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Background: Melanoma is an aggressive tumor with increasing incidence. To develop accurate prognostic markers and targeted therapies, changes leading to malignant transformation of melanocytes need to be understood. In the Xiphophorus melanoma model system, a mutated version of the EGF receptor Xmrk (Xiphophorus melanoma receptor kinase) triggers melanomagenesis. Cellular events downstream of Xmrk, such as the activation of Akt, Ras, B-Raf or Stat5, were also shown to play a role in human melanomagenesis. This makes the elucidation of Xmrk downstream targets a useful method for identifying processes involved in melanoma formation. Methods: Here, we analyzed Xmrk-induced gene expression using a microarray approach. Several highly expressed genes were confirmed by realtime PCR, and pathways responsible for their induction were revealed using small molecule inhibitors. The expression of these genes was also monitored in human melanoma cell lines, and the target gene FOSL1 was knocked down by siRNA. Proliferation and migration of siRNA-treated melanoma cell lines were then investigated. Results: Genes with the strongest upregulation after receptor activation were FOS-like antigen 1 (Fosl1), early growth response 1 (Egr1), osteopontin (Opn), insulin-like growth factor binding protein 3 (Igfbp3), dual-specificity phosphatase 4 (Dusp4), and tumor-associated antigen L6 (Taal6). Interestingly, most genes were blocked in presence of a SRC kinase inhibitor. Importantly, we found that FOSL1, OPN, IGFBP3, DUSP4, and TAAL6 also exhibited increased expression levels in human melanoma cell lines compared to human melanocytes. Knockdown of FOSL1 in human melanoma cell lines reduced their proliferation and migration. Conclusion: Altogether, the data show that the receptor tyrosine kinase Xmrk is a useful tool in the identification of target genes that are commonly expressed in Xmrk-transgenic melanocytes and melanoma cell lines. The identified molecules constitute new possible molecular players in melanoma development. Specifically, a role of FOSL1 in melanomagenic processes is demonstrated. These data are the basis for future detailed analyses of the investigated target genes.
Background: The epidermal growth factor receptor (Egfr) with its numerous ligands has fundamental roles in development, cell differentiation and physiology. Dysfunction of the receptor-ligand system contributes to many human malignancies. Consistent with such various tasks, the Egfr gene family has expanded during vertebrate evolution as a consequence of several rounds of whole genome duplication. Of particular interest is the effect of the fish-specific whole genome duplication (FSGD) on the ligand-receptor system, as it has supplied this largest group of vertebrates with additional opportunities for sub- and/or neofunctionalization in this signaling system. Results: We identified the predicted components of the Egf receptor-ligand signaling system in teleost fishes (medaka, platyfish, stickleback, pufferfishes and zebrafish). We found two duplicated egfr genes, egfra and egfrb, in all available teleost genomes. Surprisingly only one copy for each of the seven Egfr ligands could be identified in most fishes, with zebrafish hbegf being the only exception. Special focus was put on medaka, for which we more closely investigated all Egf receptors and Egfr ligands. The different expression patterns of egfra, egfrb and their ligands in medaka tissues and embryo stages suggest differences in role and function. Preferential co-expression of different subsets of Egfr ligands corroborates the possible subfunctionalization and specialization of the two receptors in adult tissues. Bioinformatic analyses of the ligand-receptor interface between Egfr and its ligands show a very weak evolutionary conservation within this region. Using in vitro analyses of medaka Egfra, we could show that this receptor is only activated by medaka ligands, but not by human EGF. Altogether, our data suggest a lineage-specific Egfr/Egfr ligand co-evolution. Conclusions: Our data indicate that medaka Egfr signaling occurs via its two copies, Egfra and Egfrb, each of them being preferentially coexpressed with different subsets of Egfr ligands. This fish-specific occurrence of Egf receptor specialization offers unique opportunities to study the functions of different Egf receptor-ligand combinations and their biological outputs in vertebrates. Furthermore, our results strongly support the use of homologous ligands in future studies, as sufficient cross-specificity is very unlikely for this ligand/receptor system.
