Richard Bartfai

Genetic analysis of two common carp broodstocks by RAPD and microsatellite markers

The whole broodstock of two Hungarian common carp farms—80 and 196 individuals—was analyzed by using random amplified polymorphic DNA (RAPD) assay and microsatellite analysis. Ten polymorphic RAPD markers and four microsatellites were selected to genotype both of the stocks. As expected, microsatellite analysis revealed more detailed information on genetic diversities than RAPD assay. Results obtained with both types of DNA markers showed lack of major differences between the genetic structure of the two stocks: heterozygosity values and allele frequencies were very similar. Dendrograms created from both sets of data did not show grouping of individuals according to stocks. Genotypes from the two stocks were also compared to those from a limited number of samples collected from other hatcheries and two rivers. Allele frequencies in the groups were similar, with the exception of wild carps. An interesting observation was that three private microsatellite alleles were found in the eight wild carp individuals, compared to the seven detected in the rest of the samples tested (372 individuals).

Transcriptomic Analyses Reveal Novel Genes with Sexually Dimorphic Expression in the Zebrafish Gonad and Brain

Background Our knowledge on zebrafish reproduction is very limited. We generated a gonad-derived cDNA microarray from zebrafish and used it to analyze large-scale gene expression profiles in adult gonads and other organs. Methodology/Principal Findings We have identified 116638 gonad-derived zebrafish expressed sequence tags (ESTs), 21% of which were isolated in our lab. Following in silico normalization, we constructed a gonad-derived microarray comprising 6370 unique, full-length cDNAs from differentiating and adult gonads. Labeled targets from adult gonad, brain, kidney and ‘rest-of-body’ from both sexes were hybridized onto the microarray. Our analyses revealed 1366, 881 and 656 differentially expressed transcripts (34.7% novel) that showed highest expression in ovary, testis and both gonads respectively. Hierarchical clustering showed correlation of the two gonadal transcriptomes and their similarities to those of the brains. In addition, we have identified 276 genes showing sexually dimorphic expression both between the brains and between the gonads. By in situ hybridization, we showed that the gonadal transcripts with the strongest array signal intensities were germline-expressed. We found that five members of the GTP-binding septin gene family, from which only one member (septin 4) has previously been implicated in reproduction in mice, were all strongly expressed in the gonads. Conclusions/Significance We have generated a gonad-derived zebrafish cDNA microarray and demonstrated its usefulness in identifying genes with sexually dimorphic co-expression in both the gonads and the brains. We have also provided the first evidence of large-scale differential gene expression between female and male brains of a teleost. Our microarray would be useful for studying gonad development, differentiation and function not only in zebrafish but also in related teleosts via cross-species hybridizations. Since several genes have been shown to play similar roles in gonadogenesis in zebrafish and other vertebrates, our array may even provide information on genetic disorders affecting gonadal phenotypes and fertility in mammals.

Male-specific DNA markers from African catfish (Clarias gariepinus)

We searched for sex-specific DNA sequences in the male and female genomes of African catfish, Clarias gariepinus (Burchell, 1822) by comparative random amplified polymorphic DNA (RAPD) assays performed on pooled DNA samples. Two sex-linked RAPD markers were identified from the male DNA pool and confirmed on individual samples, showing good agreement with phenotypic sex. Both markers were isolated, cloned and characterized. The first marker (CgaY1) was nearly 2.6 kb long, while the length of second one (CgaY2) was 458 bp. Southern blot analysis with a CgaY1 probe showed strong hybridizing fragments only in males and not in females under stringent conditions, indicating the presence of multiple copies of CgaY1 in the male genome. When tested by zoo blot on the genomes of two closely related species from the Clariidae family, CgaY1 hybridized to the DNA of Heterobranchus longifilis and generated a faint male-specific band at low stringency. CgaY2 produced similar hybridization pattern in both sexes of C. gariepinus, C. macrocephalus and H. longifilis. Specific primers were designed to the sequences and the markers were amplified in multiplex PCR reactions together with a control band common to all individuals. This allowed for rapid, molecular sexing of the species on the basis of a simple three band (male) versus one band (female) pattern. According to our knowledge these are the first sex-specific DNA markers isolated from a siluroid fish species.

