chapter 5.2 --- Effects of OC pesticides on biomarker gene expressions --- p.113 / Chapter 5.3 --- "Effects of chlorpyrifos, aldicarb and cypermetrhin on biomarker gene expressions" --- p.116 / Chapter 5.4 --- Effect of chlorpyrifos and aldicarb on AChE activity --- p.116 / References --- p.118

Cell culture systems have provided many insights into eukaryotic gene expression and other biochemical mechanisms. Since the cell represents the smallest living unit of any organism it provides a desirable in vitro system, allowing biochemical studies without the complex physiology of an entire animal. However, processes involving intracellular mechanisms, such as development, aging or carcinogenis, eventually require the analysis of the intact organism. Transgenic animals are a very promising tool to approach questions of this magnitude. Fish in general and the zebrafish (Brachydanio rerio) in particular are an excellent model system for transgenic research, mainly due to their extramaternal fertilization and development and their short generation cycle throughout the year. The recent derivation of zebrafish cell lines has opened up possibilities for in vitro analysis of this popular model species, and expression of heterologous genes under the influence of promoter and other regulatory nucleic acid aequences. In contrast to mammalian expression systems, little nucleic acid sequences controlling gene expression in fish are known. Therefore we examined mammalian expression systems in fish cells in order to determine their efficiency quantitatively. Emphasis was given to zebrafish cultures with the goal of eventually injecting in vitro manipulated embryo cells into host embryos and thereby creating transgenic chimera. / Graduation date: 1993

Thrombocytes are functional equivalents of mammalian platelets and also possess megakaryocyte features. It has been shown earlier that hox genes play a role in megakaryocyte development. Our earlier microarray analysis showed five hox genes, hoxa10b, hoxb2a, hoxc5a, hoxc11b and hoxd3a, were upregulated in zebrafish thrombocytes. However, there is no comprehensive study of genome wide scan of all the hox genes playing a role in megakaryopoiesis. I first measured the expression levels of each of these hox genes in young and mature thrombocytes and observed that all the above hox genes except hoxc11b were expressed equally in both populations of thrombocytes. hoxc11b was expressed only in young thrombocytes and not in mature thrombocytes. The goals of my study were to comprehensively knockdown hox genes and identify the specific hox genes involved in the development of thrombocytes in zebrafish. However, the existing vivo-morpholino knockdown technology was not capable of performing such genome-wide knockdowns. Therefore, I developed a novel cost- effective knockdown method by designing an antisense oligonucleotides against the target mRNA and piggybacking with standard control morpholino to silence the gene of interest. Also, to perform knockdowns of the hox genes and test for the number of thrombocytes, the available techniques were both cumbersome or required breeding and production of fish where thrombocytes are GFP labeled. Therefore, I established a flow cytometry based method of counting the number of thrombocytes. I used mepacrine to fluorescently label the blood cells and used the white cell fraction. Standard antisense oligonucleotide designed to the central portion of each of the target hox mRNAs, was piggybacked by a control morpholino and intravenously injected into the adult zebrafish. The thrombocyte count was measured 48 hours post injection. In this study, I found that the knockdown of hoxc11b resulted in increased number of thrombocytes and knockdown of hoxa10b, hoxb2a, hoxc5a, and hoxd3a showed reduction in the thrombocyte counts. I then screened the other 47 hox genes in the zebrafish genome using flow sorting method and found that knockdown of hoxa9a and hoxb1a also resulted in decreased thrombocyte number. Further, I used the dye DiI, which labels only young thrombocytes at specific concentrations and observed that the knockdown of hoxa10b, hoxb2a, hoxc5a, hoxd3a, hoxa9a and hoxb1a, lead to a decrease in young thrombocytes; whereas hoxc11b knockdown lead to increase in number of young thrombocytes. Using bromodeoxyuridine, I also showed that there is increase in release of young thrombocytes into peripheral circulation in hoxc11b knockdown fish which suggests that hoxc11b significantly promotes cell proliferation rather effecting apoptosis. In conclusion, I found six hox genes that are positive regulators and one hox gene is a negative regulator for thrombocyte development.

