Abstract
Purpose:
The olfm1 gene encodes a secreted glycoprotein highly conserved in vertebrates. There are two olfm1 genes in zebrafish, olfm1a and olfm1b. Both of these genes are expressed in the brain and retina starting from 16 h post fertilization to adults. We generated a null mutant of both olfm1a and olfm1b genes and analyzed its retinal structure and visual function.
Methods:
Olfm1a and olfm1b mutant alleles with nonsense point mutations were obtained from the Wellcome Trust Sanger Institute. Olfm1a and olfm1b null mutants were bred to generate double null mutant (olfm1a/b null). Spontaneous movement, optokinetic and optomotor responses, behavioral responses to light increments and decrements and electroretinogram (ERG) to increased (ON) and decreased (OFF) illumination were compared between olfm1a/b null and wild-type larvae 7 days post fertilization (dpf). The retinal morphology was examined by immunostaining of frozen sections. Total RNA was isolated from 3 and 7 dpf larvae for RNA sequencing analysis.
Results:
Body shape, behavior and fertility of adult olfm1a/b null fish appeared to be normal. At 7 dpf, the thickness of the retinal ganglion cell and inner plexiform layers was reduced while the outer nuclear layer was thicker in olfm1a/b null retina as compared with wild-type. The size of other retinal layers was similar in olfm1a/b null and wild-type larvae. The optomotor response of olfm1a/b null larva to OFF stimulation was normal. However, the optokinetic response and behavioral responses to light increments was significantly reduced in olfm1a/b null as compared with wild-type larvae. The ERG response of olfm1a/b null larvae was slightly reduced in both ON and OFF stimulation conditions as compared with wild-type. RNAseq analysis of 3 dpf larvae showed reductions in expression of genes encoding some transcription factors (pax6), calcium channels (cacna1a), AMPA receptors, the MAP kinase pathway, and genes involved in axon growth (neurotrophic factor receptors and neurofilaments) for olfm1a/b null larvae compared with wild-type. Many of these changes in the gene expression levels were recovered to the normal levels at 7 dpf. Nevertheless, down-regulation of indicated genes at early developmental stages may contribute to the observed defects in olfm1a/b null retina.
Conclusions:
Olfm1a and olfm1b genes are involved in retinal development and visual functions in zebrafish.
Keywords: 533 gene/expression •
753 vision and action •
698 retinal development