In vivo Eyes Development of Drosophila Embryo in Early Stage Studied by Three-dimensional Time-lapsed Imaging via Single Plane Illumination Microscopy (SPIM)

• Main Authors: Chin-Hao Chang, 張晉豪
• Other Authors: Arthur Chiou
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/91694569634553514732

碩士 === 國立陽明大學 === 生醫光電研究所 === 102 === Although fluorescent staining and imaging have been used to study the eyes development at each developmental stage of Drosophila from larva to adult, the development process of Eye-Antennal Disc Primordium (EADP) in the embryonic stage still remains unclear. In our study, we applied Single Plane Illumination Microscopy (SPIM) to acquire in vivo time-lapsed images of Drosophila eyes development in early stage with single cell resolution. Our main objectives include the elucidation of the dynamic formation of EADP and a critical examination of the contribution of engrailed (en) gene to the formation of EADP. In our experiments, Drosophila embryos were labeled with three kinds of fluorescence: (1) CD-GFP(II)/S-T; which labeled Green Fluorescent Protein (GFP) on CD enhancer; (2) en-GAL4; UAS-H2B-RFP; which labeled Red Fluorescent Protein (RFP) on en gene; and (3) CD-GFP/en-Gal4; UAS-H2B-RFP/+; which labeled red and green fluorescent protein on en gene and CD enhancer, respectively, in the same sample. Via single plane illumination microscopy, we acquired and reconstructed 3-d time-lapse image sequences of the eyes development of Drosophila. Our results revealed that CD enhancer expressed on Drosophila embryo from stage 14 to stage 17. The 3-d cells dynamics during the EADP development process were recorded and analyzed. A comparison of the expression of CD enhancer and en gene at stage 16 indicated that the en gene expressed only on a small fraction of EADP and that the role and the detail spatial-temporal distribution of en gene on EADP development still need to be verified through more imaging evidences with better spatial and temporal resolutions.