von Willebrand factor (VWF) protein acts in the intrinsic coagulation pathway by stabilizing FVIII from proteolytic clearance and at the site of injury, by promoting the adhesion and aggregation of platelets to the exposed subendothelial wall. von Willebrand disease (VWD) results from quantitative and qualitative deficiencies in VWF protein. The variability expressivity in phenotype presentations is in partly caused by the action of modifier genes. Zebrafish has been used as hemostasis animal model. However, it has not been used to evaluate VWD. Here, we report the development of a heterozygote VWF mutant zebrafish using the genome editing CRISPR/Cas9 system to screen for modifier genes involved in VWD. We designed CRISPR oligonucleotides and inserted them into pT7-gRNa plasmid. We then prepared VWF gRNA along with the endonuclease Cas9 RNA from Cas9 plasmid. We injected these two RNAs into 1-4 cell-stage zebrafish embryos and induced a mutation in VWF exon 29 of the zebrafish with a mutagenesis rate of 16.6% (3/18 adult fish). Also, we observed a germline transmission with an efficiency rate of 5.5% (1/18 adult fish). We obtained a deletion in exon 29 which should result in truncated VWF protein.

Bioinformatic searching on the zebrafish genome indicates that there are five members of this hormone family (namely GH, SLalpha, SLbeta, PRL1 and PRL2) and four receptors (namely GHR1, GHR2, PRLR1 and PRLR2). However, it should be noted that these ligands and receptors are only named according to their sequence homology with those in other species. There is so far no systematic study to unravel the relationship among the ligands and receptors. The last point is particularly relevant as some of the ligands and receptors are duplicated in the fish genome. In addition, there is much controversy regarding whether one of the two GHRs is in fact the receptor for SL. A systematic study on the interaction among the ligands and receptors in zebrafish would help to resolve these issues. / In fish, growth hormone (GH), prolactin (PRL) and somatolactin (SL) are members of a gene family of polypeptide hormones which share homology in protein sequence and structure. To date, numerous functions have been attributed to this family of hormones such as growth, immune response, protein metabolism and ion regulation. The biological functions of GHlPRL are mediated through binding of the ligands on their respective receptors. It is believed that this gene family arose as the result of multiple gene duplications and subsequent divergent evolution, co-evolving with their corresponding receptors. Despite the above mentioned similarities in their structures, their cognate receptors and their signaling mechanisms, important differences among this gene family of polypeptide hormones can be recognized in their biological functions. / In the present study, the luciferase reporter assay, His-tag pulldown assay and signaling pathway activation were employed to investigate the interaction among the ligands and their receptors. It was shown that recombinant zebrafish GH, PRLI and PRL2 could only interact with their cognate receptors, i.e. GHRl, GHR2, PRLRI and PRLR2 respectively. In comparison, zebrafish SLalpha and SLbeta could neither interact with GHR1, GHR2, PRLR1 and PRLR2 in the binding study, nor could these two SLs activate the receptor-mediated downstream signaling and transcriptional activities of the four receptors in zebrafish. These data argue against the hypothesis that GHRI is the SL receptor. / The role of PRLR in early development of zebrafish was also explored. Whole mount in situ hybridization (WISH) study showed that PRLR1 was mainly expressed in the pancreas and pronephric duct, while PRLR2 was expressed in the pronephric duct only. In the PRLR1 morpholino (MO) knockdown embryos, the yolk extension (YE), the formation of which was reported to be associated with pronephric duct development, disappeared at 24 hours post fertilization. This phenotype could not be observed in the PRLR2 MO knockdown or control embryos. Real time quantitative RT-PCR and WISH data revealed that several genes expressed in the pronephric duct were up or down-regulated. The protein expression pattern of pronephric duct marker atplal was also affected in the embryos injected with PRLRI MO. In addition, histological studies showed that structure of the pronephric duct was destroyed in the PRLRI MO embryos. These results suggest that PRLRI plays an important role in the development of the pronephric duct in