Forkhead domain基因家族編碼了很多對於胚胎發育至關重要的轉錄因子，而Foxp4則屬於p-subtype forkhead轉錄因子其中一員。Foxp4在胚胎發育期間的表達十分活躍，在發育中的腦部的不同地方表達，但其於中樞神經系統發育中的調控角色並不清楚。Foxp4基因剔除小鼠在出生前死於心臟的缺陷表型(心二分支) ，在此時間段，腦部的發育才剛剛開始，因此我們無法利用Foxp4基因剔除小鼠作為研究中樞神經系統發育的動物模型。最近，我們的團隊利用小腦組織培養技術及siRNA發佈的研究顯示，Foxp4在小鼠小腦中的蒲金氏細胞(Purkinje cell)中擔當著重要的維持作用。這項研究結果加深了我們對研究Foxp4在中樞神經系統發育中的調控角色的決心。 / 本論文旨在利用斑馬魚作為實驗模型，研究foxp4在斑馬魚中樞神經系統發育中的表達及調控角色。RT-PCR結果顯示foxp4在斑馬魚發育中的bud stage開始表達，並在及後的階段維持其表達水平。利用原位雜交技術 (whole mount in-situ hybridization)，我們發現foxp4表達的地區主要集中於發育中的腦部。在成年斑馬魚中，foxp4表達在不同組織和器官，包括腦部，眼睛和心臟。成年斑馬魚腦部切片原位雜交 (sectioned in-situ hybridization)則顯示，foxp4在小腦的蒲金氏細胞和視頂蓋(optic tectum)的periventricular gray zone表達。 / 為了進一步探究foxp4對於胚胎發育過程中的功能，我們利用微注射技術，把反義嗎啉 (morpholino) MO1注射到斑馬魚胚胎中，大幅度抑制foxp4的表達水平。胚胎受精後48小時，MO1注入的胚胎顯示出第四腦室腦積水的缺陷表型。組織學分析顯示，第四腦室以下的延髓被壓縮致形態異常。此外，利用原位雜交技術及不同的分子標記，我們發現胚胎的中後腦邊界也會出現輕度畸形，而後腦的神經元數量及排列亦受到影響。 / 本項研究展示foxp4在胚胎中樞神經系統的發展的重要性，亦提供了新的見解。我們認為foxp4可能是調控腦室發育的重要成員，但在此方面與foxp4相關的分子機制仍須作更深入的研究。 / The forkhead domain gene family encodes a large group of transcription factors that play essential roles in development. Foxp4 is one of the members in the Foxp subfamily that expressed in different parts of developing central nervous system (CNS) and its function is less characterized. Previous study on Foxp4-knockout mice resulted in early embryonic lethality due to defective heart tube development that hindered the functional study of Foxp4 in CNS development. Recently, our laboratory reported that Foxp4 functions as a maintenance role in the Purkinje cell in the mouse cerebellum. Nevertheless, the role of foxp4 in CNS development was still unclear. / In this study, we used zebrafish as a model to study the expression pattern and functional study of foxp4 in the developing CNS. RT-PCR analysis showed that foxp4 transcript was expressed at the bud stage and maintained in the later embryonic stages. Whole-mount in-situ hybridization showed that foxp4 expressed in the cephalic region during embryonic development. In adult zebrafish, foxp4 expresses in different tissues and organs including brain, eye and heart. Sectioned in-situ hybridization of the adult zebrafish brain showed that foxp4 was specifically expressed in the Purkinje cell and the periventricular gray zone of optic tectum. / To further investigate the function of foxp4 during embryonic development, we injected antisense morpholino, MO1 into the zebrafish embryo to knockdown foxp4. By 48 hour post fertilization (hpf), MO1-injected embryos displayed hydrocephalus in the 4th ventricle. Histological analysis revealed that the medulla oblongata below the 4th ventricle was compressed by the edema resulting in abnormal morphology of medulla oblongata in the MO1-injected morphant. In addition, a mild malformation of the mid-hindbrain boundary, disrupted hindbrain patterning was observed in MO1-injected morphant. / Our findings provide new insight into the function of foxp4 in embryonic CNS development. We suggested that foxp4 may be essential in regulating the brain ventricle development while the molecular mechanism underlying the functional role of foxp4 requires further investigation. