Knockdown of best1 Gene in Zebrafish Caused Abnormal Neuronal and Skeletal Development -A Subtype of Craniovertebral Junction Malformation

Objective: To investigate the developmental defects caused by knockdown of best1 gene in zebrafish as a model for a subtype of craniovertebral junction (CVJ) malformation. Methods: Two antisense morpholinos were designed targeting zebrafish best1 to block translation (ATG-MO) or to disrupt splicing (I3E4-MO). Morpholinos were microinjected into fertilized one-cell embryos. Efficacy of splicing morpholino was confirmed by RT-PCR. Phenotypes were analyzed and quantified by microscopy at multiple developmental stages. Neuronal outgrowth was assessed in transgenic zebrafish expressing GFP in neurons. Skeletal ossification was visualized by calcein staining. Results: Knockdown of best1 resulted in zebrafish embryos with shorter body length, curved axis, low survival rate, microcephaly, reduced eye size, smaller head and brain, impaired neuronal outgrowth, and reduced ossification of craniofacial and vertebral bone. Conclusion: Best1 gene plays critical roles in ophthalmologic, neurological and skeletal development in zebrafish. A patient with a premature stop codon in BEST1 gene exhibited simillar phenotypes, implying a subtype of CVJ malformation.