| Summary: | 碩士 === 國立臺灣大學 === 臨床醫學研究所 === 92 === Purpose: ZENK gene has been proposed to be associated with abnormal eye growth in chickens. The aim of this study is to identify the change in early ocular development after Egr1 (early growth response 1) gene, the homologue of ZENK gene, knockdown in zebrafish by morpholino oligo-nucleotides (MO), which is a sequence-specific anti-sense mRNA inhibitor.
Methods: Various doses of Egr1-MO were microinjected into the 1- to 4-cell zebrafish embryos. In addition, chordin MO and the standard control MO were also injected as positive control and negative control respectively. After correction with the death rate at 24 hours post-fertilization (hpf) and abnormality rate at 72 hpf of the wildtype, the expression rate of morphants was calculated according to their specific morphologic changes at 72 hpf. The most favorable Egr1-MO dose was selected to induce the highest expression rate and an acceptable lethal rate. The phenotypes were observed and photographed with a stereo dissecting microscope equipped with a digital camera. The 72-hpf morphants were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned into 5 �慆 slices, stained with hematoxylin and eosin, and observed with a light microscope. Immunohistochemical staining was applied to identify the location of glutamate receptor 1 which represents retinal ganglion cells and amacrine cells, and the acetylated ��-tubulin which indicates axonogenesis. In order to make sure that the phenotypes were caused by Egr1 gene knockdown, Western blot was performed to check the amount of Egr1 protein which should be significantly decreased in morphants. Besides, various dosage of in vitro synthetic Egr1 mRNA together with the most favorable dose of Egr1-MO found in this study was microinjected into embryos, and then the morphants were examined whether they could be rescued by the supplementary Egr1 mRNA.
Results: Four different doses of Egr1-MO were tried, and the most appropriate one was 8 ng per embryo. As many as 84% of survival embryos at 72 hpf had altered appearance, yet only <1% of live negative control did. The most common defects of Egr1 morphants were enlarged pericardium, small eyes, and deformed body axis. The more severely deformed larvae had smaller eyes and pupils. To rescue the morphants, 8 different doses of synthetic Egr1 mRNA have been tried. Concomitantly injecting 12 pg of Egr1 mRNA and 8 ng of Egr1-MO showed nearly 50% decrement of expression rate. And the severity of phenotype also decreased markedly. Western blot analysis demonstrated significantly decreased 55-kD Egr1 protein in morphants, and Egr1 protein appeared apparently in the Egr1 mRNA-rescued morphants. Histological examination revealed that morphants’ retinae had thinner inner and outer plexiform layers. Retinal cells arranged compactly and disorderly. Immunohistochemical study suggested a decreased surface marker of retinal ganglion cells and amacrine cells, and also decreased axonogenesis in morphants.
Conclusion: Egr1 gene plays an important role in the early ocular development. At 72 hpf, the morphants’ retinal neurons seemed primitive, and arranged compactly with fewer neural processes and synapses, which resulted in the appearance of microphthalmia.
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