ELOVL4 Expression and Morpholino Knock-Down in Zebrafish

Purpose: ELOVL4 has been linked to early onset autosomal dominant macular degeneration. The Zebrafish genome contains a gene encoding an ortholog of the human ELOVL4, zfElovl4. The goals of this project are to determine the zfElovl4 expression patterns in the developing zebrafish and monitor the developmental consequences of zfElovl4 knockdown using morpholino injections. Methods: The expression of zfElovl4 mRNA was investigated using in-situ hybridization with a zfElovl4 probe, followed by an anti-Dig Fab coupled to alkaline phosphatase. zfElovl4 protein expression was monitored by the anti ELOVL4 E60 antibody. Morpholinos were made corresponding to zfElolv4 cDNA sequence immediately upstream from the start codon (MO2, as a negative control) and immediately downstream from the start codon (MO1, translation blocking morpholino), as well as the exon1-intron splice site (MOsp3) by Gene Tool, LLC, Philomath, OR. Results: zfElovl4 expression is found throughout the brain from early stages in development (24hpf) until at least day 5 of embryonic development. Areas around the eye, the brain, and the otic vesicle seem to have the highest amount of expression. This is quite similar to expression levels reported in mouse studies. Morpholino knockdown of ELOVL4 has resulted in a variety of phenotypes. The most noticeable is an invagination of the developing retina. High morpholino injection concentrations (>5ng/nl) appear to be lethal, analogous to ELOVL4 homozygous knock-out mice. Conclusions: We have characterized the expression and function of zfElovl4. Zebrafish may provide a good model organism for studying and characterizing the developmental consequences related to ELOVL4, including the retinal degeneration phenotype.