Sexual development dysgenesis in interspecific hybrids of Medaka fish.

Fish are amongst vertebrates the group with the highest diversity of known sex-determining genes. Particularly, the genus Oryzias is a suitable taxon to understand how different sex determination genetic networks evolved in closely related species. Two closely related species, O. latipes and O. curvinotus, do not only share the same XX/XY sex chromosome system, but also the same male sex-determining gene, dmrt1bY. We performed whole mRNA transcriptomes and morphology analyses of the gonads of hybrids resulting from reciprocal crosses between O. latipes and O. curvinotus. XY male hybrids, presenting meiotic arrest and no production of sperm were sterile, and about 30% of the XY hybrids underwent male-to-female sex reversal. Both XX and XY hybrid females exhibited reduced fertility and developed ovotestis while aging. Transcriptome data showed that male-related genes are upregulated in the XX and XY female hybrids. The transcriptomes of both types of female and of the male gonads are characterized by upregulation of meiosis and germ cell differentiation genes. Differences in the parental species in the downstream pathways of sexual development could explain sex reversal, sterility, and the development of intersex gonads in the hybrids. We hypothesize that male-to-female sex reversal may be connected to a different development time between species at which dmrt1bY expression starts. Our results provide molecular clues for the proximate mechanisms of hybrid incompatibility and Haldane's rule.

The AP-1 transcription factor FOSL1 causes melanocyte reprogramming and transformation.

The MAPK pathway is activated in the majority of melanomas and is the target of therapeutic approaches. Under normal conditions, it initiates the so-called immediate early response, which encompasses the transient transcription of several genes belonging to the AP-1 transcription factor family. Under pathological conditions, such as continuous MAPK pathway overactivation due to oncogenic alterations occurring in melanoma, these genes are constitutively expressed. The consequences of a permanent expression of these genes are largely unknown. Here, we show that FOSL1 is the main immediate early AP-1 member induced by melanoma oncogenes. We first examined its role in established melanoma cells. We found that FOSL1 is involved in melanoma cell migration as well as cell proliferation and anoikis-independent growth, which is mediated by the gene product of its target gene HMGA1, encoding a multipotent chromatin modifier. As FOSL1 expression is increased in patient melanoma samples compared to nevi, we investigated the effect of enhanced FOSL1 expression on melanocytes. Intriguingly, we found that FOSL1 acts oncogenic and transforms melanocytes, enabling subcutaneous tumor growth in vivo. During the process of transformation, FOSL1 reprogrammed the melanocytes and downregulated MITF in a HMGA1-dependent manner. At the same time, AXL was upregulated, leading to a shift in the MITF/AXL balance. Furthermore, FOSL1 re-enforced pro-tumorigenic transcription factors MYC, E2F3 and AP-1. Together, this led to the enhancement of several growth-promoting processes, such as ribosome biogenesis, cellular detachment and pyrimidine metabolism. Overall, we demonstrate that FOSL1 is a novel reprogramming factor for melanocytes with potent tumor transformation potential.

Genomic and Transcriptomic Approaches to Study Cancer in Small Aquarium Fish Models.

Zebrafish and medaka that develop tumors have become valuable tools for experimental cancer research. With the advent of microarrays and new sequencing technologies it has become feasible to perform whole genome, exome, and transcriptome analyses in these fish models. Analyses that compare the two fish models with each other and with data from human tumors have revealed a plethora of important insights. An unexpected high degree of comparability of molecular features of fish and human tumors has been detected. Furthermore, analyses of the fish model data have uncovered molecules that have not received appropriate attention in studies on their human tumor counterparts and thus have provided valuable candidates for novel biomarkers and therapeutic targets.

In vitro evidence for senescent multinucleated melanocytes as a source for tumor-initiating cells.

Oncogenic signaling in melanocytes results in oncogene-induced senescence (OIS), a stable cell-cycle arrest frequently characterized by a bi- or multinuclear phenotype that is considered as a barrier to cancer progression. However, the long-sustained conviction that senescence is a truly irreversible process has recently been challenged. Still, it is not known whether cells driven into OIS can progress to cancer and thereby pose a potential threat. Here, we show that prolonged expression of the melanoma oncogene N-RAS(61K) in pigment cells overcomes OIS by triggering the emergence of tumor-initiating mononucleated stem-like cells from senescent cells. This progeny is dedifferentiated, highly proliferative, anoikis-resistant and induces fast growing, metastatic tumors. Our data describe that differentiated cells, which are driven into senescence by an oncogene, use this senescence state as trigger for tumor transformation, giving rise to highly aggressive tumor-initiating cells. These observations provide the first experimental in vitro evidence for the evasion of OIS on the cellular level and ensuing transformation.

