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Several species of the genus Xiphophorus are polymorphic for specific pigment patterns. Same of these give rise to malignant melanoma following the appropriate crossings. For one of these pattern Iod from the platyfish Xiphophorus maculatus the melanoma-inducing gene has been doned and found to encode a novel receptor tyrosine kinase, designated Xmrk. Using molecular probes from this gene in Southern blot analyses on single fish DNA preparations from 600 specimens of different populations of various species of the genus Xiphophorus and their hybrids, either with or without melanomapredisposing pattern, it was shown that all individuals contain the Xmrk gene as a proto-oncogene. It is located on the sex chromosome. All fish that carry a melanoma-predisposing locus which has been identified by Mendelian genetics contain an additional copy of Xmrk, closely linked to a specific melanophore pattern locus on the sex chromosome. The melanoma-inducing loci of the different species and populations are homologous. The additional copy of Xmrk obviously arose by a geneduplication event, thereby acquiring the oncogenic potential. The homology of the melanomainducing Iod points to a similar mechanism of tumor suppression in all feral fish populations of the different species of the genus Xiphophorus.
Ligand-dependent tumor induction in medakafish embryos by a Xmrk receptor tyrosine kinase transgene
(1994)
Xmrk encodes a subclass 1 receptor tyrosine kinase (RTK) which has been cloned from the melanomainducing locus Tu of the poeciliid fish Xiphophorus. To demonstrate a high oncogenic potential in vivo we transferred the gene into early embryos of the closely related medakafish. Ectopic expression of the Xmrk oncogene under the control of a strong, constitutive promoter (CMVTk) led to the induction of embryonic tumors with high incidence, after short latency periods, and with a specific pattern of affected tissues. We demonstrate ligand-dependent transformation in vivo using a chimeric receptor consisting of the extracellular and transmembrane domains of the human EGF receptor (HER) and the cytoplasmatic domain of Xmrk. Expression of the chimeric receptor alone does not lead to ldnase activation or induction of tumors. Coexpression of the chimera with its corresponding ligand, human transforming growth factor alpha (bTGF(X), however, results in the activation of the chimeric RTK. In injected fish embryos the induction of the neoplastic growth is observed with similar incidence and tissue distribution as in embryos carrying the native Xmrk oncogene suggesting that the ligand as well as factors downstream of tbe RTK are required for tumor formation. In this study we show single-step induction of tumors by ectopic expression of RTKs in vivo substantiating tbe significance of autocrine stimulation in RTK induced tumors in vertebrales.
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.
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.
The expression of the c-src gene in embryonie and adult tissue of the teleost fish Xiphophorus helleri was analyzed by in-situ hybridization. The highly conserved fish c-src gene was found to be expressed at high levels in midterm embryos, where c-src mRNA was localized in developing neurons of the sensory layer of the differentiating retina and in the developing brain. In adult tissues the expression of c-src was found to persist in certain cell types of the brain and the neural retina, especially in the bipolar cells of the inner nuclear layer, which are postmitotic, fully differentiated mature neurons. Thus c-src in Xiphophorus appears to be a developmentally regulated proto-oncogene which is important for neuronal differentiation during organogenesis, but whose persistence of expression in certain terminally differentiated neurons strongly suggests a particular maintenance function for c-src in these cells as well.
The remarkable diversity of sex determination mechanisms known in fish may be fuelled by exceptionally high rates of sex chromosome turnovers or transitions. However, the evolutionary causes and genomic mechanisms underlying this variation and instability are yet to be understood. Here we report on an over 30-year evolutionary experiment in which we tested the genomic consequences of hybridisation and selection between two Xiphophorus fish species with different sex chromosome systems. We find that introgression and imposing selection for pigmentation phenotypes results in the retention of an unexpectedly large maternally derived genomic region. During the hybridisation process, the sex-determining region of the X chromosome from one parental species was translocated to an autosome in the hybrids leading to the evolution of a new sex chromosome. Our results highlight the complexity of factors contributing to patterns observed in hybrid genomes, and we experimentally demonstrate that hybridisation can catalyze rapid evolution of a new sex chromosome.
The livebearing all-female fish Poecilia formosa reproduces by gynogenesis, a modified form of parthenogenesis. P. formosa forms at least two breeding complexes: in its northern range it exists sympatrically with Poecilia latipinna and in its southern range with Poecilia mexicana. Differences between these complexes and their possible origin are discussed. Embryogenesis is triggered by sperm of males of these closely related sympatric species. Because inheritance is stricdy maternal, from the male point of view energy and time invested are totally lost. In this study we wanted to elucidate whether males are able to distinguish between conspecific and parasitic females. It could be shown that males are able to distinguish females optically, but that this ability was obscured as soon as chemical and/or tactile contact was possible. Furthermore, we found that females in an attractive phase of their sexual cycle are always preferred, regardless of species. This is possibly the mechanism by which parasitic females obtain the matings they need to reproduce.
The male-polymorphic poeciliid fish, Limia perugiae, a small teleostean endemic to the southeast of the Caribbean island Hispafiola, consists of three male size morphs with uniform females. Large males differentiate at a size va:rying between 25 and 38 mm; intermediate males, between 21 and 25 mm. Under competition, !arge males exhibit an elaborate courtship display, whereas small males show only a sneak-chase behavior. Intermediate males adapt their tactics to the respective competitors. However, all malemorphs can switch from courtship display to sneak-chase behavior. In large mating groups with four males of different size and five or six virgin females, large dominant a-males as weil as small subordinate \(\delta\)-males did not produce any offspring. Unexpectedly, all progeny were sired exclusively by the intemediate subordinate ß- and \(\gamma\)-males. Breeding experiments with the three male morphs can best be explained by a model of Y -linked genes for small and !arge size which are both suspended by the activity of an autosomal recessive repressor responsible for the development of intermediate males. The dominant allele of the recessive repressor, in either its homoorits heterozygous state, activates the Y-chromosomal genes for !arge or small size, respectively. Accordingly, intermediate males may produce male offspring of all size classes, depending on the presence of either the Y-linked gene or the autosomal repressor.