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wong, Wai Kei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 92-102). / Abstracts also in Chinese. / Abstract --- p.iii / 摘要 --- p.v / Acknowledgement --- p.vii / Figure and table list --- p.xi / Abbreviation --- p.xii / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Zebrafish as a developmental model --- p.1 / Chapter 1.2 --- Zebrafish development with highlights --- p.3 / Chapter 1.2.1 --- CNS development --- p.3 / Chapter 1.3 --- Forkhead domain gene in development --- p.5 / Chapter 1.3.1 --- History of forkhead domain gene --- p.5 / Chapter 1.3.2 --- Functional roles of forkhead domain genes in development --- p.6 / Chapter 1.4 --- Foxp subfamily --- p.8 / Chapter 1.4.1 --- Diverse functions of Foxp1, 2, 3 and 4 --- p.8 / Chapter 1.4.2 --- Relationship between Foxp subfamily members --- p.10 / Chapter 1.5 --- Foxp4 --- p.11 / Chapter 1.5.1 --- Genomic organization and protein structure of mFoxp4 --- p.11 / Chapter 1.5.2 --- Previous studies of mFoxp4 --- p.14 / Chapter 1.5.3 --- Foxp4 studies in other model organisms --- p.14 / Chapter 1.5.3.1 --- Rat --- p.15 / Chapter 1.5.3.2 --- Xenopus --- p.16 / Chapter 1.5.3.3 --- C. elegans --- p.16 / Chapter 1.5.4 --- Zebrafish foxp4 --- p.17 / Chapter 1.5.5.1 --- Genomic organization and protein structure of foxp4 --- p.17 / Chapter 1.5.5.2 --- Sequence alignment of foxp4 with other models --- p.19 / Chapter 1.6 --- Hypothesis, aim and strategy of the study --- p.22 / Chapter Chapter 2 --- Expression of foxp4 in zebrafish embryo and adult zebrafish brain / Chapter 2.1 --- Introduction --- p.24 / Chapter 2.2 --- Materials and methods --- p.25 / Chapter 2.2.1 --- Animals --- p.25 / Chapter 2.2.2 --- Materials --- p.26 / Chapter 2.2.3 --- Semi-quantitative PCR --- p.35 / Chapter 2.2.3.1 --- cDNA of zebrafish embryo --- p.35 / Chapter 2.2.3.2 --- Isolation of adult zebrafish organs --- p.36 / Chapter 2.2.3.3 --- RNA extraction and reverse transcription --- p.36 / Chapter 2.2.3.4 --- Polymerase chain reaction --- p.37 / Chapter 2.2.4 --- Subcloning of DNA fragment / Chapter 2.2.4.1 --- Preparation of cloning vectors --- p.40 / Chapter 2.2.4.2 --- Subcloning of DNA fragments --- p.40 / Chapter 2.2.4.3 --- Transformation of DNA into competent cells --- p.40 / Chapter 2.2.4.4 --- Preparation of recombinant plasmid DNA --- p.41 / Chapter 2.2.5 --- Whole mount in-situ hybridization of zebrafish embryo --- p.45 / Chapter 2.2.5.1 --- Preparation of equipment --- p.45 / Chapter 2.2.5.2 --- Preparation of zebrafish embryos --- p.45 / Chapter 2.2.5.3 --- Preparation of RNA probe --- p.46 / Chapter 2.2.5.4 --- Whole-mount in-situ hybridization --- p.48 / Chapter 2.2.6 --- Sectioned in-situ hybridization of adult zebrafish brain --- p.49 / Chapter 2.2.6.1 --- Histology of adult zebrafish brain --- p.49 / Chapter 2.2.6.2 --- Sectioned in-situ hybridization --- p.50 / Chapter 2.3 --- Results --- p.51 / Chapter 2.3.1 --- Expression profile of foxp4 in different stages of zebrafish embryo --- p.51 / Chapter 2.3.2 --- Expression pattern of foxp4 in different stages of zebrafish embryo --- p.54 / Chapter 2.3.3 --- Expression profile of foxp4 in different zebrafish organs and tissues --- p.57 / Chapter 2.3.4 --- Expression pattern of foxp4 in adult zebrafish brain --- p.59 / Chapter 3.4 --- Discussion --- p.61 / Chapter Chapter 