Gene expression dosage regulation in an allopolyploid fish.

How allopolyploids are able not only to cope but profit from their condition is a question that remains elusive, but is of great importance within the context of successful allopolyploid evolution. One outstanding example of successful allopolyploidy is the endemic Iberian cyprinid Squalius alburnoides. Previously, based on the evaluation of a few genes, it was reported that the transcription levels between diploid and triploid S. alburnoides were similar. If this phenomenon occurs on a full genomic scale, a wide functional ''diploidization'' could be related to the success of these polyploids. We generated RNA-seq data from whole juvenile fish and from adult livers, to perform the first comparative quantitative transcriptomic analysis between diploid and triploid individuals of a vertebrate allopolyploid. Together with an assay to estimate relative expression per cell, it was possible to infer the relative sizes of transcriptomes. This showed that diploid and triploid S. alburnoides hybrids have similar liver transcriptome sizes. This in turn made it valid to directly compare the S. alburnoides RNA-seq transcript data sets and obtain a profile of dosage responses across the S. alburnoides transcriptome. We found that 64% of transcripts in juveniles' samples and 44% in liver samples differed less than twofold between diploid and triploid hybrids (similar expression). Yet, respectively 29% and 15% of transcripts presented accurate dosage compensation (PAA/PA expression ratio of 1 instead of 1.5). Therefore, an exact functional diploidization of the triploid genome does not occur, but a significant down regulation of gene expression in triploids was observed. However, for those genes with similar expression levels between diploids and triploids, expression is not globally strictly proportional to gene dosage nor is it set to a perfect diploid level. This quantitative expression flexibility may be a strong contributor to overcome the genomic shock, and be an immediate evolutionary advantage of allopolyploids.

Cystathionase mediates senescence evasion in melanocytes and melanoma cells.

The development of malignant melanoma is a highly complex process, which is still poorly understood. A majority of human melanomas are found to express a few oncogenic proteins, such as mutant RAS and BRAF variants. However, these oncogenes are also found in nevi, and it is now a well-accepted fact that their expression alone leads to senescence. This renders the understanding of senescence escape mechanisms an important point to understand tumor development. Here, we approached the question of senescence evasion by expressing the transcription factor v-myc myelocytomatosis viral oncogene homolog (c-MYC), which is known to act synergistically with many oncogenes, in melanocytes. We observed that MYC drives the evasion of reactive-oxygen stress-induced melanocyte senescence, caused by activated receptor tyrosine kinase signaling. Conversely, MIZ1, the growth suppressing interaction partner of MYC, is involved in mediating melanocyte senescence. Both, MYC overexpression and Miz1 knockdown led to a strong reduction of endogenous reactive-oxygen species (ROS), DNA damage and senescence. We identified the cystathionase (CTH) gene product as mediator of the ROS-related MYC and MIZ1 effects. Blocking CTH enzymatic activity in MYC-overexpressing and Miz1 knockdown cells increased intracellular stress and senescence. Importantly, pharmacological inhibition of CTH in human melanoma cells also reconstituted senescence in the majority of cell lines, and CTH knockdown reduced tumorigenic effects such as proliferation, H2O2 resistance and soft agar growth. Thus, we identified CTH as new MYC target gene with an important function in senescence evasion.

Hyperactivation of constitutively dimerized oncogenic EGF receptors by autocrine loops.

The epidermal growth factor (EGF) receptor (EGFR) has a key role in normal embryonic development, adult tissue homeostasis and many pathological processes, in particular tumour formation. Aberrant EGFR activation occurs in many cancer types, and inhibition of this receptor is a promising anti-tumour strategy. Besides overexpression of the wild-type receptor, mutated oncogenic EGFR variants are often associated with malignant transformation. In human non-small-cell lung cancers, kinase mutants of the EGFR are rather common. Human glioblastoma often express the truncated EGFRvIII version as well as other dimerized and permanently activated mutants of the receptor, which are considered as tumour drivers. Similarly, the mutated and dimerized EGFR variant Xiphophorus melanoma receptor kinase (Xmrk) is causative for the development of malignant pigment cell tumours in medaka and Xiphophorus melanoma models. It is generally believed that oncogenic receptors that are active due to dimerizing mutations are ligand independent. Here, we show that different EGFR variants from fish and human efficiently induce autocrine loops by inducing EGFR ligands such as amphiregulin and HB-EGF. Importantly, the pre-dimerized oncogenic EGFR versions Xmrk from Xiphophorus and human EGFR(C600F), though already active in absence of ligands, respond to ligand stimulation with enhanced oncogenic signalling. In summary, our data show that autocrine or paracrine loops are still acting on pre-dimerized oncogenic EGFRs and contribute to their pro-tumorigenic signalling.

