Olfactomedin1 Gene Expression and the Functional Analysis in Developing Zebrafish Embryos

Purpose: Olfactomedin1 (Olfm1/Noelin/Pancortin) is a secreted glycoprotein belonging to the family of olfactomedin domain-containing proteins. It is implicated in neurogenesis in Xenopus and generation of the neural crest in chicken. This gene is actively expressed in the developing and adult retina. Here we investigate the functions of Olfm1 in zebrafish development. Methods: Expression of Olfm1 was analyzed by wholemount in situ hybridization. Injection of morpholino oligonucleotides against Olfm1 (Olfm1-MO) were used to knock-down Olfm1 gene expression. Quantitative RT-PCR was used to evaluate changes in the gene expression levels. Results: Two orthologs of mouse Olfm1 located on chromosomes 5 and 24 were identified in the zebrafish genome by a blast search and were named Olfm1a and Olfm1b, respectively. Similar to the mouse Olfm1 gene, two alternative 5'-exons were predicted for transcripts from each zebrafish gene. TR1 and TR2 transcripts of the Olfm1a gene were detected from 12 hours after fertilization (hpf); TR3 and TR4 transcripts of the Olfm1b gene were detected from 20 and 14 hpf, respectively. In the course of development, the Olfm1a and Olfm1b genes were expressed in neurons in the telencephalon, dorsal-posterior retina, trigeminal ganglion, and dorsal and ventral neural tube. The expression corresponded to that of Islet1/2, suggesting that Olfm1 was expressed in partially differentiated neuronal cells. Olfm1-MO injections caused damage to the brain and eyes, reduced pigmentation, distorted body axis, expanded heart, shorter body length and cell dissociations from the head and dorsal part of the body. The effects of Olfm1-MO were dose-dependent and partially rescued by the co-injection with mouse Olfm1 RNA. Expressions of E-cadherin (Cdh1) mRNA and Cdh1 protein were reduced in embryos treated with Olfm1-MO. Expression of Snail1, a major repressor of Cdh1 mRNA expression in mammals, was increased at 10 hpf prior to Cdh1 down-regulation by Olfm1-MO. Expression of a transcriptional activator of the Cdh1 gene, microphilia-associated transcription factor, Mitf-a, was dramatically reduced at 20-30 hpf by Olfm1-MO. Furthermore, preliminary analysis using zebrafish 22K Agilent array showed 150 up-regulated genes and 58 down-regulated genes in Olfm1-MO treated embryos. <truncated: Gene Tools database size restriction>