Background: Melanoma cells are usually characterized by a strong proliferative potential and efficient invasive migration. Among the multiple molecular changes that are recorded during progression of this disease, aberrant activation of receptor tyrosine kinases (RTK) is often observed. Activation of matrix metalloproteases goes along with RTK activation and usually enhances RTK-driven migration. The purpose of this study was to examine RTKdriven three-dimensional migration of melanocytes and the pro-tumorigenic role of matrix metalloproteases for melanocytes and melanoma cells. Results: Using experimental melanocyte dedifferentiation as a model for early melanomagenesis we show that an activated EGF receptor variant potentiates migration through three-dimensional fibrillar collagen. EGFR stimulation also resulted in a strong induction of matrix metalloproteases in a MAPK-dependent manner. However, neither MAPK nor MMP activity were required for migration, as the cells migrated in an entirely amoeboid mode. Instead, MMPs fulfilled a function in cell cycle regulation, as their inhibition resulted in strong growth inhibition of melanocytes. The same effect was observed in the human melanoma cell line A375 after stimulation with FCS. Using sh- and siRNA techniques, we could show that MMP13 is the protease responsible for this effect. Along with decreased proliferation, knockdown of MMP13 strongly enhanced pigmentation of melanocytes. Conclusions: Our data show for the first time that growth stimuli are mediated via MMP13 in melanocytes and melanoma, suggesting an autocrine MMP13-driven loop. Given that MMP13-specific inhibitors are already developed, these results support the evaluation of these inhibitors in the treatment of melanoma.
Background
The dmrt1 and sox9 genes have a well conserved function related to testis formation in vertebrates, and the group of fish presents a great diversity of species and reproductive mechanisms. The lambari fish (Astyanax altiparanae) is an important Neotropical species, where studies on molecular level of sex determination and gonad maturation are scarce.
Methods
Here, we employed molecular cloning techniques to analyze the cDNA sequences of the dmrt1 and sox9 genes, and describe the expression pattern of those genes during development and the male reproductive cycle by qRT-PCR, and related to histology of the gonad.
Results
Phylogenetic analyses of predicted amino acid sequences of dmrt1 and sox9 clustered A. altiparanae in the Ostariophysi group, which is consistent with the morphological phylogeny of this species. Studies of the gonad development revealed that ovary formation occurred at 58 days after hatching (dah), 2 weeks earlier than testis formation. Expression studies of sox9 and dmrt1 in different tissues of adult males and females and during development revealed specific expression in the testis, indicating that both genes also have a male-specific role in the adult. During the period of gonad sex differentiation, dmrt1 seems to have a more significant role than sox9. During the male reproductive cycle dmrt1 and sox9 are down-regulated after spermiation, indicating a role of these genes in spermatogenesis.
Conclusions
For the first time the dmrt1 and sox9 were cloned in a Characiformes species. We show that both genes have a conserved structure and expression, evidencing their role in sex determination, sex differentiation and the male reproductive cycle in A. altiparanae. These findings contribute to a better understanding of the molecular mechanisms of sex determination and differentiation in fish.
In one of the simplest metazoan organisms, the sponge Spongilla lacustris, at least four different src-related kin ase genes (srkl-4) are expressed, aD of which show a high degree of similarity to the c-src genes of vertebrates. Whereas srk2 and srk3 are c1early unrelated at the nucleic acid level, srkl and srk4 share identical sequences in the 5' parts of their cDNAs. The cloning of several primer extension clones and genomic polymerase chain re action experiments confirmed the hypo thesis of an alternative splicing of tandemly arranged carboxyterminal parts of srkl and srk4. The genomic sequence encoding both proteins was found to be interrupted at the splice point by an intron which is located in the same position as one of the introns in the chicken src gene, which is the only gene conserved in invertebrates and vertebrates. All four srk genes are expressed in adult sponges as mRNA transcripts of about 2.2 kb. Tyrosine kin ase activity of a src-related kin ase could be detected in adult sponges but not in their resting form (gemmulae), and may reflect the activity of the srk protein products. Spongilla lacustris is the simplest organism from which a pro tein tyrosine kinase gene has been isolated. The presence of at least four such genes in the evolutionary ancient and primitive phylum Porifera suggests that tyrosine kinase genes arose concomitantly with or shortly after the appearance of multicellular organisms and that their activity may be involved in aggregation and cell-cell recognition.
DURING vertebrale development, many neurons depend for survival and differentiation on their target cells\(^{1-3}\). The best documented mediator of such a retrograde trophic action is the neurotrophin nerve growth factor (NGF)\(^1\). NGF and the other known members of tbe neurotrophin family, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT -3) and neurotrophin-4/5 (NT -4/5) are conserved as distinct genes over large evolutionary distances\(^{4 -6}\). Here we report the cloning of neurotrophin-6 (NT -6), a new member of this family from the teleost fish Xiphophorus. NT -6 distinguishes itself from the other known neurotrophins in that it is not found as a soluble protein in the medium of producing cells. The addition of heparin (but not chondroitin) effects the release of NT -6 from cell surface and extracellular matrix molecules. Recombinant purified NT -6 has a spectrum of actions similar to NGF on chick sympathetic and sensory neurons, albeit with a lower potency. NT -6 is expressed in tbe embryonie valvulla cerebelli; expression persists in some adult tissues. The interaction of NT-6 with heparin-binding molecuJes may modulate its action in the nervous system .