Effects of short read quality and quantity on a de novo vertebrate transcriptome assembly.

For many researchers, next generation sequencing data holds the key to answering a category of questions previously unassailable. One of the important and challenging steps in achieving these goals is accurately assembling the massive quantity of short sequencing reads into full nucleic acid sequences. For research groups working with non-model or wild systems, short read assembly can pose a significant challenge due to the lack of pre-existing EST or genome reference libraries. While many publications describe the overall process of sequencing and assembly, few address the topic of how many and what types of reads are best for assembly. The goal of this project was use real world data to explore the effects of read quantity and short read quality scores on the resulting de novo assemblies. Using several samples of short reads of various sizes and qualities we produced many assemblies in an automated manner. We observe how the properties of read length, read quality, and read quantity affect the resulting assemblies and provide some general recommendations based on our real-world data set.

Isolation of a cancer-associated microchromosome in the sperm-dependent parthenogen Poecilia formosa.

In the asexual all-female fish species Poecilia formosa, the Amazon molly, supernumerary chromosomes have frequently been found in both laboratory-reared and wild-caught individuals. While wild-caught individuals with B chromosomes are phenotypically indifferent from conspecifics, individuals carrying B chromosomes from recent introgression events in the laboratory show phenotypic changes. Former analyses showed that the expression of a pigment cell locus is associated with the presence of these B chromosomes. In addition, they contain a so far unidentified locus that confers a higher susceptibility to tumor formation in the presence of pigmentation pattern. Isolation by microdissection and hybridization to metaphase chromosomes revealed that they contain one or several sequences with similarity to a highly repetitive pericentromeric and subtelomeric sequence in A chromosomes. Isolation of one particular sequence by AFLP showed that the B chromosomes contain at least 1 copy of an A-chromosomal region which is highly conserved in the whole genus Poecilia, i.e. more than 5 million years old. We propose it to be a single copy sequence.

Morphology, testes development and behaviour of unusual triploid males in microchromosome-carrying clones of Poecilia formosa.

In a microchromosome-carrying laboratory stock of the normally all-female Amazon molly Poecilia formosa triploid individuals were obtained, all of which spontaneously developed into males. A comparison of morphology of the external and internal insemination apparatus and the gonads, sperm ploidy and behaviour, to laboratory-bred F(1) hybrids revealed that the triploid P. formosa males, though producing mostly aneuploid sperm, are partly functional males that differ mainly in sperm maturation and sexual motivation from gonochoristic P. formosa males.

Evidence for hermaphroditism in the Squalius alburnoides allopolyploid fish complex.

Fish show an amazing variety of mechanisms of sex determination and modes of reproduction. Of these, simultaneous hermaphroditism has rarely been described. Here, we report a novel case of this phenomenon in adults of the Squalius alburnoides allopolyploid cyprinid complex. We found evidence for the simultaneous presence of mature male and female gonads on the basis of gonad gross morphology and histological analyses. Different stages of male and female germ cell maturation were identified, including motile spermatocytes. We hypothesize on the genetic and/or environmental causes of this phenomenon.

Sex determination diversity and sex chromosome evolution in poeciliid fish.

Poeciliids, a family of live-bearing freshwater fish, including among others platyfish, swordtails and guppies, fully illustrate the diversity of genetic sex determination mechanisms observed in teleosts. Besides unisexuality, a variety of sex-determining systems has been described in this group of fish, including male and female heterogamety with or without autosomal influence, as well as more complicated situations such as multichromosomal and polyfactorial sex determination. Due to the presence of different mechanisms in closely related species or even between populations within a same species, poeciliids are a very attractive model to study the evolutionary dynamics of sex determination. For one species, the Southern platyfish Xiphophorus maculatus, positional cloning of the master sex-determining gene has been initiated through the construction and sequencing of bacterial artificial chromosome contigs covering the region differentiating the X from the Y chromosome. Initial analysis revealed a high plasticity of the sex-determining region and the absence of synteny with other fish and vertebrate sex chromosomes, indicating an independent evolutionary origin.

