Egr1 gene knockdown affects embryonic ocular development inzebrafish

摘要

Purpose: To identify the changes in zebrafish embryonic oculardevelopment after early growth response factor 1 (Egr1) gene knockdownby Egr1-specific translation inhibitor, morpholino oligonucleotides(MO).Methods: Two kinds of Egr1-MO were microinjected separately withvarious dosages into one to four celled zebrafish embryos to find anoptimal dose generating an acceptable mortality rate and high frequencyof specific phenotype. Chordin-MO served as the positive control; a 5mismatch MO of Egr1-MO1 and a nonspecific MO served as negativecontrols. We graded the Egr1 morphants according to their grossabnormalities, and measured their ocular dimensions accordingly. Westernblot analysis and synthetic Egr1 mRNA rescue experiments confirmedwhether the deformities were caused by Egr1 gene knockdown. Histologicalexamination and three kinds of immunohistochemical staining were appliedto identify glutamate receptor one expression in retinal ganglion cellsand amacrine cells, to recognize acetylated α-tubulin expressionwhich indicated axonogenesis, and to label photoreceptor cells withzpr-1 antibody.Results: After microinjection of 8 ng Egr1-MO1 or 2 ng Egr1-MO2,81.8% and 97.3% of larvae at 72 h postfertilization had specificdefects, respectively. The gross phenotype included string-like heart,flat head, and deformed tail. The more severely deformed larvae hadsmaller eyes and pupils. Co-injection of 8 ng Egr1-MO1 and supplementary12 pg synthetic Egr1 mRNA reduced the gross abnormality rate from 84.4%to 29.7%, and decreased the severity of deformities. Egr1 proteinappeared in the wildtype and rescued morphants, but was lacking in theEgr1 morphants with specific deformities. Lenses of Egr1 morphants weresmaller and had some residual nucleated lens fiber cells. Morphants'retinal cells arranged disorderly and compactly with thin plexiformlayers. Immunohistochemical studies showed that morphants had a markedlydecreased number of mature retinal ganglion cells, amacrine cells, andphotoreceptor cells. Retinal axonogenesis was prominently reduced inmorphants.Conclusions: The Egr1 gene plays an important role in zebrafishembryonic oculogenesis. Ocular structures including lens and retina wereprimitive and lacked appropriate differentiation. Such arrested retinaland lenticular development in Egr1 morphants resulted inmicrophthalmos.