Sumoylated Pax6 Regulates Lens Stalk Regression During Lens Development

Abstract

Purpose : Pax6 is a master regulator governing development of both brain and eye. Our previous studies have shown that SUMO1-mediated sumoylation is an indispensable step towards activation of p32 Pax-6 (Yan et al. 2010. PNAS). However, how does the sumoylated Pax6 regulate lens development is largely unknown. Here, we present evidence to show how sumoylated Pax6 regulates lens stalk regression during lens development.

Methods : CRISPR/Cas9 technology is used to create the mouse model where the conserved amino acid, 90E was deleted so that the conserved sumoylation site was altered. RNAseq analysis was used to analyze the transcriptional activity of the mutated Pax6. In situ hybridization and QRT-PCR were used to confirm mRNA expression of Pax6 and other downstream genes in both wild type and mutant mice. Cycloheximide and MG132 treatments were used to determine the half life of wild type and mutant Pax6 protein. H.E. staining was used to record the lens morphology.

Results : Deletion of the conserved 90E amino acid caused significant changes in the sumoylation pattern of the mutant Pax6, which changes the stability of Pax6. The mutated pax6 displays fundamental changes in regulating expression of the downstream genes. The expression patterns of over 20 genes involved in control of apoptosis in the embryonic lens were altered, leading to inhibition of apoptosis in the lens stalk region of the mutant mouse. As a result, lens stalk regression was halted, causing microphthalmia.

Conclusions : Sumoylated Pax6 regulates apoptotic genes to control lens stalk regression. Supported by National Natural Science Foundation of China (Grants #81970787, #82000876, #81770910) and Natural Science Foundation of Guangdong Province and Guangdong City Joint Program of China (Grant # 2019B1515120014)，and the Fundamental Funds, 3030901010110 of the State Key Laboratory of Ophthalmology of Zhongshan Ophthalmic Center.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.