Transcriptional Rewiring of the Sex Determining dmrt1 Gene Duplicate by Transposable Elements
(2010)
Control and coordination of eukaryotic gene expression rely on transcriptional and posttranscriptional regulatory networks. Evolutionary innovations and adaptations often require rapid changes of such networks. It has long been hypothesized that transposable elements (TE) might contribute to the rewiring of regulatory interactions. More recently it emerged that TEs might bring in ready-to-use transcription factor binding sites to create alterations to the promoters by which they were captured. A process where the gene regulatory architecture is of remarkable plasticity is sex determination. While the more downstream components of the sex determination cascades are evolutionary conserved, the master regulators can switch between groups of organisms even on the interspecies level or between populations. In the medaka fish (Oryzias latipes) a duplicated copy of dmrt1, designated dmrt1bY or DMY, on the Y chromosome was shown to be the master regulator of male development, similar to Sry in mammals. We found that the dmrt1bY gene has acquired a new feedback downregulation of its expression. Additionally, the autosomal dmrt1a gene is also able to regulate transcription of its duplicated paralog by binding to a unique target Dmrt1 site nested within the dmrt1bY proximal promoter region. We could trace back this novel regulatory element to a highly conserved sequence within a new type of TE that inserted into the upstream region of dmrt1bY shortly after the duplication event. Our data provide functional evidence for a role of TEs in transcriptional network rewiring for sub- and/or neo-functionalization of duplicated genes. In the particular case of dmrt1bY, this contributed to create new hierarchies of sex-determining genes.
Background
Squalius alburnoides is an Iberian cyprinid fish resulting from an interspecific hybridisation between Squalius pyrenaicus females (P genome) and males of an unknown Anaecypris hispanica- like species (A genome). S. alburnoides is an allopolyploid hybridogenetic complex, which makes it a likely candidate for ploidy mosaicism occurrence, and is also an interesting model to address questions about gene expression regulation and genomic interactions. Indeed, it was previously suggested that in S. alburnoides triploids (PAA composition) silencing of one of the three alleles (mainly of the P allele) occurs. However, not a whole haplome is inactivated but a more or less random inactivation of alleles varying between individuals and even between organs of the same fish was seen.
In this work we intended to correlate expression differences between individuals and/or between organs to the occurrence of mosaicism, evaluating if mosaics could explain previous observations and its impact on the assessment of gene expression patterns.
Results
To achieve our goal, we developed flow cytometry and cell sorting protocols for this system generating more homogenous cellular and transcriptional samples. With this set-up we detected 10% ploidy mosaicism within the S. alburnoides complex, and determined the allelic expression profiles of ubiquitously expressed genes (rpl8; gapdh and β-actin) in cells from liver and kidney of mosaic and non-mosaic individuals coming from different rivers over a wide geographic range.
Conclusions
Ploidy mosaicism occurs sporadically within the S. alburnoides complex, but in a frequency significantly higher than reported for other organisms. Moreover, we could exclude the influence of this phenomenon on the detection of variable allelic expression profiles of ubiquitously expressed genes (rpl8; gapdh and β-actin) in cells from liver and kidney of triploid individuals. Finally, we determined that the expression patterns previously detected only in a narrow geographic range is not a local restricted phenomenon but is pervasive in rivers where S. pyrenaicus is sympatric with S. alburnoides.
We discuss mechanisms that could lead to the formation of mosaic S. alburnoides and hypothesise about a relaxation of the mechanisms that impose a tight control over mitosis and ploidy control in mixoploids."
Enormous amounts of data are being generated by modern methods such as transcriptome or exome sequencing and microarray profiling. Primary analyses such as quality control, normalization, statistics and mapping are highly complex and need to be performed by specialists. Thereafter, results are handed back to biomedical researchers, who are then confronted with complicated data lists. For rather simple tasks like data filtering, sorting and cross-association there is a need for new tools which can be used by non-specialists. Here, we describe CrossQuery, a web tool that enables straight forward, simple syntax queries to be executed on transcriptome sequencing and microarray datasets. We provide deepsequencing data sets of stem cell lines derived from the model fish Medaka and microarray data of human endothelial cells. In the example datasets provided, mRNA expression levels, gene, transcript and sample identification numbers, GO-terms and gene descriptions can be freely correlated, filtered and sorted. Queries can be saved for later reuse and results can be exported to standard formats that allow copy-and-paste to all widespread data visualization tools such as Microsoft Excel. CrossQuery enables researchers to quickly and freely work with transcriptome and microarray data sets requiring only minimal computer skills. Furthermore, CrossQuery allows growing association of multiple datasets as long as at least one common point of correlated information, such as transcript identification numbers or GO-terms, is shared between samples. For advanced users, the object-oriented plug-in and event-driven code design of both server-side and client-side scripts allow easy addition of new features, data sources and data types.
In the initial phase of development of fish embryos, a prominent and critical event is the midblastula transition (MBT). Before MBT cell cycle is rapid, highly synchronous and zygotic gene transcription is turned off. Only during MBT the cell cycle desynchronizes and transcription is activated. Multiple mechanisms, primarily the nucleocytoplasmic ratio, are supposed to control MBT activation. Unexpectedly, we find in the small teleost fish medaka (Oryzias latipes) that at very early stages, well before midblastula, cell division becomes asynchronous and cell volumes diverge. Furthermore, zygotic transcription is extensively activated already after the 64-cell stage. Thus, at least in medaka, the transition from maternal to zygotic transcription is uncoupled from the midblastula stage and not solely controlled by the nucleocytoplasmic ratio.
