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Cell culture and protein target-based compound screening strategies, though broadly utilized in selecting candidate compounds, often fail to eliminate candidate compounds with non-target effects and/or safety concerns until late in the drug developmental process. Phenotype screening using intact research animals is attractive because it can help identify small molecule candidate compounds that have a high probability of proceeding to clinical use. Most FDA approved, first-in-class small molecules were identified from phenotypic screening. However, phenotypic screening using rodent models is labor intensive, low-throughput, and very expensive. As a novel alternative for small molecule screening, we have been developing gene expression disease profiles, termed the Transcriptional Disease Signature (TDS), as readout of small molecule screens for therapeutic molecules. In this concept, compounds that can reverse, or otherwise affect known disease-associated gene expression patterns in whole animals may be rapidly identified for more detailed downstream direct testing of their efficacy and mode of action. To establish proof of concept for this screening strategy, we employed a transgenic strain of a small aquarium fish, medaka (Oryzias latipes), that overexpresses the malignant melanoma driver gene xmrk, a mutant egfr gene, that is driven by a pigment cell-specific mitf promoter. In this model, melanoma develops with 100% penetrance. Using the transgenic medaka malignant melanoma model, we established a screening system that employs the NanoString nCounter platform to quantify gene expression within custom sets of TDS gene targets that we had previously shown to exhibit differential transcription among xmrk-transgenic and wild-type medaka. Compound-modulated gene expression was identified using an internet-accessible custom-built data processing pipeline. The effect of a given drug on the entire TDS profile was estimated by comparing compound-modulated genes in the TDS using an activation Z-score and Kolmogorov-Smirnov statistics. TDS gene probes were designed that target common signaling pathways that include proliferation, development, toxicity, immune function, metabolism and detoxification. These pathways may be utilized to evaluate candidate compounds for potential favorable, or unfavorable, effects on melanoma-associated gene expression. Here we present the logistics of using medaka to screen compounds, as well as, the development of a user-friendly NanoString data analysis pipeline to support feasibility of this novel TDS drug-screening strategy.
Dmrt1 is a highly conserved transcription factor, which is critically involved in regulation of gonad development of vertebrates. In medaka, a duplicate of dmrt1—acting as master sex-determining gene—has a tightly timely and spatially controlled gonadal expression pattern. In addition to transcriptional regulation, a sequence motif in the 3′ UTR (D3U-box) mediates transcript stability of dmrt1 mRNAs from medaka and other vertebrates. We show here that in medaka, two RNA-binding proteins with antagonizing properties target this D3U-box, promoting either RNA stabilization in germ cells or degradation in the soma. The D3U-box is also conserved in other germ-cell transcripts, making them responsive to the same RNA binding proteins. The evolutionary conservation of the D3U-box motif within dmrt1 genes of metazoans—together with preserved expression patterns of the targeting RNA binding proteins in subsets of germ cells—suggest that this new mechanism for controlling RNA stability is not restricted to fishes but might also apply to other vertebrates.
In accordance with the Red Queen hypothesis, the lower genotypic diversity in clonally reproducing species should make them easier targets for pathogen infection, especially when closely related sexually reproducing species occur in close proximity. We analyzed two populations of clonal P. formosa and their sexual parental species P. mexicana by correlating individual parasite infection with overall and immune genotype. Our study revealed lower levels of overall genotypic diversity and marginally fewer MHC class I alleles in P. formosa individuals compared to sexually reproducing P. mexicana. Parasite load, however, differed only between field sites but not between species. We hypothesize that this might be due to slightly higher genotypic diversity in P. formosa at the innate immune system (toll like receptor 8) which is likely due to the species’ hybrid origin. In consequence, it appears that clonal individuals do not necessarily suffer a disadvantage compared to sexual individuals when fighting parasite infection.
MicroRNAs play a crucial role in eukaryotic gene regulation. For a long time, only little was known about microRNA-based gene regulatory mechanisms in polyploid animal genomes due to difficulties of polyploid genome assembly. However, in recent years, several polyploid genomes of fish, amphibian, and even invertebrate species have been sequenced and assembled. Here we investigated several key microRNA-associated genes in the recently sequenced sterlet (Acipenser ruthenus) genome, whose lineage has undergone a whole genome duplication around 180 MYA. We show that two paralogs of drosha, dgcr8, xpo1, and xpo5 as well as most ago genes have been retained after the acipenserid-specific whole genome duplication, while ago1 and ago3 genes have lost one paralog. While most diploid vertebrates possess only a single copy of dicer1, we strikingly found four paralogs of this gene in the sterlet genome, derived from a tandem segmental duplication that occurred prior to the last whole genome duplication. ago1,3,4 and exportins1,5 look to be prone to additional segment duplications producing up to four-five paralog copies in ray-finned fishes. We demonstrate for the first time exon microsatellite amplification in the acipenserid drosha2 gene, resulting in a highly variable protein product, which may indicate sub- or neofunctionalization. Paralogous copies of most microRNA metabolism genes exhibit different expression profiles in various tissues and remain functional despite the rediploidization process. Subfunctionalization of microRNA processing gene paralogs may be beneficial for different pathways of microRNA metabolism. Genetic variability of microRNA processing genes may represent a substrate for natural selection, and, by increasing genetic plasticity, could facilitate adaptations to changing environments.
