Knockout of myoc Provides Evidence for the Role of Myocilin in Zebrafish Sex Determination Associated with Wnt Signalling Downregulation

SIMPLE SUMMARY: Myocilin is a protein with an incompletely understood function, mainly known because of its role in glaucoma. In this study we have analysed the normal role of this protein in vivo. To that end, we generated the first myocilin knockout zebrafish line reported to date. This zebrafish line did not show any apparent gross morphological anomaly, but unexpectedly, we observed that all knockout animals were males. Detailed analyses revealed the existence of apoptosis in the immature juvenile gonad, which is associated with male differentiation. Moreover, we demonstrate that adult knockout differentially expressed key genes involved both in male sex determination and the Wnt signalling pathway, which also plays a role in zebrafish gonad differentiation. Altogether, these results indicate that myocilin is a novel key protein involved in sex determination in zebrafish. ABSTRACT: Myocilin is a secreted glycoprotein with a poorly understood biological function and it is mainly known as the first glaucoma gene. To explore the normal role of this protein in vivo we developed a myoc knockout (KO) zebrafish line using CRISPR/Cas9 genome editing. This line carries a homozygous variant (c.236_239delinsAAAGGGGAAGGGGA) that is predicted to result in a loss-of-function of the protein because of a premature termination codon p.(V75EfsX60) that resulted in a significant reduction of myoc mRNA levels. Immunohistochemistry showed the presence of myocilin in wild-type embryonic (96 h post-fertilization) anterior segment eye structures and caudal muscles. The protein was also detected in different adult ocular and non-ocular tissues. No gross macroscopic or microscopic alterations were identified in the KO zebrafish, but, remarkably, we observed absence of females among the adult KO animals and apoptosis in the immature juvenile gonad (28 dpf) of these animals, which is characteristic of male development. Transcriptomic analysis showed that adult KO males overexpressed key genes involved in male sex determination and presented differentially expressed Wnt signalling genes. These results show that myocilin is required for ovary differentiation in zebrafish and provides in vivo support for the role of myocilin as a Wnt signalling pathway modulator. In summary, this myoc KO zebrafish line can be useful to investigate the elusive function of this protein, and it provides evidence for the unexpected function of myocilin as a key factor in zebrafish sex determination.