zebrafish embryos. / Chen, Mingliang. / "October 2010." / Adviser: Cheung Wing-Tai. / Source: Dissertation Abstracts International, Volume: 73-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 140-179). / 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, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Clitellate annelids display conserved mechanisms of segmental ectodermal and mesodermal patterning. These tissues are generated by asymmetric divisions of large stem cells called teloblasts, elongating the ectoderm and mesoderm of the embryo. Each teloblast-derived lineage makes highly stereotyped contributions to the leech: the N, O, P, and Q contribute specific neurons, epidermis, and other ectodermal tissues along the ventral-to-dorsal axis of the embryo, respectively. The N and Q ectodermal lineages appear to be specified autonomously, but specification of the O and P lineages depends upon interactions with other, neighboring teloblast lineages. Until quite recently, there have been precious few teloblast lineage-specific markers, and virtually no molecular candidates for genes influencing the proper differentiation of any of these lineages. Here, I explore the possibility that members of the Pax-Six-Eyes absent-Dachshund network are involved in leech ectodermal patterning. I show that the leech Helobdella sp. Austin has two Pax6 paralogs, and demonstrate that Hau-Pax6A is expressed early in a subset of N-derived cells and O-derived cells. Next, I demonstrate that an ortholog of the six gene family, Hau-six1/2a, is expressed in the P lineage. I show through a series of cell ablations that Hau-six1/2a expression is regulated by neighboring teloblasts in a manner consistent with P fate induction, hinting that this transcription factor may be involved in P specification. The identification of these genes is a first step towards dissecting the molecular mechanisms of ectodermal teloblast differentiation in the leech embryo. The evolutionary context of the deployment of these genes is also discussed. In the appendices, I present two projects on the evolution of pigment patterns in Danio rerio and its relatives. In the first, I show that the larval melanin-containing pigment cells of Danio nigrofasciatus are uniquely redeployed into the adult pigment pattern, in contrast to seven related fishes. In the second, I show that variation in yellow pigment cell populations in different danio species may be dependent on variable signaling through the receptor tyrosine kinase fms pathway. / text

As a functional unit involved in both maintaining endocrine homeostasis and also producing mature eggs, the ovary plays a central role in female reproduction. The development and function of the ovarian follicles are controlled by gonadotropins released from the pituitary. It is widely accepted that the action of gonadotropins on ovarian follicles is mediated by paracrine/autocrine factors produced by the somatic cells surrounding the oocyte. Increasing evidence indicates that the Igf system is involved in mediating the action of gonadotropins in the ovary. Previously, we identified a gonad-specific Igf subtype (Igf3) distinct from Igf1 and Igf2. This fmding further highlights the importance of the Igf system in the fish ovary. In this thesis, efforts were made to understand the role of the Igf system in ovary using zebrafish as the model organism, and attention was focused on Igf3. / Because the expression of Igf3 is correlated with the LH receptor in zebrafish follicles, the regulation of igf3 by gonadotropins was subsequently studied in the ovary. The expression of igf3 was significantly up-regulated in both ovarian fragments and isolated follicles upon treatment with hCG in dose- and time-dependent manners. Treatment with 8-Br-cAMP or IBMX mimicked the effects of hCG on the expression of igf3 in follicles of different stages. / Four Igfs are present in zebrafish, and our results show that all four igfs are expressed in the ovary of zebrafish and exhibit the differential expression profiles during folliculogenesis. Using a primary culture of zebrafish follicle cells, we demonstrated that hCG stimulated igf2b and igf3 expression but suppressed igf2a expression. Moreover, the effect of gonadotropin could be mimicked by IBMX, which increased the intracellular levels of cAMP, suggesting the possible involvement of cAMP in the gonadotropin-based regulation and differential expression of igf2a, igf2b and igf3. These results also show that the Igf3 is the Igf subtype most