Loss of Nlz2 Expression Results in Coloboma and Abnormal RPE Development in Mouse

Abstract

Purpose: Failure of the optic fissure—a transiently open gap in the ventral optic cup during normal eye development—results in uveal coloboma, a cause of childhood blindness. We have previously shown that nlz2 is important for optic fissure closure in zebrafish. In this study, we try to uncover the role of Nlz2 in optic fissure closure in a mouse knockout (KO) model.

Methods: Embryos were evaluated grossly and with histology. Nlz2 expression was evaluated during the relevant stages of eye morphogenesis in wild-type (WT) and Nlz2 KO mice. The expression patterns of other developmental genes (e.g., Pax2, Pax6, Vsx2, Mitf and Otx2) were also evaluated. Western blots were used to evaluate the levels of Nlz2 expression in human fetal RPE cells and ARPE-19 cells.

Results: Nlz2 ^(-/-) embryos exhibit coloboma on gross and histologic examination, as well as a striking decrement in RPE pigmentation. Nlz2 was dynamically expressed in the optic stalk/vesicle/cup in WT embryos at E10.5, E11.5 and E12.5; KO mice showed no expression. At later stages in embryonic development, Nlz2 was expressed in the RPE and optic stalk. In KO mice, Pax6 expression was abnormally expanded into the optic stalk, while Pax2 expression was expanded in the RPE. Both Otx2 and Mitf levels were decreased in the RPE in the KO compared to control, whereas Otx2 levels were increased in the dorsal neural retina in the KO embryo. Both dorsal and ventral proximal RPE layer was thickened and positive for VSX2 staining. Western blot and immunostaining show Nlz2 expression in hfRPE and ARPE-19 cells.

Conclusions: Loss of Nlz2 expression leads to coloboma in mice and is associated with dysregulation of several key transcription factors. We posit that the hypopigmentation and abnormal morphology of KO embryos RPE is related to the reduced levels of Mitf expression. Vsx2 staining in the thickened presumptive RPE suggests that these cells may be differentiating along a more neural-retina-path. These data suggest that Nlz2 is critical in mammalian eye development, optic fissure closure and RPE cell differentiation/melanogenesis.