Introduction

The platyfish Xiphophorus maculatus (photo courtesy of Manfred Schartl, Würzburg)

    Xiphophorus is a freshwater fish genus belonging to the family Poeciliidae like the guppies (Poecilia reticulata) and mollies (Poecilia latipinna). The name Xiphophorus itself derives from the Greek words xiphos (sword) and phoros (bearer). Xiphophorus includes 23 species that live in eastern drainages in Mexico, Guatemala, Belize and Honduras and are classified into Northern swordtails, Southern swordtails and platyfishes. Xiphophorus are live-bearers, meaning that females retain the eggs inside the body and give birth to live, free-swimming young fishes. More precisely, Xiphophorus is ovoviviparous: while eggs are retained inside the body of the female for protection, they are essentially independent of the mother and she does not provide them with any nutrients. Xiphophorus is very popular (the definition of Xiphophorus by P. Desproges) and commonly reared by aquarium hobbyists.

Hereditary melanoma and sex-chromosomal cancer genes in Xiphophorus

Fig2.: Crossing scheme showing the segregation of Tu (tumour locus) and R (regulatory locus) in Xiphophorus interspecific hybrids. The crossing depicted here is the so-called "Gordon-Kosswig" model, where the platyfish X. maculatus and the swordtail X. hellerii are used as parental fishes.

Melanoma are among the most aggressive forms of human cancer. In the 1920's, it was realized by scientists in Germany and the USA that melanoma can occur without any exogenous treatment in Xiphophorus simply by crossing different species, for example the platyfish Xiphophorus maculatus and the swordtail Xiphophorus hellerii (Fig. 2). These melanomas are derived from dysregulated melanin-producing pigment cells called the macromelanophores. Subsequently, Xiphophorus became a well-established model for the study of hereditary cancer.
In Xiphophorus, melanoma development is induced by generating a regulatory imbalance between a dominant sex chromosomal tumour-inducing locus (Tu) and an autosomal Tu-repressing regulatory locus (R), which are present in the platyfish but absent from the swordtail. The presence of R and Tu on different chromosomes allow for their separation through selective breeding. When X. maculatus is crossed with X. hellerii, the F1 progeny is heterozygous for both R and Tu. Further crossing of F1 animals with X. hellerii produces 25% offspring heterozygous for the Tu locus, but devoid of R. In this situation, Tu is out of control in pigment cells, where it is overexpressed and performs its oncogenic function. This results in the formation of highly malignant, invasive and exophytic melanomas, which are generally fatal to the fish.
Xmrk, the gene responsible for tumour development at the Tu locus, encodes a subclass I receptor tyrosine kinase belonging to the epidermal growth factor receptor (Egfr) family (Wittbrodt et al. 1989, Nature 341:415). The Xmrk oncogene was generated through duplication of the egfrb proto-oncogene early during the evolution of the genus Xiphophorus (Adam et al. 1993, Science 259:816). Compared to its proto-oncogenic counterpart, Xmrk displays a completely new transcriptional regulation as well as activating mutations conferring a ligand-independent constitutive activity to the receptor. Xmrk and egfrb are located in the subtelomeric region of the sex chromosomes of X. maculatus. Both genes are closely linked to the master sex-determining gene SD of the platyfish on the X and Y chromosomes. Xmrk is potentially hazardous, non-essential and located in a very unstable genomic region. Nevertheless, Xmrk has been maintained functional in divergent Xiphophorus species. Hence, Xmrk has probably a beneficial function under certain conditions. The analysis of this function is a major challenge for future research in the Xiphophorus model.
Another sex-chromosomal locus of high relevance for the formation of melanoma in Xiphophorus is Mdl, the macromelanophore-determining locus. Macromelanophores are large melanin-containing cells that produce highly polymorphic pigment patterns. They are the cellular progenitors of Xmrk-induced melanomas. Mdl determines not only the phenotype of macromelanophore patterns, but also, probably in combination with Xmrk, the location, onset and malignancy of melanoma. Therefore, Mdl can be defined as a tumour modifier. Mdl is intimately linked to Xmrk but has not been identified so far at the molecular level.

