Embryonic expression of zebrafish b3glct genes.

(A) RT-PCR analysis of b3glct expression demonstrates robust expression of both b3glcta (left panel) and b3glctb (middle panel) at different stages of development in whole embryos as well as various embryonic tissues at 48-hpf (right panel). Controls included pitx2c as negative control for 0-hpf, rhodopsin as negative control for the lens, beta-actin as positive control for all tissues and H₂O as negative contamination control for all reactions. (B) In-situ hybridization analysis of b3glcta and b3glctb expression demonstrates broad expression in 24-120-hpf embryos with enrichment in the developing eyes, fins, brain, craniofacial region and somites. aer–apical ectodermal ridge, ase–anterior segment of the eye, b–brain, cmz–ciliary marginal zone, crc–craniofacial cartilage, e–eye, f–fins, h–heart, le–lens, sm–skeletal muscles.

Genetic disruption of b3glct.

(A) Schematic of b3glct genes indicating TALEN target sites (exon 1 and 12 for b3glcta and exon 12 for b3glctb, black arrows). The predicted protein product resulting from TALEN mediated disruption is shown. Editing events in the first exon of b3glcta are predicted to disrupt nearly the entire coding region of the transcript. For both b3glcta and b3glctb, editing in the 12^th exon is predicted to result in loss of most of the catalytic domain including the catalytic core and KDEL-like ER retention signal. Blue (SP)- Signal Peptide, Green (SR)- Stem Region, Orange (CD)- Catalytic Domain. (B) Images of zebrafish embryos at 5-dpf and adult zebrafish showing no gross morphological defects associated with loss of b3glct. (C) Functional evaluation of wild-type and mutant b3glct by in vitro β3-glucosyltransferase assays. Left panel- the endogenous β3-glucosyltransferase activity toward O-fucosylated TSR3 is dependent on the amount of protein in the wild type zebrafish homogenate; control reaction with 10 μg homogenate was performed using unmodified TSR3. Right panel- the endogenous β3-glucosyltransferase activity toward O-fucosylated TSR3 in the homogenate of double homozygous b3glct embryos is profoundly reduced compared with that in the wild type zebrafish homogenate; control reactions were performed using unmodified TSR3. Assays were performed in triplicate. Error bars indicate s.d.

In situ hybridization with sense probes for b3glcta and b3glctb.