葉酸(維生素B9)對於穩定基因的正常表現、調控表觀遺傳以及維持胞內氧化還原平衡相當重要。研究證實葉酸缺乏會影響神經脊發育且與神經管缺損、先天性心臟病和巨球性貧血的發生相關，然而引發葉酸缺乏相關疾病的機制仍待研究。本研究利用可誘發葉酸缺乏的基因轉殖斑馬魚Tg(hsp:EGFP-γGH)來探討葉酸缺乏如何造成神經脊細胞衍生之心臟與黑色素細胞的發育缺陷。首先，我們探討葉酸缺乏對於斑馬魚循環系統發育的影響，包含心臟、造血及血管的發育。葉酸缺乏的斑馬魚胚胎出現血紅素量降低、紅血球數目減少、紅血球體積變大和c-myb訊號表現改變等紅血球生成異常現象，以及心臟腔室變小、心跳速率增加、心室射出分率減少和cmlc2訊號表現改變等心臟缺陷。給予葉酸缺乏的斑馬魚胚胎還原態葉酸(5-CHO-THF)或抗氧化劑(NAC)能減輕其造血及心臟的缺陷，然而核苷酸或Erk抑制劑的補充僅能緩解造血異常，而氧化態葉酸(FA)僅能緩解心臟缺陷。此研究突顯出因葉酸缺乏而異常增加的氧化壓力是導致斑馬魚胚胎造血及心臟缺陷的重要原因，而還原態葉酸對於緩解葉酸缺乏引發氧化壓力而導致的造血及心臟缺陷有較好的效果。接著，我們探討葉酸缺乏對黑色素細胞形態及功能的影響。黑色素細胞導致皮膚色素沈著是抵抗紫外光傷害不可或缺的機制。我們發現葉酸缺乏斑馬幼魚的皮膚黑色素細胞呈現黑色素減少、黑素體運送異常以及胞內黑色素異常分散的現象。給予葉酸缺乏斑馬幼魚5-CHO-THF補充可以緩解上述所列黑色素細胞的異常，然而補充FA僅能增加黑色素製造異常但無法改善黑素體運送功能。此外，葉酸缺乏斑馬幼魚胞內黑色素異常分散的現象可藉腎上腺素的補充改善。腎上腺素能透過刺激黑素體向細胞核週邊集中而調控胞內黑素體運送的平衡。透過給予腎上腺素及其前驅物並評估其對黑素體運送的矯正功能，同時分析斑馬幼魚腎上腺素及其前驅物含量，我們發現腎上腺素前驅物多巴胺的減少可能是導致葉酸缺乏斑馬幼魚胞內黑色素異常分散的原因。Dopa decarboxylase (Ddc)是合成多巴胺的酵素，而葉酸缺乏斑馬幼魚的Ddc表現減少。進一步對葉酸缺乏斑馬魚胚胎注射ddc表現質體可以改善其胞內黑色素異常分散的現象。上述發現顯示葉酸缺乏會導致黑色素細胞製造及運送黑色素的功能異常，同時揭示葉酸缺乏會藉由抑制Ddc的表現量導致胞內黑色素異常分散。鑒於葉酸缺乏導致黑色素細胞發育缺陷，我們進一步檢視葉酸含量對於紫外光傷害感受性的影響。葉酸缺乏斑馬幼魚在紫外光照射後產生相較對照組更顯著的尾鰭損傷及氧化壓力上升，顯示葉酸缺乏導致個體對紫外光傷害的敏感性增加。此外，5-CHO-THF對於減緩黑色素細胞缺陷以及紫外光傷害的能力優於FA。上述結果顯示，葉酸缺乏引發之發育缺陷的致病機轉是具組織特異性的。此外，在減緩葉酸缺乏引發之發育缺陷上，補充5-CHO-THF的效果優於FA，顯示出5-CHO-THF與FA的生物有效性是有差異的。

Folate (vitamin B9) is crucial for genome stability, epigenetic regulation and redox balance. Folate deficiency (FD) impedes neural crest development and has been correlated with many diseases such as neural tube defects (NTDs), congenital heart diseases (CHDs) and megaloblastic anemia. However, the mechanisms underlying these FD-associated disorders remain elusive. In this study, how FD impairs the development of heart and melanocyte, two of the neural crest lineages, was examined using FD-inducible zebrafish transgenic line Tg(hsp:EGFP-γGH). In the first part of this study, the impact of FD on circulation development, including cardiac development, hematopoiesis, and vascular integrity was examined. We found impaired hematopoiesis including decreased hemoglobin level, reduced erythrocyte number, enlarged erythrocyte size and altered c-myb expression, as well as cardiac defects including smaller chamber size, increased heart rate, reduced ejection fraction and altered cmlc2 expression in FD embryos/larvae. Treating FD embryos with reduced form of folate, 5-formyltetrahydrofolate (5-CHO-THF) or antioxidant N-acetyl-L-cysteine (NAC) ameliorated both hematopoietic and cardiac defects, whereas nucleotide or Erk inhibitor ameliorated only the hematopoietic defects and the oxidized form of folate, folic acid (FA) ameliorated only the cardiac defects. These