Hiroshi Hori

Polycystic Kidney Disease in the Medaka (Oryzias latipes) pc Mutant Caused by a Mutation in the Gli-Similar3 (glis3) Gene

Polycystic kidney disease (PKD) is a common hereditary disease in humans. Recent studies have shown an increasing number of ciliary genes that are involved in the pathogenesis of PKD. In this study, the Gli-similar3 (glis3) gene was identified as the causal gene of the medaka pc mutant, a model of PKD. In the pc mutant, a transposon was found to be inserted into the fourth intron of the pc/glis3 gene, causing aberrant splicing of the pc/glis3 mRNA and thus a putatively truncated protein with a defective zinc finger domain. pc/glis3 mRNA is expressed in the epithelial cells of the renal tubules and ducts of the pronephros and mesonephros, and also in the pancreas. Antisense oligonucleotide-mediated knockdown of pc/glis3 resulted in cyst formation in the pronephric tubules of medaka fry. Although three other glis family members, glis1a, glis1b and glis2, were found in the medaka genome, none were expressed in the embryonic or larval kidney. In the pc mutant, the urine flow rate in the pronephros was significantly reduced, which was considered to be a direct cause of renal cyst formation. The cilia on the surface of the renal tubular epithelium were significantly shorter in the pc mutant than in wild-type, suggesting that shortened cilia resulted in a decrease in driving force and, in turn, a reduction in urine flow rate. Most importantly, EGFP-tagged pc/glis3 protein localized in primary cilia as well as in the nucleus when expressed in mouse renal epithelial cells, indicating a strong connection between pc/glis3 and ciliary function. Unlike human patients with GLIS3 mutations, the medaka pc mutant shows none of the symptoms of a pancreatic phenotype, such as impaired insulin expression and/or diabetes, suggesting that the pc mutant may be suitable for use as a kidney-specific model for human GLIS3 patients.

Targeted reduction of the DNA methylation level with 5-azacytidine promotes excision of the medaka fish Tol2 transposable element.

The Tol2 element of the medaka fish Oryzias latipes is a member of the hAT (hobo/Activator/Tam3) transposable element family. There is evidence for rapid expansion in the genome and throughout the species in the past but a high spontaneous transposition rate is not observed with current fish materials, suggesting that the Tol2 element and its host species have already acquired an interactive mechanism to control the transposition frequency. DNA methylation is a possible contributing factor, given its involvement with many other transposable elements. We therefore soaked embryos in 5-azacytidine, a reagent that causes reduction in the DNA methylation level, and examined amounts of PCR products reflecting the somatic excision frequency, obtaining direct evidence that exposure promotes Tol2 excision. Our results thus suggest that methylation of the genome DNA is a factor included in the putative mechanisms of control of transposition of the Tol2 element.

Duplicated Abd-B class genes in medaka hoxAa and hoxAb clusters exhibit differential expression patterns in pectoral fin buds

Hox genes form clusters. Invertebrates and Amphioxus have only one hox cluster, but in vertebrates, they are multiple, i.e., four in the basal teleost fish Polyodon and tetrapods (HoxA, B, C, D), but seven or eight in common teleosts. We earlier completely sequenced the entire hox gene loci in medaka fish, showing a total of 46 hox genes to be encoded in seven clusters (hoxAa, Ab, Ba, Bb, Ca, Da, Db). Among them, hoxAa, hoxAb and hoxDa clusters are presumed to be important for fin-to-limb evolution because of their key role in forelimb and pectoral fin development. In the present study, we compared genome organization and nucleotide sequences of the hoxAa and hoxAb clusters to these of tetrapod HoxA clusters, and found greater similarity in hoxAa case. We then analyzed expression of Abd-B family genes in the clusters. In the trunk, those from the hoxAa cluster, i.e., hoxA9a, hoxA10a, hoxA11a and hoxA13a, were expressed in a manner keeping the colinearity rule of the hox expression as those of tetrapods, while those from the hoxAb cluster, i.e., hoxA9b, hoxA10b, hoxA11b and hoxA13b, were not. In the pectoral fins, the hoxAa cluster was expressed in split domains and did not obey the rule. By contrast, those from the hoxAb and hoxDa clusters were expressed in a manner keeping the rule, i.e., an ancestral pattern similar to those of tetrapods. It is plausible that this differential expression of the two clusters is caused by changes occurred in global control regions after cluster duplications.

Comparative genomics of medaka and fugu.

A small freshwater fish medaka (Oryzias latipes) has been one of the most attractive experimental systems for research in genetics and developmental biology. We have formed an international consortium Medaka Genome Initiative (MGI) to collect and share various information and resources on medaka. The MGI has set an ambitious goal aiming at the complete sequencing of the medaka genome and as a feasibility study we have begun sequencing one particular chromosome, linkage group 22 (LG22) of approximately 22 Mb in size. Initial sequence analysis revealed unique features of the medaka genome in comparison to fugu genome.