Shared morphological and molecular pathways in microphthalmia using patient-derived iPSC-optic vesicles

Abstract

Purpose : Eye morphogenesis is tightly regulated by a highly conserved genetic network that when disrupted, can result in severe ocular malformation on a spectrum known as microphthalmia, anophthalmia and coloboma (MAC). While over 90 genes are associated with MAC, little is known about disease mechanisms shared between patients with differing genetic diagnoses. This study aimed to identify aberrations to pathways involved in early eye development shared between different causes of microphthalmia. Due to low genetic diagnostic rates, studying shared pathways will critically reveal new candidate genes for future diagnostics.

Methods : Patient-specific (PAX6-associated microphthalmia p.(Asn124Lys) and an unsolved microphthalmia patient) and healthy control iPSC-derived optic vesicles were generated using two clones from each line to model ocular development until day 35. Data on gene and protein expression were generated through RNA-seq, qRT-PCR and immunohistochemistry. Apoptosis, cell proliferation and vesicle diameter were quantified using ImageJ.

Results : At day 35, vesicle diameter was significantly reduced in both microphthalmia models compared to healthy controls. TUNEL staining revealed a significant increase in apoptotic cells in patient vesicles compared to healthy controls while PH3 staining revealed decreased cell proliferation. Using RNA-seq, we detected upregulation of pro-apoptotic genes in microphthalmia optic vesicles. Dysregulation of Notch signalling modulated through an ASCL1 negative feedback loop between the DLL ligand and HES effector families was also common between microphthalmia samples. This resulted in differential expression of Notch target genes such as MITF and HDAC1. Additionally, the increased production of extracellular matrix (ECM) components such as collagen and laminin, resulting in ECM abnormalities, was detected in microphthalmia optic vesicles.

Conclusions : Our study identified reduced size and proliferation, and increased apoptosis in microphthalmia patient-derived optic vesicles possibly contributing to the small eye phenotype. Disruptions to Notch signalling may contribute to a global increase of ECM production and further dysregulation of key early ocular developmental genes. Additionally, abnormally high production of ECM in microphthalmia patients may overly restrict optic vesicle growth, resulting in ocular malformations.

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