findings highlight the role of excessive oxidative stress in mediating FD-induced hematopoietic and cardiac defects, and demonstrate that folate with reducing power serves as a better reliever to oxidative stress-dependent hematopoietic and cardiac defects resulted from FD. In the second part of this study, the impact of FD on the morphology and function of melanocytes was examined. Melanocytes are the foundation of pigmentation and therefore indispensable for protecting organisms from ultraviolet (UV)-inflicted damage. Maldevelopment of skin melanocytes including decreased melanogenesis, reduced melanosome anterograde response and aberrant melanin dispersion was observed in FD larvae. Treating FD larvae with 5-CHO-THF relieved both melanogenesis and melanosome transportation defects while FA failed to rescue the melanosome anterograde defects. The aberrant melanin dispersion could be readily rectified when treating FD larvae with epinephrine, one of the melanosome retrograde stimulus involved in the homeostasis of intracellular melanosome transportation. Further examination of the melanocyte retrograde response and metabolite levels of epinephrine and its precursors suggested that reduced synthesis of dopamine, a precursor of epinephrine, contributes to the aberrant melanin dispersion observed in FD larvae. Dopa decarboxylase (Ddc) is the enzyme catalyzing dopamine synthesis from L-dopa. Decreased Ddc expression in both transcript and protein levels was observed in FD larvae. Through injecting FD embryos with ddc construct, the aberrant melanin dispersion was ameliorated. These results demonstrate that FD impairs melanogenesis and melanosome transportation, and reveal for the first time that down-regulated Ddc contributes to the aberrant melanin dispersion resulted from FD. Along with the finding that FD disturbs melanocyte development, the impact of folate statue on the vulnerability to UVB irradiation was investigated. In comparison to control larvae, FD larvae displayed more significant tail fin damage and ROS elevation after UVB irradiation, indicating that FD leads to an increased susceptibility to UVB irradiation. 5-CHO-THF, in concordance with its efficacy in relieving melanocyte defects, exerts better protection effects in ameliorating UVB-inflicted damage than FA. Taken together, these results demonstrate tissue-specific pathomechanisms resulted from FD. Moreover, the finding that 5-CHO-THF surpasses FA in relieving FD-inflicted developmental defects suggests that the biological availability is different between 5-CHO-THF and FA.

