Reduction in N-Acetylglucosaminyltransferase-I Activity Decreases Survivability and Delays Development of Zebrafish.

ABSTRACT

A lack of complex and hybrid types of N-glycans in mice is embryonically lethal due to neural tube maldevelopment. N-acetylglucosaminyltransferase-I (GnT-I; Mgat1) catalyzes a required step for converting oligomannose N-glycans into hybrid and complex N-glycans. Unlike mice, zebrafish have two Mgat1a/b genes. Herein, CRISPR/Cas9 technology was used to knockdown GnT-Ib activity in zebrafish, referred to as Mgat1b-/-, to examine the impact of a decrease in complex types of N-glycans on survival and development, and sensory and motor functions. Genotyping verified the occurrence of edited Mgat1b, and LC-ESI-MS and lectin blotting identified higher levels of oligomannose and lower levels of complex N-glycans in Mgat1b-/- relative to Wt AB. The microscopic visualization of developmental stages and locomotor studies using an automated tracking unit and manual touch assays revealed reduced survivability, and delayed motor and sensory functions in Mgat1b-/-. Moreover, embryonic staging linked reduced survivability of Mgat1b-/- to disruption in brain anlagen formation. Birefringence measurements supported delayed skeletal muscle development, which corresponded with motor and sensory function impediments in Mgat1b-/-. Furthermore, GnT-Ib knockdown hindered cardiac activity onset. Collectively, Mgat1b-/- displayed incomplete penetrance and variable expressivity, such that some died in early embryonic development, while others survived to adulthood, albeit, with developmental delays. Thus, the results reveal that reducing the amount of complex-type N-glycans is unfavorable for zebrafish survival and development. Moreover, our results support a better understanding of human congenital disorders of glycosylation.