利用基因轉殖過量表現或抑制方法探討Secreted Frizzled-Related Protein 1和Dickkopf 1 在斑馬魚顱顏組織眼睛發育之功能

Abstract

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.