Analysis of functional domains in zebrafish Ugly duckling (Udu) protein

目前已知斑馬魚Ugly duckling (Udu)蛋白具有許多功能，包含紅血球發育相關、細胞凋亡調控及有可能參與在DNA複製及細胞週期的過程中。Udu蛋白具有多個功能區域 (functional domain)，包含兩個YY1-binding (YY1BD)、PAH及SANT 區域。udu具有高度保守性，在許多物種中都有它的同源基因。其中，udu在人類的同源基因Gon4l，在演化過程中發生部分複製的現象，產生一個全新的蛋白YY1-associated protein (YY1AP)。YY1AP的結構跟Udu/Gon4l比起來，只包含了兩個YY1BD，並且能夠與轉錄因子YY1結合，在人類細胞中增強下游基因的表現。同樣地，我們實驗室之前的研究發現，斑馬魚Udu也可以透過其YY1BD和YY1結合。因此我們假設，在發育過程中，Udu的YY1BD、PAH及SANT 區域可能各自具有獨立的功能。 我們利用兩種斑馬魚的udu突變種，udutu24及udusq3進行實驗。udusq3的突變位置跟udutu24比較，多了一個YY1BD的區域。形態方面udusq3和udutu24同樣具有較短的體長、彎曲尾巴及不正常的體節發育。然而udusq3的形態改變情形比udutu24和緩，因此我們認為YY1BD具有獨立的功能。我們透過原位雜合實驗 (in situ hybridization)，發現血液發育、血管發育等基因在udutu24及udusq3中分別有不同的表現。另外，我們透過顯微注射的方式，分別將udu N-terminal mRNA (只包含兩個YY1BD) 及udu C-terminal mRNA (只包含PAH、SANT區域) 注射到udutu24或是udusq3的胚胎當中，發現對於udutu24和udusq3擁有不同的效果。大部分的發育缺陷形態透過udu N-terminal mRNA即可挽救，證明了在Udu上面的YY1BD具有獨立的功能。 另外，我們發現在udu mutants當中，除了血液發育缺陷之外，同時也有血管發育缺陷的情形。同時，我們也發現在udu mutants中，Notch signaling的配體 (ligand)─ dll4的表現量是下降的。因此，我們推測這種血管發育缺陷的情形，可能和Notch-VEGF pathway有關。另外，由於在udusq3中，肌節間血管 (intersomitic vessels，ISV)的發育情形比udutu24良好，因此我們推測肌節間血管的發育可能藉由Udu的YY1BD和YY1結合來調控。我們將yy1 morpholino注射到udusq3的胚胎當中，發現其肌節間血管的發育缺陷更為嚴重。這也證明了在Udu當中的YY1BD具有獨立的功能。It has been shown that zebrafish Udu plays a role in many developmental processes. Our previous data showed that Udu has diverse functions, including erythrocyte development and may be involved in modulating DNA replication or cell cycle. Gon4l is one of the homolog of zebrafish Udu in human and mouse. During evolution, Gon4l was partially duplicated and produced a novel protein, YY1AP (YY1-associated protein). It has been demonstrated that when YY1AP binds to YY1, it will enhance the transcriptional activation of YY1-targeted promoter in human cells. Likewise, our previous data showed that YY1 can associate with YY1-binding domains (YY1BD) of Udu. According to these results, I hypothesize that Udu YY1BD, PAH and SANT domains might have respective or independent functions during development. I used udu mutants, udutu24 and udusq3 to study whether N-terminal segment of Udu has distinct functions or not. Compared to udutu24, udusq3 remains one of YY1BD sequence in udu gene structure. By using hematopoietic and angiogenic markers for in situ hybridization, I found that there are different expression patterns in udutu24 and udusq3. Furthermore, I injected udu N-terminal or full-length mRNA into one-cell stage udutu24 or udusq3 embryos separately. I have shown that both udu N-terminal and full-length mRNA could partially rescue phenotypes in udu mutants. Moreover, I discovered that ISV defect in udusq3 is much more severe after yy1 MO injection. It suggests that YY1BD of Udu has distinct functions in zebrafish. To sum up, these data shows that YY1BD, PAH and SANT domains of Udu may play independent roles in different developmental processes.誌謝 i 中文摘要 iii Abstract v Table of Contents vii Index of Figures viii Index of Tables ix Chapter 1. Introduction 1 1.1 ugly duckling (udu) gene functions and its important domains 1 1.2 Zebrafish as a model animal to study vertebrate development 3 1.3 Hematopoietic development in zebrafish 5 1.4 YY1AP (YY1-associated protein) 9 1.5 Specific aims 11 Chapter 2. Materials and Methods 12 2.1 Maintenance of zebrafish 12 2.2 Embryo staging 12 2.3 Genotyping of zebrafish udutu24 and udusq3 embryos 12 2.4 Amplication of cDNA by RT-PCR 14 2.5 Microinjection experiments 15 2.6 Antisense probe synthesis 16 2.7 Whole-mount in situ hybridization (WISH) 17 2.8 Microscopy 19 2.9 Real-time PCR 19 Chapter 3. Results 24 3.1 Phenotypic observations of udutu24 and udusq3 embryos 24 3.2 Real-time PCR results of hematopoiesis- and apoptosis-related genes in udutu24 and udusq3 embryos 24 3.3 Rescue experiments by micro-injection and analyzed by in situ hybridization of p53 in udutu24 mutants 25 3.4 Comparison of gata1, hbae1 (hematopoiesis-related markers) in situ hybridization results in udutu24 and udusq3 mutants 28 3.5 ISV angiogenic phenotype in udutu24 and udusq3 mutants 30 Chapter 4. Discussion 54 4.1 Accuracy of real-time PCR in this study 54 4.2 Functional domains in zebrafish Udu protein 55 4.3 YY1-Udu-NICD complex ? 56 4.4 The angioblast precusor migration defect in udu mutants 57 4.5 Udu may be involved in Notch-VEGF pathway 58 References 60 List of Abbreviations 6