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Our previous studies analyzing medaka mutants defective in primordial germ cell (PGC) migration identified cxcr4b and cxcr7, which are both receptors of the chemokine sdf1/cxcl12, as key regulators of PGC migration. Among PGC migration mutants, naruto (nar) is unique in that the mutant phenotype includes gross morphological abnormalities of embryos, suggesting that the mutation affects a broader range of processes. A fine genetic linkage mapping and genome sequencing showed the nar gene encodes Cleavage and Polyadenylation Specificity Factor subunit 6 (CPSF6/CFIm68). CPSF6 is a component of the Cleavage Factor Im complex (CFIm) which plays a key role in pre-mRNA 3'-cleavage and polyadenylation. 3'RACE of sdf1a/b and cxcr7 transcripts in the mutant embryos indicated shorter 3’UTRs with poly A additions occurring at more upstream positions than wild-type embryos, suggesting CPSF6 functions to prevent premature 3’UTR cleavage. In addition, expression of the coding region sequences of sdf1a/b in nar mutants was more anteriorly extended in somites than wild-type embryos, accounting for the abnormally extended distribution of PGCs in nar mutants. An expected consequence of shortening 3'UTR is the escape from the degradation mechanism mediated by microRNAs interacting with distal 3’UTR sequence. The abnormal expression pattern of sdf1a coding sequence may be at least partially accounted for by this mechanism. Given the pleiotropic effects of nar mutation, further analysis using the nar mutant will reveal processes in which CPSF6 plays essential regulatory roles in poly A site selection and involvement of 3'UTRs in posttranscriptional gene regulation in various genes in vivo. [ABSTRACT FROM AUTHOR]

Abstract: Cyp19 is expressed at a high level in the gonad of the female tadpole of the frog Rana rugosa during sex determination. To identify sequence elements important for expression of Cyp19, we isolated a genomic clone (∼40 kbp) carrying R. rugosa Cyp19 and analyzed the nucleotide sequence of the 5′-flanking region to search for potential transcription factor binding sites. Sox (SRY-related HMG box) protein and Sf1 binding sites were found in the ovary-specific promoter region of Cyp19. Because Sox3 is located on the sex chromosome in R. rugosa, we conducted the luciferase reporter assay in Xenopus A6 cells using the promoter region. Sox3 drove the reporter gene in the cells, but Sf1 did not. When sequential deletion of the 2.7 kbp Cyp19-promoter region was undertaken, a fragment spanning nucleotides −191 to +48 was sufficient to drive the transcription of the reporter gene. In site-directed mutagenesis, the binding site at −57 in the region was critical for Sox3 responsiveness. Sox3 lacking the HMG box had no ability to promote Cyp19 transcription. In addition, a chromatin immunoprecipitation (ChIP) assay showed that DNA fragments were enriched 8-fold, as determined by real-time PCR, when chromatin was immunoprecipitated with the anti-His antibody against His-tagged Sox3. The results, taken together, suggest that Sox3 activates Cyp19 transcription by its direct binding to the binding site of the Cyp19 promoter region. Sox3 appears to be a factor that directs indifferent gonads to develop into an ovary in R. rugosa. [Copyright &y& Elsevier]

Teleosts comprise more than half of all vertebrate species and have adapted to a variety of marine and freshwater habitats. Their genome evolution and diversification are important subjects for the understanding of vertebrate evolution. Although draft genome sequences of two pufferfishes have been published, analysis of more fish genomes is desirable. Here we report a high-quality draft genome sequence of a small egg-laying freshwater teleost, medaka (Oryzias latipes). Medaka is native to East Asia and an excellent model system for a wide range of biology, including ecotoxicology, carcinogenesis, sex determination and developmental genetics. In the assembled medaka genome (700 megabases), which is less than half of the zebrafish genome, we predicted 20,141 genes, including ∼2,900 new genes, using 5′-end serial analysis of gene expression tag information. We found single nucleotide polymorphisms (SNPs) at an average rate of 3.42% between the two inbred strains derived from two regional populations; this is the highest SNP rate seen in any vertebrate species. Analyses based on the dense SNP information show a strict genetic separation of 4 million years (Myr) between the two populations, and suggest that differential selective pressures acted on specific gene categories. Four-way comparisons with the human, pufferfish (Tetraodon), zebrafish and medaka genomes revealed that eight major interchromosomal rearrangements took place in a remarkably short period of ∼50 Myr after the whole-genome duplication event in the teleost ancestor and afterwards, intriguingly, the medaka genome preserved its ancestral karyotype for more than 300 Myr. [ABSTRACT FROM AUTHOR]

A cDNA clone encoding the soluble guanylyl cyclase α[sub2] subunit was isolated from medaka fish (Oryzias latipes) and designated as OIGCS-α[sub2]. The O/GCS-α[sub2]. cDNA was 3192 bp in length and the open reading frame (ORF) encodes a protein of 805 amino acids. The deduced amino acid sequence has high similarity to that of the mammalian α[sub2] subunit gene except for the N-terminal regulatory domain. The C-terminal 5 ammo acids, "RETSL". which have been reported to interact with the post synaptic density protein (PSD)-95 were conserved. An RNase protection assay with adult fish organs showed that OIGCS-α[sub2] was expressed mainly in the brain and testis. The complete nucleotide sequence (about 41 kbp) of the OlGCS-α[sub2] genomic DNA clone isolated from a medaka fish BAC library indicated that the OlGCS-α[sub2] gene consisted of 9 exons and 8 introns. The 5′-flanking region and larger introns, such as introns 1. 4, and 7, contained the several fragments conserved in the nucleotide sequences of Rex6 (non-long terminal repeat retrotransposon), MHC class I genomic region, and OIGC1. the medaka fish homolog of the mammalian guanylyl cyclase B gene. Linkage analysis on the medaka fish chromosome demonstrated that the OIGCS-α[sub2] gene was mapped to LG13; this mapping position was different from those for the OIGCS-α[sub1] and O/GCS-β[sub1], genes (LG1). [ABSTRACT FROM AUTHOR]