Sex determination and sex chromosome evolution: insights from medaka.

Among fish that exhibit a variety of sex-determining systems, medaka (Oryzias latipes) has been used as an ideal model to study sex determination and differentiation. Medaka sex differentiation starts with germ cell migration and proliferation during development, and the earliest sexual difference can be seen in germ cell number at stage 38 (1 day before hatching). Differentiation continues in young larvae. Medaka has an XX-XY genetic system, and the male sex-determining gene dmrt1bY (dmy) has been identified. Along with a sister species O. curvinotus, these species are the only 2 fish species whose sex-determining gene has been identified so far. The medaka sex chromosome is of recent origin, and the Y chromosome was formed through generation of the Y-specific region by duplication and insertion of an autosomal region into the proto-Y chromosome. Thus, the dmrt1bY gene is a duplicate of the autosomal dmrt1a gene. This event is estimated to have occurred about 10 million years ago. Sex-determining systems of other Oryzias species are under investigation, and a variety of sex-determining systems seem to exist. These findings may provide new insights into the sex-determining system of medaka.

Major histocompatibility complex variability in the clonal Amazon molly, Poecilia formosa: is copy number less important than genotype?

The evolution of sex is still a major unsolved puzzle in biology. One of the most promising theoretical models to answer this question is the Red Queen hypothesis. The Red Queen hypothesis proposes a fast adaptation of pathogens to common genotypes and therefore a negative frequency-dependent selection against common genotypes. Clonal organisms should be especially endangered when co-occurring with closely related sexual species. In this context, major histocompatibility (MHC) genes have been discussed to be auspicious candidates that could provide the genetic basis on which selection for immune competence could act. In this study, we investigated MHC variability in a clonal teleost fish: the Amazon molly, Poecilia formosa. The Amazon molly is an ideal candidate to test the Red Queen hypothesis as it is a clonal species but co-occurs with a closely related sexual species and should therefore be especially susceptible to pathogen infection. We found that allele numbers did in general not differ between sexual and clonal 'species' but that genotypic variability is reduced in the clonally reproducing fish, especially in the polyploids. We conclude that in clonal organisms, genotype frequency might be more important for immune competence than MHC allele number. Amazon mollies and their co-occurring parental species clearly fulfil a prerequisite of the Red Queen hypothesis and should therefore provide an ideal system to experimentally test this basic principle probably underlying the evolution of sex.

Cross-species chromosome painting corroborates microchromosome fusion during karyotype evolution of birds.

The stone curlew, also known as thick-knee (Burhinus oedicnemus, BOE), represents a phylogenetically young species of the shorebirds (Charadriiformes) that exhibits one of the most atypical genome organizations known within the class of Aves, due to an extremely low diploid number (2n = 42) and only 6 pairs of microchromosomes in its complement. This distinct deviation from the 'typical' avian karyotype is attributed to repeated fusions of ancestral microchromosomes. In order to compare different species with this atypical avian karyotype and to investigate the chromosome rearrangement patterns, chromosome-specific painting probes representing the whole genome of the stone curlew were used to delineate chromosome homology between BOE and 5 species belonging to 5 different avian orders: herring gull (Charadriiformes), cockatiel (Psittaciformes), rock pigeon (Columbiformes), great gray owl (Strigiformes) and Eurasian coot (Gruiformes). Paints derived from the 20 BOE autosomes delimited 28 to 33 evolutionarily conserved segments in the karyotypes of the 5 species, similar to the number recognized by BOE paints in such a basal lineage as the chicken (28 conserved segments). This suggests a high degree of conservation in genome organization in birds. BOE paints also revealed some species-specific rearrangements. In particular, chromosomes BOE1-4 and 14, as well as to a large extent BOE5 and 6, showed conserved synteny with macrochromosomes, whereas homologous regions for BOE7-13 are found to be largely distributed on microchromosomes in the species investigated. Interestingly, the 6 pairs of BOE microchromosomes 15-20 appear to have undergone very few rearrangements in the 5 lineages investigated. Although the arrangements of BOE homologous segments on some chromosomes can be explained by complex fusions and inversions, the occurrence of homologous regions at multiple sites may point to fission of ancestral chromosomes in the karyotypes of the species investigated. However, the present results demonstrate that the ancestral microchromosomes most likely experienced fusion in the stone curlew lineage forming the medium-sized BOE chromosomes, while they have been conserved as microchromosomes in the other neoavian lineages.

STAT3 and SMAD1 signaling in Medaka embryonic stem-like cells and blastula embryos.

