臺灣博碩士論文加值系統

Akt1是一個在斑馬魚血液循環系統中最為廣泛研究的基因之一，並且常被指出可能在血癌與其他血液血管的疾病當中扮演重要的角色。然而目前大多數的研究主要是利用過度表現的變異基因來進行體外試驗，而這些方法無法反映出Akt1在真實的生理功能中造成的影響。在這裡，我們分析了Akt1在發育中的斑馬魚血液循環系統中的影響，並發現斑馬魚Akt1對於血液生成作用扮演著必需的角色。利用morpholino或是對Akt1磷酸化位點進行點突變的RNA來競爭抑制內生性Akt1的生成，皆會造成造血前趨細胞與血管內皮前趨細胞的減少，進而導致所有成熟血球的數量下降。而造成此現象可能是Akt1受到抑制之後，間接抑制了CyclinD1的活性，並導致不經由p53活化的細胞凋亡作用增加所造成。相反地，持續活化態的Akt1可促進造血幹細胞的分化。總而言之，我們在斑馬魚的研究闡明了Akt1對於血液血管共同前趨細胞的細胞存活扮演著必需的角色，並且可促進造血幹細胞的分化。此外，我也對於Akt1在造血細胞的存活中，提出了一個新的調控機制，進而對於Akt1在斑馬魚血液生成作用中所扮演的角色提供了更進一步的了解，也對於利用斑馬魚作為研究血癌與其他血液血管疾病的方法提供了更詳細的資訊。

Akt1 is one of the most characterized genes that plays fundamental role in hematopoiesis and vessel formation and is implicated in leukemias and blood/vessel disorders. However, most of the studies to date were performed by in vitro analysis mainly relies on the mis-expression of mutated constructs, which may not truly reflect the physiological role of Akt1. Here, I analyzed the role of Akt1 in developing circulatory system in zebrafish and found that zebrafish Akt1 is required for hematopoiesis. Abolishing endogenous Akt1 function by morpholino antisense oligos or a mutated-negative form resulted in depletion of hematovascular progenitors and consequently reduced all hematopoietic derivatives. And further analysis showed that this effect was caused by p53-independent cell apoptosis, intriguingly, through CyclinD1 activation. On the contrary, constitutive activation of Akt1 is sufficient to induce the differentiation of hematopoietic stem cells. In conjunction with previous in vitro studies, my study in zebrafish embryos clarified that Akt1 is required for the survival of hematovascular progenitors and is sufficient to induce hematopoietic stem cell differentiation, and in addition, it revealed a novel mechanism for the role of Akt1 in hematopoietic cell survival. My results therefore provide important information towards a better understanding for the role of Akt1 in hematopoiesis and outlined a useful model for studying hematopoietic disorders and leukemias.

Table of Contents
摘要……………………………………………………………………..vii
Abstract……………………………………………………………….viii
Table of Contents………………………………………………………ix
Table and Figure List……………………………………………………xii
CHAPTER I Introduction
1.1 Circulatory system……………………………………………1
1.2 Akt signaling pathway………………………………………2
1.3 Zebrafish circulatory system…………………………………4
1.4 Aim………………………………………….…….…….…….5
CHAPTER II Materials and Methods
2.1 Collection and maintenance of zebrafish embryos……………7
2.2 Whole mount in situ hybridization..………………………….7
2.3 Microinjection………………………………………………9
2.4 In vitro transcription for mRNA……………………………9
2.5 Immunohistochemistry and immunocytochemistry…………9
2.6 TdT-mediated dUTP nick end labeling (TUNEL) assay……11
2.7 Western blotting……………………………………………11
2.8 Flow cytometry………………………………………………12

CHAPTER III Results
3.1 The expression patterns of akt1 in zebrafish……………. …13
3.2 Zebrafish Akt1 knockdown resulted in reduction of hemangioblasts, HSCs, erythroid progenitors, granulocytes and lymphocytes…………………………………………………14
3.3 Zebrafish Akt1 knockdown increases p53-independent cell apoptosis during hematopoiesis………………………………17
3.4 Zebrafish Akt1 knockdown decreases cell proliferation during hematopoiesis…………………………………………………18
3.5 Gain of AKT1 function in zebrafish embryos does not affect the population of hemangioblasts and HSCs, but increase the population of erythroid progenitors, granulocytes and lymphocytes…………………………………………………19
3.6 Higher phosphorylation level of Akt1 is needed at 36 hpf…20
3.7 The expression patterns of ccnd1 is similar with akt1in zebrafish………………………………………………………20
3.8 CyclinD1T282A mRNA partially rescues akt1 morphants on scl and gata2 expression…………………………………………22
3.9 Akt1 positively regulate CyclinD1 on cell cycle distribution and may reduce cell proliferation rate………………………23
3.10 Akt1 inhibit cell apoptosis through CyclinD1 activation…23
3.11 Akt1 protein, but not its kinase activity is required for normal angiogenesis…………………………………………………24

CHAPTER IV Discussions and Future Works
4.1 Summary……………………………………………………26
4.2 The expression of Akt1 in the developmental circulatory system is evolutionarily conserved…………………………26
4.3 The specificity of Akt1 morpholino…………………………27
4.4 The role of Akt1 in zebrafish hemangioblasts………………28
4.5 The role of Akt1 in zebrafish hematopoietic stem cells (HSCs)………………………………………………………29
4.6 The interaction of Akt1 and CyclinD1 on hematopoiesis……31
4.7 The role of Akt1 in zebrafish erythropoiesis, granulopoiesis and lymphopoiesis……………………………………………34
4.8 The role of Akt1 in zebrafish vasculogenesis and angiogenesis…………………………………………………36

References………………………………………………………….……38
Figures…………………………………………………………………50
 
Table and Figure List
Figure 1. akt1 expression patterns in zebrafish…………………………38
Figure 2. Knockdown of Akt1 by akt1 MO and akt1T302A/S467A mRNA showed morphological defects of developing circulatory system…………………………………………………………40
Figure 3. Knockdown of Akt1 by akt1 MO and akt1T302A/S467A mRNA cause the reduction of hemangioblasts and hematopoietic cells……………………………………………………………42
Figure 4. akt1 mRNA partially rescues akt1 morphants………………44
Figure 5. Knockdown of Akt1 increases cell apoptosis………………46
Figure 6. The reduction of hemangioblasts and hematopoietic cells in akt1 morphants could not be rescued by co-injection with p53 MO……………………………………………………………47
Figure 7. Knockdown of Akt1 decreases cell proliferation during hematopoiesis and causes cell cycle arrest at G0/G1 and G2/M phase…………………………………………………………49
Figure 8. Over-expression of hu caAKT1 mRNA in zebrafish embryos increases the population of erythroid progenitors, granulocytes and lymphocytes………………………………………………51
Figure 9. Developmental regulation of Akt1 kinase activity…………………………………………………………53
Figure 10. ccnd1 expression patterns in zebrafish………………………54
Figure 11. ccnd1T282A mRNA partially rescues akt1 morphants in hemangioblasts and HSCs……………………………………55
Figure 12. ccnd1T282A mRNA rescues akt1 morphants on cell apoptosis………………………………………………………57
Figure 13. ccnd1T282A mRNA increases the S phase distribution in akt1 morphants……………………………………………………59
Figure 14. Knockdown of Akt1 by Akt1 MO, over-expression of akt1T302A/S467A mRNA and caAKT1 mRNA cause angiogenesis defects…………………………………………………………61

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