sensitive to gonadatropin and cAMP. / In addition, the expression patterns of igf1, igf2a, igf2b, igf3, igf1ra and igf1rb were also studied during zebrafish embryogenesis. The unique temporal and spatial expression patterns of igf1, igf2a, igf2b, igf3, igf1ra and igf1rb were revealed by both real-time PCR and whole mount in situ hybridization, the results suggest divergent functions for these Igfs in early zebrafish development. / Taken together, the present studies provide substantial information about the Igf system, especially that of Igf3 in the zebrafish ovary. Data were gathered regarding Igf3 expression, regulation and functions, which is not only helpful for the understanding of the role of the Igf system in fish reproduction, but also contributes toward uncovering the ovarian signaling network involved in oocyte maturation across vertebrates. This study of igfs gene expression provides direct information to the study of Igf signaling in zebrafish. / To study the function of Igf3, bioactive recombinant Igf3 proteins were prepared using a bacterial expression system. Incubation of follicles with recombinant zebrafish Igf3 significantly enhanced oocyte maturation in time-, dose- and stage-dependent manners. The potential mechanisms of Igf3-induced oocyte maturation were then investigated. Igf3 stimulated oocyte maturation via a steroid-independent manner. Igf3 induced oocyte maturation through Igf1rs and the PI3 kinase, PDE3 and MAP kinase were necessary for Igf3-mediated oocyte maturation in zebrafish. / We first examined the gene expression patterns of Igf3 in the ovary. The igf3 gene in zebrafish was found to be alternatively spliced into two transcripts, with transcript variant 1 exclusively expressed in the gonads and transcript variant 2 only expressed during early development. Using specific antibodies developed for zebrafish Igf3, both the prepropeptide and the mature peptide forms of Igf3 were found to be predominantly expressed in the zebrafish ovary. Real-time PCR and in situ hybridization revealed that igf3 mRNA levels were relatively low in the early follicles but significantly increased after the mid vitellogenic stage (midstage III) and were high in the full grown follicles. In the full grown follicles, igf3 mRNA was detected primarily in the somatic follicular cells, with a low level of expression in the oocytes. Igf3 immunoreactivity was confined to the follicular cells alone. / Li, Jianzhen. / Advisers: Hui Zhao; Hon Ki Christopher Cheng. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 122-150). / 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, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Lin Sze-Wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 111-127). / Abstracts in English and Chinese. / Abstract (in English) --- p.i / Abstract (in Chinese) --- p.iii / Acknowledgements --- p.iv / Table of Contents --- p.v / Abbreviations --- p.x / Scientific Names --- p.xii / Chapter CHAPTER 1 --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- Gonadotropins --- p.1 / Chapter 1.1.1 --- Structure --- p.1 / Chapter 1.1.2 --- Function --- p.1 / Chapter 1.1.3 --- Regulation --- p.2 / Chapter 1.1.3.1 --- Gonadotropin-releasing hormone (GnRH) --- p.3 / Chapter 1.1.3.2 --- Dopamine --- p.4 / Chapter 1.1.3.3 --- Sex steroids --- p.5 / Chapter 1.1.3.3.1 --- Functions --- p.5 / Chapter 1.1.3.3.2 --- Working mechanism´ؤEstrogen signaling pathway --- p.7 / Chapter 1.1.3.4 --- Gonadal peptides --- p.9 / Chapter 1.1.3.4.1 --- Functions --- p.9 / Chapter 1.1.3.4.2 --- Working mechanism一Activin signaling pathway --- p.11 / Chapter 1.2 --- Transcriptional regulation of pituitary gonadotropin subunit genes at the promoter level --- p.13 / Chapter 1.2.1 --- Transcriptional regulation of mammalian glycoprotein a subunits --- p.13 / Chapter 1.2.1.1 --- GnRH --- p.14 / Chapter 1.2.1.2 --- Activin --- p.15 / Chapter 1.2.1.3 --- Steroids --- p.15 / Chapter 1.2.2 --- Transcriptional regulation of mammalian FSHβ and LHβ subunits --- p.16 / Chapter 1.2.2.1 --- Regulation of LHβ expression by GnRH --- p.17 / Chapter 1.2.2.1.1 --- Roles of SP-1 binding sites on LHβ promoter --- p.17 / Chapter 1.2.2.1.2 --- Effect of SF-1 on LHp expression --- p.17 / Chapter 1.2.2.1.3 --- Effect of Egr-1 on LHp expression --- p.18 / Chapter 1.2.2.1.4 --- "Synergistic effect ofSP-1, SF-1 and Egr-1 on LHp expression." --- p.18 / Chapter 1.2.2.1.5 --- Effect of Pitx-1 on LHβ expression --- p.19 / Chapter 1.2.2.1.6 --- "Effect of SF-1, Egr-1 and Pitx-1 on LHβ expression