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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.
DARWIN\(^1\) believed that sexual selection accounts for the evolution of exaggerated male ornaments, such as the sword-like caudal fin extensions of male fishes of the genus Xiphophorus, that appear detrimental to survival. Swordtails continue to feature prominently in empirical work and theories of sexual selection; the pre-existing bias hypothesis has been offered as an explanation for the evolution of swords in these fishes\(^{2,3}\). Based upon a largely morphological phylogeny, this hypothesis suggests that female preference to mate with sworded males arose in ancestrally swordless species, thus pre-dating the origin of the sword itself and directly driving its evolution. Here we present a molecular phylogeny (based on mitochondrial and nuclear DNA sequences) of Xiphophorus which differs from the traditional one: it indicates that the sword originated and was lost repeatedly. Our phylogeny suggests that the ancestor of the genus is more likely to have possessed a sword than not, thus questioning the applicability of the pre-existing bias hypothesis as an explanation for the cvolution of this sexually selected trait.
Hierarchical structures among male individuals in a population are frequently reflected in differences in aggressive and reproductive behaviour and access to the females. In general social dominance requires large investments which in turn may have to be compensated for by high reproductive success. However, this hypothesis has so far only been sufficiently tested in small mating groups due to the difficulties of determining paternity by classical methods using non-molecular markers. DNA fingerprinting overcomes these problems offering the possibility to determine genetic relationships and mating patterns within larger groups. Using this approach we have recently shown (Schartl et al., 1993) that in the poeciliid fish Limia perugiae in small mating groups the dominant male has 100% mating success, while in larger groups its contribution to the offspring unexpectedly drops to zero. The reproductive failure under such social conditions is explained by the inability of the ex-male to protect all the females simultaneously against mating attempts of his numerous subordinate competitors.
In Xiphophorus melanoma formation has been attributed by classical genetic findings to the overexpression of a cellular oncogene (Tu) due to elimination of the corresponding regulatory gene locus in hybrids. We have attempted to elucidate this phenomenon on the molecular biological level. Studies on the structure and expression of known proto-oncogenes revealed that several of these genes, especially the c-src gene of Xiphophorus, may act as effectors in establishing the neoplastic phenotype of the melanoma cells . However, these genes appear more to participate in secondary steps of tumorigenesis. Another gene, being termed Xmrk, which represents obviously a so far unknown proto-oncogene but with a cons iderably high similarity to the epidermal growth-factorreceptor gene, was mapped to the Tu-containing region of the chromosome. This gene shows features with respect to its structure and expression that seem to justify it to be regarded as a candidate for a gene involved in the primary processes leading to neoplastic transformation of pigment cells in Xiphophorus.
Melanoma formation in Xiphophorus hybrids is mediated by a growth factor receptor tyrosine kinase oncogene encoded by the Tu locus. In the wild-type parental fish no tumors occur due to the activity of a locus that regulates the activity of the melanoma oncogene. Molecu/ar identification of this regulatory locus (R) requires a precise physical map of the chromosomal region. Therefore we studied esterase isozymes in Xiphophorus, two of which have been previously reported to be linked to locus R. We confinn that ES 1 is a distant marker for R ( approx. 30cM), and contrary to earlier studies, we show that this isozyme is present in all species of the genus and at similar activity Ievels in all organs tested. ES4, which has also been reported to be linked to R, was found to be a misclassification of liver ES1. In an attempt to identify markersthat bridge the large distance between ESl and R, we have generated DNA probes which are highly polymorphic. They will be useful in finding Iandmarks on a physical map of the R-containing chromosomal region.
The observation of a slower migrating form of pp6oc-src in neural tissue of chicken and mouse has recently been shown to be due to an alternative transcript form of tbe c-src gene (Martinez et al.: Science 237:411-415, 1987; Levy et al.: Mol Cell Bio17:4142- 4145, 1987). An insertion of 18 basepairs between exons 3 and 4, presumed to be due to alternative splicing of a mini-exon, gives rise to six amino acid residues not found in the non-neuronal (termed flbroblastic) form of pp60\(^{c-src}\). Wehave addressed the question of the evolutionary origin of the c-src neuronal insert · and its functional signiflcance regarding neural-speciflc expression of the c-src gene. To this end we have investigated whether the c-src gene of a lower verlebrate (the teleost fish Xiphophorus) gives rise to a neural-specific transcript in an analogous manner. We could show that the fish c-src gene does encode for a "fibroblastic" and a "neuronal" form of transcript and that the neuronal transcript does indeed arise by way of alternative splicing of a mini-exon. The miniexon is also 18 basepairs long and we could demoostrate directly that this exon lies within the intron separating exons 3 and 4. For comparative purposes we have examined whether the fish c-yes gene, the member of the src gene family most closely related to c-src, also encodes a neural tissue-specific transcript. No evidence for a second transcript form in brain was obtained. This result suggests that the mini-exon arose within the c-src gene lineage sometime between the srclyes gene duplication event and the divergence of the evolutionary lineage giving rise to the teleost fish. Published genomic sequence of src-related genes in Drosophila and our own results with Hydra demoostrate no intron in these species at the analogous location, consistent with first appearance of this mini-exon sometime between 550 and 400 million years ago.
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.