Abstract
CYP1B1 loss of function (LoF) is the main known genetic alteration present in recessive primary congenital glaucoma (PCG), an infrequent disease characterized by delayed embryonic development of the ocular iridocorneal angle; however, the underlying molecular mechanisms are poorly understood. To model CYP1B1 LoF underlying PCG, we developed a cyp1b1 knockout (KO) zebrafish line using CRISPR/Cas9 genome editing. This line carries the c.535_667del frameshift mutation that results in the 72% mRNA reduction with the residual mRNA predicted to produce an inactive truncated protein (p.(His179Glyfs*6)). Microphthalmia and jaw maldevelopment were observed in 23% of F0 somatic mosaic mutant larvae (144 hpf). These early phenotypes were not detected in cyp1b1-KO F3 larvae (144 hpf), but 27% of adult (four months) zebrafish exhibited uni- or bilateral craniofacial alterations, indicating the existence of incomplete penetrance and variable expressivity. These phenotypes increased to 86% in the adult offspring of inbred progenitors with craniofacial defects. No glaucoma-related phenotypes were observed in cyp1b1 mutants. Transcriptomic analyses of the offspring (seven dpf) of cyp1b1-KO progenitors with adult-onset craniofacial defects revealed functionally enriched differentially expressed genes related to extracellular matrix and cell adhesion, cell growth and proliferation, lipid metabolism (retinoids, steroids and fatty acids and oxidation-reduction processes that include several cytochrome P450 genes) and inflammation. In summary, this study shows the complexity of the phenotypes and molecular pathways associated with cyp1b1 LoF, with species dependency, and provides evidence for the dysregulation of extracellular matrix gene expression as one of the mechanisms underlying the pathogenicity associated with cyp1b1 disruption.