Functional Analyses of Secreted Frizzled-Related Protein 1 (sFRP1) and Dickkopf 1 (Dkk1) during Eye Development of Zebrafish by Transgenic Over-expression and Down-regulation Assays

Wnt訊息傳遞路徑在早期生長發育過程中扮演重要的角色。當Wnt配體(ligands)與細胞膜上的Frizzled (Fz)受體(receptors)及LRP5/6 (low-density lipoprotein receptor-related protein)結合後，會促使細胞質中β-catenin增加並進入細胞核中調控特定基因之轉錄。 本論文以斑馬魚為材料，探討Wnt訊息傳遞過程中位於細胞外分泌性之負面調控(拮抗)分子Secreted Frizzled-Related Protein 1 (sFRP1)、Dickkopf1 (Dkk1)在眼睛發育過程中之功能。以超量表現和抑制基因表現，剖析sFRP1及Dkk1對於眼睛組織發育所扮演之角色。我利用gain-of-function及loss-of-function的方法，透過斑馬魚βB1-Crystallin 1.3 kb啟動子的驅動，經IRES-GFP的載體，以基因轉殖方式在水晶體過量表現sFRP1或Dkk1蛋白，或以antisense RNA抑制內生性mRNA的方式降低sFRP1、Dkk1蛋白的製造；同時藉由IRES系統觀察綠色螢光蛋白之表現，篩選轉殖斑馬魚。 實驗結果發現，在過渡轉殖實驗中，具有綠色螢光表現之斑馬魚F0親代並無明顯之表現型(phenotype)。在篩選轉殖品系後，篩選到三個抑制內生性sFRP1之轉殖恆定品系No.1、No.9及No.54；二個過量表現sFRP1之轉殖恆定品系No.6及No.11；一個抑制內生性Dkk1轉殖恆定品系No.74。 觀察F1子代發現轉殖恆定品系外觀上也無明顯之表現型。而後，經由樹脂包埋切片，發現在抑制內生性sFRP1轉殖恆定品系No.9中，水晶體上皮細胞在出生後第四天和第六天有增厚呈菱形不規則排列的現象，然而在第八天此現象變得不明顯；此外，在抑制內生性Dkk1轉殖恆定品系No.74中，在第四天、第六天水晶體上皮細胞也有增厚之現象發生，同樣地在第八天變得不明顯。另一方面，於過量表現sFRP1 No.6和No.11的品系中，在第四天和第六天無顯著變化之表現型。 為了加強Wnt訊息對水晶體細胞的作用，我將抑制內生性sFRP1轉殖恆定品系No.9和抑制內生性Dkk1轉殖恆定品系No.74互相配對，挑選雙轉殖品系之子代。觀察切片之結果，發現在第六天、第八天時，水晶體上皮細胞仍然呈增厚菱形不規則排列。因此，不同於單一轉殖品系(Antisense sFRP1 No.9或Antisense Dkk1 No.74)中，在第八天其基因轉殖之外表型巳消失。初步推測，此結果為加強Wnt訊號效果所造成。關於Wnt訊息如何影響水晶體之發育，其詳細之相關機制必須以in situ hybridization、immunohistochemistry和BrdU等方法作進一步分析，才能對於水晶體上表皮細胞不正常的形態作更完整之了解，以及釐清其分子間相互調控機制。The Wnt signaling plays a crucial role during early development. Wnt ligands activate responding cells by interacting with the receptor of transmembrane protein Frizzled (Fz) and LRP5/6 (low-density lipoprotein receptor-related protein), leading to augmentation of β- catenin concentration and formation of nuclear transcriptional complexes. Zebrafish was chosen as a model animal in this study. The aim of the study was to elucidate the function of sFRP1 and Dkk1, extracellular negative regulators of Wnt singaling pathway, during early eye development by transgenic over-expression or down-regulation assays. I use lens-specific βB1-crystallin promoter to drive the expression of full-length or antisense sFRP1 and Dkk1 cDNA in the lens. Through IRES system, it facilitates the screening of transgenic fish containing these constructs by expression of GFP. In transient transgenic assays, there were no conspicuous phenotypes in the F0 transgenic fish. Afterwards, three stable transgenic zebrafish lines were obtained (No.1, No.9, and No.54) with the pCr1.3-Antisense sFRP1-IRES-hrGFP construct and two stable transgenic zebrafish lines were obtained (No.6 and No.11) with the pCr1.3-Full Length sFRP1- IRES-hrGFP construct. As regarding Dkk1, one stable transgenic zebrafish line was obtained (No.74) with the pCr1.3-Antisense Dkk1-IRES-hrGFP construct. By observing, still, no conspicuous phenotypes were found in the F1 transgenic fish. After resin-sectioning, I found that the epithelial cells of lens tend to be more thickening and irregularly packed in antisense sFRP1 No.9 and antisense Dkk1 No.74 strains, when compared to the cuboidal regularly packed cells of wild type at 4 and 6 dfp. Howerver, the phenomena diminished at 8 dpf. On the other hand, no evident phenotypes were observed in the full-length sFRP1 No.6 and No.11 of transgenic fish. To reinforce the transgenic effects, I utilized a double transgenic approach by crossing antisense sFRP1 No.9 and antisense Dkk1 No.74. This resulted in the prolongation of phenotype in epithelial cells of lens until 8 dpf and thickening of GCL (ganglion cell layer) around the optic nerve. In the future, in situ hybridization and other methods may be empolyed to elucidate the detailed function of Wnt signaling during the eye development of zebrafish.中文摘要 1 英文摘要 3 壹、前言 5 貳、實驗材料 30 參、實驗方法 36 肆、結果 47 伍、討論 52 陸、圖表 57 參考文獻 7