Abstract: MicroRNAs (miRNAs, miRs) are short noncoding RNA molecules that negatively control the target mRNAs by binding to the 3′ untranslated region (UTR). Previous studies have demonstrated that miR-430 is encoded by a clustered multigene family and is abundantly expressed in early development. In zebrafish, miR-430 is needed to suppress primordial germ cell (PGC)-specific genes, such as nanos1, in somatic cells. However, the molecular characteristics of the miR-430 family in other teleost species have not been reported, and it is unclear whether such a function of miR-430 in PGC specification is a conserved feature of animals or not. In medaka (Oryzias latipes), a distantly related teleost, it has been suggested that PGC might be established in a different mode of specification from that of zebrafish. We characterized 16 miR-430 precursors in the medaka genomic sequence. These miR-430 genes form clusters on chromosome 4, which might share its evolutionary origin with that of the very large miR-430 clusters in zebrafish chromosome 4. However, none of the medaka miR-430 genes are identical to the zebrafish miR-430 paralogs. Medaka miR-430 expression starts during epiboly and decreases after axis formation. Functional analysis using reporter gene constructs showed that miR-430 repressed protein expression by binding to the 3′UTR of zebrafish TDRD7. Consistently, the 3′UTR of medaka TDRD7 contains at least two significant candidates for the putative miR-430 binding site. The ubiquitous and early expression of medaka miR-430 and its ability to downregulate GFP:TDRD7 reporter mRNA imply that miR-430 has a conserved role in early embryogenesis. Smaller copy numbers of miR-430 genes and relatively brief expression in medaka might represent the characteristics of this miRNA family in the common ancestor of teleosts. Changes in the relationships between miR-430 and the target mRNA might be related to differences in the localization patterns of PGC-related genes in medaka and zebrafish. [Copyright &y& Elsevier]

At midblastula transition (MBT), zygotic gene transcription is activated, cells become motile and cell division becomes asynchronous. The onset of the medaka (Oryzias latipes) MBT was examined using expressed sequence tag (EST) markers. Among 187 randomly chosen medaka EST markers, 33 EST markers and two genes (eIF-4C and hsc70) showed polymorphisms in terms of insertion/deletions or restriction sites between the two parental inbred strains, one from the northern Japanese population and the other from the southern Japanese population. There was no evidence of zygotic expression of these EST markers before Stage 10 (early blastula stage), whereas expression of 12 genes was found from Stage 11 on. These results suggest that the medaka MBT in terms of first time transcription of paternal genes in the life of the embryo begins at Stage 11 (late blastula stage). [Copyright &y& Elsevier]

Summary: Teleosts have an asymmetrical caudal fin skeleton formed by the upward bending of the caudal-most portion of the body axis, the ural region []. This homocercal type of caudal fin ensures powerful and complex locomotion and is regarded as one of the most important innovations for teleosts during adaptive radiation in an aquatic environment []. However, the mechanisms that create asymmetric caudal fin remain largely unknown. The spontaneous medaka (teleost fish) mutant, Double anal fin (Da), exhibits a unique symmetrical caudal skeleton that resembles the diphycercal type seen in Polypterus and Coelacanth. We performed a detailed analysis of the Da mutant to obtain molecular insight into caudal fin morphogenesis. We first demonstrate that a large transposon, inserted into the enhancer region of the zic1 and zic4 genes (zic1/zic4) in Da, is associated with the mesoderm-specific loss of their transcription. We then show that zic1/zic4 are strongly expressed in the dorsal part of the ural mesenchyme and thereby induce asymmetric caudal fin development in wild-type embryos, whereas their expression is lost in Da. Comparative analysis further indicates that the dorsal mesoderm expression of zic1/zic4 is conserved in teleosts, highlighting the crucial role of zic1/zic4 in caudal fin morphogenesis. [ABSTRACT FROM AUTHOR]

Abstract: Immediate-early genes (IEGs) are useful for mapping active brain regions in various vertebrates. Here we identified a c-fos homologue gene in medaka and demonstrated that the amounts of c-fos transcripts and proteins in the medaka brain increased in relation to an artificially evoked seizure, suggesting that the homologue gene has the characteristics of IEGs, which are used as markers of neural activity. Next, quantitative reverse-transcription-polymerase chain reaction revealed that female mating behaviors upregulated c-fos transcription in some brain regions including the telencephalon, optic tectum, and cerebellum. In addition, we performed in situ hybridization with a c-fos intron probe to detect the de novo synthesis of c-fos transcripts and confirmed induction of c-fos transcription in these brain regions after mating. This is the first report of IEG induction in response to mating stimuli in teleost fish. Our results indicated that c-fos expression was induced in response to behavioral stimuli in the medaka brain and that medaka c-fos could be a useful marker of neural activity. [ABSTRACT FROM AUTHOR]