Knockdown of Zebrafish Pitx3 Gene Affects the Early Development of Lens

Abstract

Abstract: : Purpose: The homeobox gene, PITX3, is associated with the anterior segment mesenchymal dysgenesis and congenital cataract in humans. In this study, we attempt to investigate its effect on lens development by way of gene knockdown in zebrafish. Methods: We microinjected Pitx3 morpholino oligo–nucleotides (MO), a translational inhibitor specific for Pitx3, into 1– to 4–celled zebrafish embryos. In order to observe their lenses in vivo, some morphants and normal controls were treated with 0.003% 1–phenyl–2–thiourea (PTU) every day to inhibit melanogenesis. At 72 hours post–fertilization (hpf), their left eyes were photographed for measuring ocular dimensions. The results were analyzed with 2–tailed independent t–test. Some larvae untreated with PTU were fixed for histological examination. We also performed Western blot and immunohistochemical stain to show the amount and location of alpha–A–crystallin, the major lenticular structural protein, in 72–hpf controls and Pitx3 morphants. Results: After microinjection of 3, 6, 7.5, or 9 ng of Pitx3–MO per embryo, more than 94% of morphants survived at 24 hpf and less than 8% of survived morphants had deformed body axis at 72 hpf. Grossly morphants had less bulging lenses, or even were aphakic. Image analysis proved that morphants’ eyes and lenses were significantly smaller than those of the controls, but their anterior chamber depth and retinal thickness did not differ. Western blot analysis showed that the 20–kD alpha–A–crystallin was less in morphants. Histological examination revealed that morphants had smaller lenses, but their retinal structure was similar to that of controls. Immunohistochemical stain showed decreased alpha–A–crystallin in the lens of Pitx3 morphant. Conclusions: Knockdown of Pitx3 gene did not significantly affect the early gross development, except for arrested lens development and microphthalmia which was associated with inadequate lenticular alpha–A–crystallin synthesis.

Keywords: transcription factors • crystallins • in situ hybridization