Evolution of sex determination in vertebrates: the model Xiphophorus
The molecular basis of sex determination and gonad differentiation is poorly understood in non-mammalian vertebrates, and even in human important members of the sex-determining cascade are still to be identified. The Y-chromosomal gene Sry, inducing the male phenotype in human and mouse, has been formed during the evolution of mammals and is absent from other vertebrate lineages. The mammalian Y chromosome is losing its genes; this ongoing process might lead to the loss of the Y chromosome and to the extinction of Homo sapiens. Investigation of sex determination mechanisms in non-mammalian vertebrate models might help to predict the future of sex determination in human and to discover alternative sex-determining systems able to substitute for the current Sry-dependent pathway.
Fish are attractive models to study sex determination, particularly because they have developed an amazing diversity of environmental and genetic strategies to establish sexual dimorphism. No simple model of genetic sex determination can be generalized in fish. Both male (XX/XY) and female heterogamety (ZZ/ZW) has been observed, as well as more complicated situations involving multiple sex chromosomes, polygenic sex determination and autosomal modifiers. Furthermore, gonadal sex differentiation can be strongly influenced in some fishes by environmental factors including temperature, pH or fish density. Sex inversion can be obtained in several fish species spontaneously or after treatment by steroid hormones. Almost nothing is known about the molecular and evolutionary mechanisms driving the variability of sex determination. This phenomenon might play a role in speciation in fish.

Fig3.: Genes and gene loci linked to the master sex-determining gene SD on the sex chromosomes of the platyfish Xiphophorus maculatus.

Importantly, molecular sexing and manipulation of sex determination in fish are key economical challenges for the aquaculture. The necessity of the control of reproduction to avoid overcrowding, as well as the presence of sex-specific differences in fish growth, quality and behavior make monosex populations generally highly desirable in fish farming (all-male or all-female populations, depending on the species). Monosex populations can be generated after treatment of one of the parents with estrogens or androgens. The development of molecular tests to determine routinely the sex of individuals at early stages of development could allow the early selection of breeders of a chosen genotype necessary for the production of such monosex populations, and the rapid analysis of products in breeding, androgenesis and gynogenesis experiments.
Almost no information is available concerning sex determination mechanisms in most fishes, and neither sex-linked molecular markers nor sex chromosomes are identifiable in the classical fish models, the zebrafish Danio rerio and the pufferfishes Takifugu rubripes (Fugu) and Tetraodon nigroviridis. The master sex-determining gene dmrt1bY of the medaka Oryzias latipes is present only in a very restricted number of species, and is therefore not the ubiquitous sex-determining gene in fish (Matsuda et al. 2002, Nature 417:559; Nanda et al. 2002, Proc Natl Acad Sci USA 2002, 99:11778). Therefore, alternative models are needed to identify the master sex-determining gene(s) in more than 23.000 fish species.
The platyfish Xiphophorus maculatus is an ideal model to perform the positional cloning of a master sex-determining (SD) gene. In this fish, SD is located between two genes, the melanoma-inducing oncogene Xmrk and its proto-oncogenic duplicate egfrb, which are both already characterized at the molecular level. This situation should allow the identification of the master sex-determining gene of the platyfish by a positional cloning strategy. SD is also closely linked to loci involved in pigmentation and/or melanoma phenotype (Mdl and RY, for "red-yellow" locus) and in the onset of sexual maturity (P, for "puberty") (Fig. 3). The identification of these genetic loci, also aim of the project, is highly relevant for both cancer research and aquaculture.

Definition of Xiphophorus according to Pierre Desproges (in French)
Xiphophore (n.m.): Le xiphophore est un petit poisson de coloration variée, de six à dix centimètres de long et originaire du Mexique. Au moment de se reproduire, le xiphophore émet un cri strident: Christiane! pour appeler la xiphophorette qui accourt bientôt ventre à flotte, la caudale en feu. S'ensuit alors une danse d'amour effrénée dont le tendre spectacle ne peut que toucher le coeur de tout homme capable de supporter un documentaire écologique marin sans balancer ensuite une grenade offensive dans le lac d'Enghien (Dictionnaire superflu à l'usage de l'élite et des bien nantis, 1985).

References

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• Meierjohann S, Schartl M, Volff JN (2004) Genetic, biochemical and evolutionary facets of Xmrk-induced melanoma formation in the fish Xiphophorus. Comp Biochem Physiol C Toxicol Pharmacol 138: 281-289.
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• Volff JN, Schartl M (2002) Sex determination and sex chromosome evolution in the medaka, Oryzias latipes, and the platyfish, Xiphophorus maculatus. Cytogenet Genome Res 99: 170-177.