of other mammalian counterparts" --- p.19 / Chapter 1.2.2.1.7 --- Effect of other transcription factors on mammalian LHβ expression --- p.19 / Chapter 1.2.2.2 --- Regulation of LHβ expression by steroids and activin --- p.20 / Chapter 1.2.2.3 --- Regulation of FSHβ expression by activin and GnRH --- p.20 / Chapter 1.2.2.4 --- Regulation of FSHβ expression by steroids --- p.21 / Chapter 1.2.2.5 --- Regulation of FSHβ expression by other transcription factors --- p.22 / Chapter 1.2.3 --- Transcriptional regulation of fish FSHβ and LHβ subunits --- p.22 / Chapter 1.3 --- The project objectives and long-term significance --- p.24 / Chapter CHAPTER 2 --- CLONING OF ZEBRAFISH FSHB AND LHB PROMOTERS. --- p.26 / Chapter 2.1 --- Introduction --- p.26 / Chapter 2.2 --- Materials and Methods --- p.27 / Chapter 2.2.1 --- Chemicals --- p.27 / Chapter 2.2.2 --- Animals --- p.27 / Chapter 2.2.3 --- Isolation of genomic DNA --- p.28 / Chapter 2.2.4 --- Cloning of promoters of zebrafish FSHβ and LHβ from the genomic DNA --- p.28 / Chapter 2.2.5 --- Construction of the reporter plasmids containing zebrafish FSHβ and LHβ promoters --- p.30 / Chapter 2.2.6 --- Cell culture and transient transfection --- p.31 / Chapter 2.2.7 --- SEAP reporter gene assay --- p.32 / Chapter 2.2.8 --- β-galactosidase reporter gene assay --- p.32 / Chapter 2.2.9 --- Data analysis --- p.33 / Chapter 2.3 --- Results --- p.33 / Chapter 2.3.1 --- Cloning of zebrafish FSHβ and LHβ promoters --- p.33 / Chapter 2.3.2 --- Sequence characterization of zebrafish FSHβ and LHβ promoters --- p.34 / Chapter 2.3.3 --- Basal FSHp and LHβ promoter activities in LβT2 cells --- p.35 / Chapter 2.4 --- Discussion --- p.36 / Chapter CHAPTER 3 --- ROLES OF ACTIVIN/SMADS AND ESTROGEN/ERS IN THE REGULATION OF ZEBRAFISH FSHB AND LHB PROMOTER ACTIVITY --- p.51 / Chapter 3.1 --- Introduction --- p.52 / Chapter 3.2 --- Materials and Methods --- p.56 / Chapter 3.2.1 --- Chemicals --- p.56 / Chapter 3.2.2 --- Animals --- p.56 / Chapter 3.2.3 --- Isolation of total RNA --- p.57 / Chapter 3.2.4 --- Rapid amplification of full-length cDNA (RACE) --- p.57 / Chapter 3.2.5 --- Construction of expression plasmids --- p.57 / Chapter 3.2.6 --- cell culture and transient transfection --- p.59 / Chapter 3.2.7 --- SEAP reporter gene assay --- p.59 / Chapter 3.2.8 --- p-galactosidase reporter gene assay --- p.59 / Chapter 3.2.9 --- Data analysis --- p.59 / Chapter 3.3 --- Results --- p.60 / Chapter 3.3.1 --- Cloning and sequence characterization of zebrafish Smad 4 (zfSmad 4) --- p.60 / Chapter 3.3.2 --- Smads regulate FSHβ transcription in LβT2 cells --- p.61 / Chapter 3.3.3 --- Smads regulate LHβ transcription in LPβT2 cells --- p.61 / Chapter 3.3.4 --- Functionality of the two forms of Smad 4 cloned --- p.62 / Chapter 3.3.5 --- Estrogen and ERs regulate zJFSHβ transcription in LβT2 cells --- p.63 / Chapter 3.3.6 --- Estrogen and ERs regulate zfLHβ transcription in LβT2 cells --- p.63 / Chapter 3.4 --- Discussion --- p.64 / Chapter CHAPTER 4 --- PROMOTER ANALYSIS FOR SMAD RESPONSIVE ELEMENT AND ESTROGEN RESPONSIVE ELEMENT IN ZEBRAFISH FSHB AND LHB PROMOTERS --- p.82 / Chapter 4.1 --- Introduction --- p.83 / Chapter 4.2 --- Materials and Methods --- p.85 / Chapter 4.2.1 --- Chemicals and animals --- p.85 / Chapter 4.2.2 --- Construction of SEAP reporter plasmids containing different lengths of zfFSHβ promoter --- p.85 / Chapter 4.2.3 --- Construction of SEAP reporter plasmids containing different lengths of zfLHβ promoter --- p.85 / Chapter 4.2.4 --- Site-directed mutagenesis --- p.86 / Chapter 4.2.5 --- cell culture and transient transfection --- p.87 / Chapter 4.2.6 --- SEAP reporter gene assay --- p.87 / Chapter 4.2.7 --- P-galactosidase reporter gene assay --- p.87 / Chapter 4.2.8 --- Data analysis --- p.88 / Chapter 4.3 --- Results --- p.88 / Chapter 4.3.1 --- Localization of Smad-responsive element (SRE) on zfFSHβ promoter --- p.88 / Chapter 4.3.2 --- Localization of estrogen-responsive element (ERE) on zfLHβ promoter --- p.89 / Chapter 4.3.3 --- Localization of estrogen-responsive element (ERE) on zfFSHβ promoter --- p.90 / Chapter 4.3.4 --- Confirmation of SRE by site-directed mutagenesis --- p.91 / Chapter 4.3.5 --- Confirmation of ERE by site-directed mutagenesis --- p.92 / Chapter 4.4 --- Discussion --- p.92 / Chapter CHAPTER 5 --- GENERAL DISCUSSION --- p.106 / Chapter 5.1 --- Overview --- p.106 / Chapter 5.2 --- Contribution of the present research --- p.107 / Chapter 5.3 --- Future research direction --- p.108 / REFERENCE: --- p.111