The activation and transcriptional activity of signal transducer and activator of transcription 3 (STAT3) is essential for maintaining mouse embryonic stem (ES) cell cultures in an undifferentiated state. However, reports from human and monkey ES-cell culture suggest that STAT3 is dispensable for pluripotency in these systems. At the same time, BMP signaling via smad1 was shown to be able to counteract STAT3 signaling in murine ES-cell cultures, while it influences differentiation in multifaceted ways in other cellular contexts. Hence, the question arises whether the signaling situation found in mice or primates and human ES-cells represent the rule or the exception. With this study, we want to contribute an answer to this question from an evolutionary perspective. Therefore, we analyzed the expression and activation status of the Medaka (Oryzias latipes) STAT3 and SMAD1 in Medaka ES-cell-like cultures and their in vivo counterpart, the Medaka blastula embryo. While SMAD signaling is active in the culture system as well as in blastula embryos, our results indicate that STAT3 is inactive and can thus not be involved in pluripotency control of blastula cells or their derived pluripotent in vitro counterparts. These results suggest that the signaling pathways active in the mouse ES-cell culture system represent the exception, while inactivity of STAT3 is apparently the rule in vertebrate ES-cell cultures.

Synteny conservation of the Z chromosome in 14 avian species (11 families) supports a role for Z dosage in avian sex determination.

In order to determine synteny conservation of the avian Z chromosome, a chicken (Gallus gallus, GGA) Z chromosome painting probe was hybridized to the chromosomes of 14 bird species belonging to 11 different families. The GGAZ painted the Z chromosomes in all species analyzed, suggesting strong conservation of its gene content among the different avian lineages. This was confirmed by the mapping of five GGAZ-orthologous genes (DMRT1, GHR, CHRNB3, ALDOB, B4GALT1) to the Z chromosomes of eight other species. The shuffled order of these genes on different Z chromosomes can be explained by the prevalence of intrachromosomal rearrangements during avian evolution. Synteny conservation of the mammalian X is generally thought to be the result of X chromosome inactivation. The absence of Z chromosome inactivation implies sex-specific dosage differences of a highly conserved array of Z-linked genes in birds. The evolutionary conservation of the entire Z chromosome among avian lineages supports the idea that avian sex determination and/or sex-specific functions are largely based on sex chromosome dosage. We propose that the accumulation of male-specific genes on the Z chromosome confers selective pressure on the Z to conserve its synteny.

Oncogene activation in melanocytes links reactive oxygen to multinucleated phenotype and senescence.

Contrary to malignant melanoma, nevi are a benign form of melanocytic hyperproliferation. They are frequently observed as precursor lesions of melanoma, but they also feature biochemical markers of senescence. In particular, evidence for oncogene-induced melanocyte senescence as natural means to prevent tumorigenesis has been obtained in nevi with mutated B-Raf(V600E). Here, we demonstrate that strong oncogenic growth factor receptor signalling drives melanocytes into senescence, whereas weaker signals keep them in the proliferative state. Activation of oncogene-induced senescence also produces multinucleated giant cells, a long known histological feature of nevus cells. The protein levels of the senescence mediators, p53 and pRB, and their upstream activators do not correlate with senescence. However, strong oncogene signalling leads to pronounced reactive oxygen stress, and scavenging of reactive oxygen species (ROS) efficiently prevents the formation of multinucleated cells and senescence. Similarly, expression of oncogenic N-RAS results in ROS generation, DNA damage and the same multinuclear senescent phenotype. Hence, we identified oncogenic signalling-dependent ROS production as critical mediator of the melanocytic multinuclear phenotype and senescence, both of them being hallmarks of human nevus cells.

The origin and evolution of a unisexual hybrid: Poecilia formosa.

Clonal reproduction in vertebrates can always be traced back to hybridization events as all known unisexual vertebrates are hybrids between recognized species or genetically defined races. Interestingly, clonal vertebrates often also rely on interspecific matings for their reproduction because gynogenesis (sperm-dependent parthenogenesis) and hybridogenesis are common modes of propagation. While in most cases these hybridization events leave no hereditary traces in the offspring, occasionally the genome exclusion mechanism fails and either small parts of male genetic material remain inside the oocyte in the form of microchromosomes, or fusion of the sperm nucleus with the oocyte nucleus leads to polyploid individuals. In this review, we highlight the important role of hybridization for the origin and evolution of a unisexual hybrid: the Amazon molly, Poecilia formosa.