Generation and classification of the Mgat1b^−/− mutant line. Depiction of N-glycan types and the sequential processing initiated via GnT-I (Mgat1) from the simplest N-glycan type (oligomannose) to more processed hybrid and complex types. The area encased within the dashed red box indicates the common pentasaccharide core (A). Products of BclI digest, which yield either homozygous Mgat1b^−/−, heterozygous Mgat1b^+/−, or Wt AB (Mgat1b^+/+). Number adjacent to Kb ladder (500 bp) indicates band size (B). Chromatogram with coding sequence of a fragment of Mgat1b from a Wt AB fish compared to that of Mgat1b^−/−, revealing five nucleotide deletions (Δ5), which generated a premature stop codon (encased in red box) (C).

Comparison of LC-ESI-MS relative intensities of the permethylated N-glycans derived from Wt AB and the Mgat1b^−/− mutant line. LC-ESI-MS intensities of released N-glycans from Wt AB (blue bars) and Mgat1b^−/− (grey bars) (A). All molecular ions are present in sodiated form ([M + Na]+). N-glycan numbers correspond to Figures S1 and S2, and Tables S2 and S3. Breakdown of the percent of various detected N-glycan types (B) and types of oligomannose structures (C) identified by the ESI-MS spectra.

Confirmation of decreased Mgat1b expression, and an increase in oligomannose N-glycans in mutant strain of adult and larvae fish. Lectin blots of whole cell lysates of adult brain (A), and larvae at 24 dpf and 6 dpf from Wt AB and Mgat1b^−/− mutant zebrafish (B). Separated proteins were probed with Phaseolus vulgaris Erthroagglutinin (E-PHA) or Galanthus nivalis lectin (GNL). Coomassie blue (CB)-stained gel demonstrating equal protein loads among the samples. Lines adjacent to the blots denote protein markers (in KDa) 250, 150, 100, 75, 50, 37. Bar graphs represent Mgat1b mRNA levels from Wt AB and Mgat1b^−/− mutant zebrafish relative to housekeeping genes β-actin (C) and ELFα (Figure S1). Data are presented as mean ± SEM, n ≥ 3 and were all compared using Student’s t-test (** p < 0.005, * p < 0.02).

Decline in survivability of Mgat1b^−/− mutants post 75% epiboly relative to Wt AB. Survivability of Wt AB and Mgat1b^−/− zebrafish tracked up to 48 hpf (A). Percent of viable embryos at the various stages of embryogenesis (B). Data are presented as mean ± SEM, n ≥ 8 clutches, and were all compared using Student’s t-test (* p < 0.0001).

Inhibition of motor and sensory functions in Mgat1b^−/− mutants. Touch-evoked response of Wt AB (upper panels) and Mgat1b^−/− (lower panels) at 2 and 3 dpf, as indicated (A). Data are presented as mean ± SEM, n ≥ 90 larvae. Distance swam in response to vibrational stimuli exhibited in 10 dpf larvae, n ≥ 45 larvae (B). Spontaneous locomotor swimming distance recorded in 5–29 dpf larvae, n ≥ 6 fish (C). Data are presented as mean ± SEM and were compared using Student’s t-test (** p < 0.005, * p < 0.05).

Maldevelopment of skeletal and cardiac muscle in Mgat1b^−/− mutants. Normalized birefringence of Wt AB (n = 26, 31, 22 as 58 hpf, 3 dpf, and 4 dpf) and Mgat1b^−/− (n = 30, 25, and 22, at 58 hpf, 3 dpf, and 4 dpf); n denotes number of examined embryos or larvae (A). Percent of Wt AB and Mgat1b^−/− zebrafish with detected heartbeat at 25 hpf, n = 5 and n = 7 for Wt AB and Mgat1b^−/−, respectively (B). Data are presented as mean ± SEM and were compared using Student’s t-test (* p < 0.003).