Investigating the Role of Porcn in Optic Vesicle Morphogenesis

Abstract

Purpose : Congenital ocular malformations, such as microphthalmia, anophthalmia, and coloboma (MAC), are responsible for 25% of childhood blindness. Focal Dermal Hypoplasia is an x-linked dominant disorder that is often associated with MAC and caused by mutations in Porcn, a membrane bound O-acyl transferase that is required for the palmitoylation of Wnt ligands. Previous studies in frogs and zebrafish have demonstrated that the non-canonical Wnt pathway is essential for early eye formation. However, little about the role of non-canonical Wnt signaling in eye development is known in mammals. Here, we inactivate Porcn in mouse to interfere with non-canonical Wnt signaling and examine its effect on early eye formation and tissue patterning.

Methods : PORCN is inactivated in mouse embryos at embryonic day (E)6.5-E7.5 by tamoxifen-inducible, ubiquitous ROSA26^CreER. Affected male embryos with conditional deletion of Porcn are analyzed by immunohistochemistry (minimum n=3) using antibodies for eye field developmental markers PAX6, SIX3, LHX2, RX, OTX2 and the optic vesicle markers MITF and VSX2.

Results : Porcn mutants show a range of defects, depending on the time of inactivation. Tamoxifen treatment at E7.5 results in arrested eye morphogenesis in less severely affected Porcn mutants; the optic vesicle is reduced and does not invaginate to form an optic cup. At E10.5, MITF and VSX2 labeling reveals lack of expression in the RPE and neural retina, respectively. Similarly, OTX2 is not detectable in the optic vesicle. The pan-ocular marker PAX6 is expressed at slightly lower level in the mutant optic vesicle and lens ectoderm. Surprisingly, Six3 expression is detectable in the retina and lens ectoderm of Porcn mutants, indicative of eye field formation. Currently, we are investigating expression of additional ocular genes critical for ocular morphogenesis and patterning (e.g. LHX2, RX, COUPTF2, PAX2), including at earlier stages. In addition, our goal is to rescue ocular defects in Porcn mutants in vitro by non-canonical Wnt activation.

Conclusions : Porcn is required for proper formation of the optic vesicle and differentiation into retina and RPE, possibly to regulate transition from the eye field to optic vesicle stage. We predict that this occurs via non-canonical Wnt signaling.

This is a 2021 ARVO Annual Meeting abstract.