TBP2, a Vertebrate-Specific Member of the TBP Family, Is Required in Embryonic Development of Zebrafish

TATA binding protein (TBP) is a key regulator of RNA polymerase transcription. It binds to core promoters, often in large multiprotein complexes, and nucleates RNA polymerase II (Pol II) transcription initiation. In addition to the previously described TBP-like factor present in metazoans (TLF/TRF2/TRP/TLP), we describe a third, vertebrate-specific member of the TBP protein family from zebrafish, called TBP2. Evolutionary conserved TBP2 homologs were also found in human, mouse, frog, and pufferfish. The N-terminal domains of TBP2s are divergent amongst themselves and different from those of TBPs; however, the core domain of TBP2s and TBPs are almost identical. TBP2 binds the TATA box, interacts with TFIIA and TFIIB (similarly to TBP), and can mediate Pol II transcription initiation. However, TBP2 shows contrasting expression patterns in the gonads and during embryonic development in comparison to TBP, suggesting differential function. Knockdown of zebrafish TBP2 results in specific reduction of the protein level, leading to a phenotype, which indicates the requirement of TBP2 for embryonic patterning. The presence of three different TBP family members in vertebrates suggests the existence of developmental stage- and tissue-specific preinitiation complexes with specific requirements for different TBP family members.

Gonad Differentiation in Zebrafish Is Regulated by the Canonical Wnt Signaling Pathway

ABSTRACT Zebrafish males undergo a “juvenile ovary-to-testis” gonadal transformation process. Several genes, including nuclear receptor subfamily 5, group A (nr5a) and anti-Müllerian hormone (amh), and pathways such as Tp53-mediated germ-cell apoptosis have been implicated in zebrafish testis formation. However, our knowledge of the regulation of this complex process is incomplete, and much remains to be investigated about the molecular pathways and network of genes that control it. Using a microarray-based analysis of transforming zebrafish male gonads, we demonstrated that their transcriptomes undergo transition from an ovary-like pattern to an ovotestis to a testis-like profile. Microarray results also validated the previous histological and immunohistochemical observation that there is high variation in the duration and extent of commitment to the juvenile ovary phase among individuals. Interestingly, global gene expression profiling of diverging zebrafish juvenile ovaries and transforming ovotestes revealed that some members of the canonical Wnt/beta-catenin signaling pathway were differentially expressed between these two phases. To investigate whether Wnt/beta-catenin signaling plays a role in zebrafish gonad differentiation, we used the Tg (hsp70l:dkk1b-GFP)w32 line to inhibit Wnt/beta-catenin signaling during gonad differentiation. Activation of dkk1b-GFP expression by heat shock resulted in an increased proportion of males and corresponding decrease in gonadal aromatase gene (cyp19a1a) expression. The Wnt target gene, lymphocyte enhancer binding factor 1 (lef1), was also down-regulated in the process. Together, these results provide the first functional evidence that, similarly to mammals, Wnt/beta-catenin signaling is a “pro-female” pathway that regulates gonad differentiation in zebrafish.

Comparative analysis of the testis and ovary transcriptomes in zebrafish by combining experimental and computational tools.

Studies on the zebrafish model have contributed to our understanding of several important developmental processes, especially those that can be easily studied in the embryo. However, our knowledge on late events such as gonad differentiation in the zebrafish is still limited. Here we provide an analysis on the gene sets expressed in the adult zebrafish testis and ovary in an attempt to identify genes with potential role in (zebra)fish gonad development and function. We produced 10 533 expressed sequence tags (ESTs) from zebrafish testis or ovary and downloaded an additional 23 642 gonad-derived sequences from the zebrafish EST database. We clustered these sequences together with over 13 000 kidney-derived zebrafish ESTs to study partial transcriptomes for these three organs. We searched for genes with gonad-specific expression by screening macroarrays containing at least 2600 unique cDNA inserts with testis-, ovary- and kidney-derived cDNA probes. Clones hybridizing to only one of the two gonad probes were selected, and subsequently screened with computational tools to identify 72 genes with potentially testis-specific and 97 genes with potentially ovary-specific expression, respectively. PCR-amplification confirmed gonad-specificity for 21 of the 45 clones tested (all without known function). Our study, which involves over 47 000 EST sequences and specialized cDNA arrays, is the first analysis of adult organ transcriptomes of zebrafish at such a scale. The study of genes expressed in adult zebrafish testis and ovary will provide useful information on regulation of gene expression in teleost gonads and might also contribute to our understanding of the development and differentiation of reproductive organs in vertebrates.