CONTENT
ABSTRACT ..........................................I
摘要.............................................III
ACKNOWLEDGEMENT ................................. V
CONTENT OF FIGURES AND TABLES ........................ X
ABBREVIATION.................................... XII
GENERAL INTRODUCTION .............................. 1
Folate .......................................... 1
Clinical significance of folate.............................. 1
Folate and the development of neural crest cells.................. 5
Zebrafish ........................................ 5
Zebrafish in nutrient-related studies......................... 6
MATERIALS AND METHODS.............................. 7
Materials ........................................ 7
Zebrafish line and maintenance........................... 8
Induction of folate deficiency ............................. 8
Compound treatment.................................. 8
Hemoglobin staining ................................. 9
Erythrocyte analysis.................................. 9
Zebrafish larval cryostat sectioning .......................... 9
Whole mount in situ hybridization .......................... 10
Cardiac function analysis ............................... 10
Homocysteine quantification............................ 11
Melanin measurement................................. 11
RNA isolation, reverse transcription and polymerase chain reaction...... 11
Melanosome transportation assay ......................... 11
Evaluation of melanosome retrograde extent................... 12
Zebrafish metabolite analysis............................. 12
Zebrafish Ddc protein expression.......................... 14
Zebrafish ddc construct for rescue .......................... 14
Touch response analysis ............................... 14
UV exposure..................................... 15
Reactive oxygen species assay ............................ 15
Total antioxidant capacity assay .......................... 15
Statistical analysis ................................... 16
RESEARCH GOALS.................................... 17
PART I ........................................... 18
The impact of folate deficiency on the development of zebrafish heart and circulation system* ........................................... 18
INTRODUCTION ................................... 18
Folate deficiency and circulation defects ...................... 18
Zebrafish in the studies of circulation development ................ 18
Folate, oxidative stress and circulation defects .................. 19
Folate, ERK signaling and circulation defects ................... 19
SPECIFIC AIMS..................................... 20
RESULTS......................................... 21
Folate deficiency impaired cardiac development ................. 21
Folate deficiency altered the expression pattern of cmlc2 ............. 21
Folate deficiency impeded erythropoiesis ..................... 24
Folate deficiency altered the expression pattern of c-myb............. 24
Folate-deficient larvae showed no significant anomalies on vascular development ............................................. 28
Antioxidant ameliorated the hematopoietic and cardiac defects in folate-deficient larvae .......................................... 31
Erk inhibitor alleviated the defective hematopoiesis in folate-deficient larvae. 31
Nucleotide supplement restored the hemoglobin level in folate-deficient larvae 31
Folic acid supplement did not restore the hemoglobin level in folate-deficient larvae .......................................... 32
DISCUSSION ...................................... 34
PART II.......................................... 37
The impact of folate deficiency on the development of zebrafish melanocytes* ... 37
INTRODUCTION ................................... 37
Melanocytes...................................... 37
Cutaneous pigmentation ............................... 37
Folate and skin melanocytes............................. 38
Zebrafish in the studies of melanocyte biology................... 39
Dopa decarboxylase .................................. 39
SPECIFIC AIMS..................................... 41
RESULTS......................................... 42
Folate-deficient larvae displayed reduced melanin synthesis ........... 42
Folate-deficient larvae displayed aberrant melanin dispersion.......... 42
Melanosome retrograde response induced by dopamine, norepinephrine and epinephrine ameliorated the aberrant melanin dispersion observed in folate- deficient larvae..................................... 47
The expression of dopa decarboxylase was decreased in folate-deficient larvae 50
Restoring Ddc expression ameliorated the aberrant melanin dispersion in folate- deficient larvae..................................... 50
PLP supplementation rescued the melanin synthesis and motor response in folate-deficient larvae................................ 54
Folate supplementation ameliorated the UVB-induced injury in wild-type larvae ............................................. 54
Folate-deficient larvae displayed increased vulnerability to UVB-inflicted damage. ........................................ 57
Folate-deficient larvae possessed decreased antioxidant capacity........ 57
Selective folate supplementation differentially protected FD larvae from UV- inflicted damage and oxidative stress ........................ 58
DISCUSSION ...................................... 63
REFERENCES ...................................... 68
APPENDIX ........................................ 81
AUTHOR ......................................... 91

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