3 --- Functional analysis of foxp4 in zebrafish embryonic development / Chapter 3.1 --- Introduction --- p.63 / Chapter 3.2 --- Materials and methods --- p.64 / Chapter 3.2.1 --- Materials --- p.64 / Chapter 3.2.2 --- Design of morpholino --- p.68 / Chapter 3.2.3 --- Sequencing of morpholino target regions of foxp4 --- p.70 / Chapter 3.2.4 --- Microinjection --- p.70 / Chapter 3.2.4.1 --- Preparation of materials and equipment --- p.70 / Chapter 3.2.4.2 --- Preparation of injection needle --- p.70 / Chapter 3.2.4.3 --- Preparation of morpholinos --- p.70 / Chapter 3.2.4.4 --- Calibration of injection volume --- p.71 / Chapter 3.2.4.5 --- Microinjection of zebrafish embryo --- p.71 / Chapter 3.2.5 --- Western blotting to assay foxp4 translation inhibition --- p.72 / Chapter 3.2.5.1 --- Preparation of protein extracts --- p.72 / Chapter 3.2.5.2 --- Coomassie blue staining --- p.73 / Chapter 3.2.5.3 --- Western blotting --- p.74 / Chapter 3.2.6 --- Whole mount in-situ hybridization --- p.74 / Chapter 3.3 --- Results --- p.75 / Chapter 3.3.1 --- MO1 knockdown efficiency assayed by Western blotting --- p.75 / Chapter 3.3.2 --- General morphology of morphants --- p.77 / Chapter 3.3.3 --- Histology at the hindbrain region showing the phenotype --- p.79 / Chapter 3.3.4 --- Whole mount in-situ hybridization of different molecular markers --- p.81 / Chapter 3.4 --- Discussion --- p.85 / Chapter Chapter 4 --- Future directions and conclusion / Chapter 4.1 --- Future directions --- p.89 / Chapter 4.2 --- Conclusion --- p.91 / Reference --- p.92

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.

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.

Arachidonic acid (ARA) and docosahexaenoic acid (DHA) are polyunsaturated fatty acids required for proper embryonic development, specifically neurodevelopment. However, little is known regarding their conversion to other metabolites during embryogenesis. The oxidation of ARA gives rise to the biologically active eicosanoids and the oxidation of DHA gives rise to the biologically active docosanoids. The oxidation of ARA and DHA occurs through enzymatic processes, via lipoxygenase (LOX), or non-enzymatic processes, via radical-mediated lipid peroxidation. We hypothesize that oxidation of ARA and DHA via LOX is required for proper embryonic development. Additionally, we hypothesize that α-tocopherol, a potent lipid soluble antioxidant, mediates the conversion of ARA and DHA to their respective oxidized metabolites. Using zebrafish as a model of vertebrate embryogenesis, we found that the selective knockdown of either 12-LOX or 5-LOX decreased the production of docosanoids, altered fatty acid homeostasis, and increased the incidence of malformations and mortality in embryos by 24 hours post fertilization. α-Tocopherol deficiency also increased the incidence of malformations and mortality during embryogenesis, and in its absence, increased oxidized metabolites of ARA and DHA and decreased fatty acids concentrations. Therefore, oxidized metabolites of ARA and DHA perform crucial functions during embryonic development, but the production of oxidized fatty acids must be balanced with antioxidant bioavailability for proper embryogenesis. / Graduation date: 2012