Amphibians evolved in the Devonian period about 400 Mya and represent a transition step in tetrapod evolution. Among amphibians, high-throughput sequencing data are very limited for Caudata, due to their largest genome sizes among terrestrial vertebrates. In this paper we present the transcriptome from the fire bellied newt Cynops orientalis. Data here presented display a high level of completeness, comparable to the fully sequenced genomes available from other amphibians. Moreover, this work focused on genes involved in gametogenesis and sexual development. Surprisingly, the gsdf gene was identified for the first time in a tetrapod species, so far known only from bony fish and basal sarcopterygians. Our analysis failed to isolate fgf24 and foxl3, supporting the possible loss of both genes in the common ancestor of Rhipidistians. In Cynops, the expression analysis of genes described to be sex-related in vertebrates singled out an expected functional role for some genes, while others displayed an unforeseen behavior, confirming the high variability of the sex-related pathway in vertebrates.
Polyploid genomes present a challenge for cytogenetic and genomic studies, due to the high number of similar size chromosomes and the simultaneous presence of hardly distinguishable paralogous elements. The karyotype of the Siberian sturgeon (Acipenser baerii) contains around 250 chromosomes and is remarkable for the presence of paralogs from two rounds of whole-genome duplications (WGD). In this study, we applied the sterlet-derived acipenserid satDNA-based whole chromosome-specific probes to analyze the Siberian sturgeon karyotype. We demonstrate that the last genome duplication event in the Siberian sturgeon was accompanied by the simultaneous expansion of several repetitive DNA families. Some of the repetitive probes serve as good cytogenetic markers distinguishing paralogous chromosomes and detecting ancestral syntenic regions, which underwent fusions and fissions. The tendency of minisatellite specificity for chromosome size groups previously observed in the sterlet genome is also visible in the Siberian sturgeon. We provide an initial physical chromosome map of the Siberian sturgeon genome supported by molecular markers. The application of these data will facilitate genomic studies in other recent polyploid sturgeon species.
The unusual occurrence and developmental diversity of asexual eukaryotes remain a puzzle. De novo formation of a functioning asexual genome requires a unique assembly of sets of genes or gene states to disrupt cellular mechanisms of meiosis and gametogenesis, and to affect discrete components of sexuality and produce clonal or hemiclonal offspring. We highlight two usually overlooked but essential conditions to understand the molecular nature of clonal organisms, that is, a nonrecombinant genomic assemblage retaining modifiers of the sexual program, and a complementation between altered reproductive components. These subtle conditions are the basis for physiologically viable and genetically balanced transitions between generations. Genomic and developmental evidence from asexual animals and plants indicates the lack of complementation of molecular changes in the sexual reproductive program is likely the main cause of asexuals' rarity, and can provide an explanatory frame for the developmental diversity and lability of developmental patterns in some asexuals as well as for the discordant time to extinction estimations.
Sex determination (SD) is a highly diverse and complex mechanism. In vertebrates, one of the first morphological differences between the sexes is the timing of initiation of the first meiosis, where its initiation occurs first in female and later in male. Thus, SD is intimately related to the responsiveness of the germ cells to undergo meiosis in a sex-specific manner. In some vertebrates, it has been reported that the timing for meiosis entry would be under control of retinoic acid (RA), through activation of Stra8. In this study, we used a fish model species for sex determination and lacking the stra8 gene, the Japanese medaka (Oryzias latipes), to investigate the connection between RA and the sex determination pathway. Exogenous RA treatments act as a stress factor inhibiting germ cell differentiation probably by activation of dmrt1a and amh. Disruption of the RA degrading enzyme gene cyp26a1 induced precocious meiosis and oogenesis in embryos/hatchlings of female and even some males. Transcriptome analyzes of cyp26a1–/–adult gonads revealed upregulation of genes related to germ cell differentiation and meiosis, in both ovaries and testes. Our findings show that germ cells respond to RA in a stra8 independent model species. The responsiveness to RA is conferred by sex-related genes, restricting its action to the sex differentiation period in both sexes.
Arapaima gigas is one of the largest freshwater fish species of high ecological and economic importance. Overfishing and habitat destruction are severe threats to the remaining wild populations. By incorporating a chromosomal Hi-C contact map, we improved the arapaima genome assembly to chromosome-level, revealing an unexpected high degree of chromosome rearrangements during evolution of the bonytongues (Osteoglossiformes). Combining this new assembly with pool-sequencing of male and female genomes, we identified id2bbY, a duplicated copy of the inhibitor of DNA binding 2b (id2b) gene on the Y chromosome as candidate male sex-determining gene. A PCR-test for id2bbY was developed, demonstrating that this gene is a reliable male-specific marker for genotyping. Expression analyses showed that this gene is expressed in juvenile male gonads. Its paralog, id2ba, exhibits a male-biased expression in immature gonads. Transcriptome analyses and protein structure predictions confirm id2bbY as a prime candidate for the master sex-determiner. Acting through the TGF beta signaling pathway, id2bbY from arapaima would provide the first evidence for a link of this family of transcriptional regulators to sex determination. Our study broadens our current understanding about the evolution of sex determination genetic networks and provide a tool for improving arapaima aquaculture for commercial and conservation purposes.
Malignant melanoma incidence is rising worldwide. Its treatment in an advanced state is difficult, and the prognosis of this severe disease is still very poor. One major source of these difficulties is the high rate of metastasis and increased genomic instability leading to a high mutation rate and the development of resistance against therapeutic approaches. Here we investigate as one source of genomic instability the contribution of activation of transposable elements (TEs) within the tumor. We used the well-established medaka melanoma model and RNA-sequencing to investigate the differential expression of TEs in wildtype and transgenic fish carrying melanoma. We constructed a medaka-specific TE sequence library and identified TE sequences that were specifically upregulated in tumors. Validation by qRT- PCR confirmed a specific upregulation of a LINE and an LTR element in malignant melanomas of transgenic fish.