Enormous amounts of data are being generated by modern methods such as transcriptome or exome sequencing and microarray profiling. Primary analyses such as quality control, normalization, statistics and mapping are highly complex and need to be performed by specialists. Thereafter, results are handed back to biomedical researchers, who are then confronted with complicated data lists. For rather simple tasks like data filtering, sorting and cross-association there is a need for new tools which can be used by non-specialists. Here, we describe CrossQuery, a web tool that enables straight forward, simple syntax queries to be executed on transcriptome sequencing and microarray datasets. We provide deep-sequencing data sets of stem cell lines derived from the model fish Medaka and microarray data of human endothelial cells. In the example datasets provided, mRNA expression levels, gene, transcript and sample identification numbers, GO-terms and gene descriptions can be freely correlated, filtered and sorted. Queries can be saved for later reuse and results can be exported to standard formats that allow copy-and-paste to all widespread data visualization tools such as Microsoft Excel. CrossQuery enables researchers to quickly and freely work with transcriptome and microarray data sets requiring only minimal computer skills. Furthermore, CrossQuery allows growing association of multiple datasets as long as at least one common point of correlated information, such as transcript identification numbers or GO-terms, is shared between samples. For advanced users, the object-oriented plug-in and event-driven code design of both server-side and client-side scripts allow easy addition of new features, data sources and data types.
Myc is a global transcriptional regulator and one of the most frequently overexpressed oncoproteins in human tumors. It is well established that activation of Myc leads to enhanced cell proliferation but can also lead to increased apoptosis. The use of animal models expressing deregulated levels of Myc has helped to both elucidate its function in normal cells and give insight into how Myc initiates and maintains tumorigenesis. Analyses of the medaka (Oryzias latipes) genome uncovered the unexpected presence of two Myc gene copies in this teleost species. Comparison of these Myc versions to other vertebrate species revealed that one gene, myc17, differs by the loss of some conserved regulatory protein motifs present in all other known Myc genes. To investigate how such differences might affect the basic biological functions of Myc, we generated a tamoxifeninducible in vivo model utilizing a natural, fish-specific Myc gene. Using this model we show that, when activated, Myc17 leads to increased proliferation and to apoptosis in a dose-dependent manner, similar to human Myc. We have also shown that long-term Myc17 activation triggers liver hyperplasia in adult fish, allowing this newly established transgenic medaka model to be used to study the transition from hyperplasia to liver cancer and to identify Myc-induced tumorigenesis modifiers.
Aberrations in gene expression are a hallmark of cancer cells. Differential tumor-specific transcript levels of single genes or whole sets of genes may be critical for the neoplastic phenotype and important for therapeutic considerations or useful as biomarkers. As an approach to filter out such relevant expression differences from the plethora of changes noted in global expression profiling studies, we searched for changes of gene expression levels that are conserved. Transcriptomes from massive parallel sequencing of different types of melanoma from medaka were generated and compared to microarray datasets from zebrafish and human melanoma. This revealed molecular conservation at various levels between fish models and human tumors providing a useful strategy for identifying expression signatures strongly associated with disease phenotypes and uncovering new melanoma molecules.
Recent studies show that combinations of defined key developmental transcription factors (TFs) can reprogram somatic cells to pluripotency or induce cell conversion of one somatic cell type to another. However, it is not clear if single genes can define a cells identity and if the cell fate defining potential of TFs is also operative in pluripotent stem cells in vitro. Here, we show that ectopic expression of the neural TF Neurogenin2 (Ngn2) is sufficient to induce rapid and efficient differentiation of embryonic stem cells (ESCs) into mature glutamatergic neurons. Ngn2-induced neuronal differentiation did not require any additional external or internal factors and occurred even under pluripotency-promoting conditions. Differentiated cells displayed neuron-specific morphology, protein expression, and functional features, most importantly the generation of action potentials and contacts with hippocampal neurons. Gene expression analyses revealed that Ngn2-induced in vitro differentiation partially resembled neurogenesis in vivo, as it included specific activation of Ngn2 target genes and interaction partners. These findings demonstrate that a single gene is sufficient to determine cell fate decisions of uncommitted stem cells thus giving insights into the role of key developmental genes during lineage commitment. Furthermore, we present a promising tool to improve directed differentiation strategies for applications in both stem cell research and regenerative medicine.