In vivo long-term continuous observation of gene expression in zebrafish embryo nerve systems by using harmonic generation microscopy and morphant technology.
Gene expression plays an important role in embryo development and organ function. Previous studies have shown that harmonic generation microscopy (HGM) can be used as a fluorescence signal-independent, minimally invasive method with a subcellular 3-D resolution and a penetration depth in the order of millimeters for long-term continuous imaging of vertebrate embryos. We show that it is ideal to combine in vivo HGM with the morphant technology for minimally invasive, long-term continuous observation of gene expression in the nervous system of vertebrate embryos. Since second- and third-harmonic generations (SHG, THG) are virtual-state-transition-based systems that depend only on the structure of the organisms, they are not temporally limited by the expression of the fluorescence proteins. We successfully identified the expression of the zarnt2a and the hif-1alpha, 2alpha, and 3alpha genes in the nervous system of zebrafish embryos with specific knockdown genes by microscopically observing the embryos from the early stages of embryogenesis. The results from a combination of the two different modalities, i.e., SHG microscopy and THG microscopy, successfully revealed the weak cell adhesion, cell apoptosis, nerve formation reduction, and neural tube distortion in the morphant zebrafish embryos.
Hsieh
Cho-Shuen
CS
National Taiwan University, Department of Electrical Engineering, and, Graduate Institute of Photonics and Optoelectronics, Taipei 10617, Taiwan.
Ko
Ching-Yi
CY
Chen
Szu-Yu
SY
Liu
Tzu-Ming
TM
Wu
Jian-Shiung
JS
Hu
Chin-Hwa
CH
Sun
Chi-Kuang
CK
D000818:Animals; D030801:Animals, Genetically Modified / Q000378:metabolism*; D001709:Biotechnology / Q000379:methods; D001921:Brain / Q000196:embryology* / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods*; D018507:Gene Expression Regulation, Developmental / Q000502:physiology; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D015027:Zebrafish / Q000196:embryology / Q000235:genetics* / Q000378:metabolism*