Using a Zebrafish Animal Model to Identify the First Candidate Gene for Pigmentary Glaucoma

Abstract

Premelanosome protein (PMEL) was identified as a candidate gene for the development of pigment dispersion syndrome (PDS) and pigmentary glaucoma (PG) in humans. Mutations in PMEL were shown to cause pigment and ocular defects in several animals but there is currently no known PMEL associated defects in humans. We hypothesize that mutations in PMEL are associated with PG/PDS. To elucidate the pathology of PG/PDS, we analyzed the impact that mutations in a zebrafish homolog of PMEL (pmela) have on the structure and function of the zebrafish eye. Zebrafish are an excellent animal model for studying an ocular disease because of its genetic tractability and the zebrafish’s similar ocular structure to the human eye.We deployed morpholinos (MOs) and CRISPR/Cas9 to generate transient pmela knockdowns and pmela knockout zebrafish, respectively, to assess the requirement for PMEL in early development and ocular maintenance. We assayed ocular pigmentation, ocular structure, and anterior segment structure.MOs targeted at a PMEL paralog in zebrafish (pmela) significantly reduced global pigmentation and ocular pigmentation. The disruption of pmela by CRISPR/Cas9 created two stable alleles. One allele, ua5022, when homozygous, caused significant global pigment reduction, enlarged anterior segments, microphthalmia, and a change in eye shape. The mRNA transcripts of pmela in the ua5022 homozygotes were reduced by 20-fold compared to wild type fish.Using zebrafish as an animal model to ascertain the role of PMEL in the etiology of PG/PDS will help elucidate the mechanisms of the disease, leading to novel diagnosis and treatment avenues.