The genetic basis of spontaneous melanoma formation in spotted dorsal (Sd) Xiphophorus platyfishswordtail hybrids has been studied for decades, and is adequately explained by a two-gene inheritance model involving a sex-linked oncogene, Xmrk, and an autosomal tumor suppressor, DIFF. The Xmrk oncogene encodes a receptor tyrosine kinase related to EGFR; the nature of the DIFF tumor suppressor gene is unknown. We analyzed the genetic basis of UV-B-induced melanoma formation in closely related, spotted side platyfish-swordtail hybrids, which carry a different sex-linked pigment pattern locus, Sp. We UV-irradiated spotted side Xiphophorus platyfish-swordtail backcross hybrids to induce melanomas at frequencies 6-fold higher than occur spontaneously in unirradiated control animals. To identify genetic determinants of melanoma susceptibility in this UVinducible Xiphophorus model, we genotyped individual animals from control and UV-irradiated experimental regimes using allozyme and DNA restriction fragment length polymorphisms and tested for joint segregation of genetic markers with pigmentation phenotype and UV-induced melanoma formation. Joint segregation results show linkage of a CDKN2-like DNA polymorphism with UV-B-induced melanoma formation in these hybrids. The CDKN2-like polymorphism maps to Xiphophorus linkage group V and exhibits recombination fractions with ES1 and MDH2 allozyme markers consistent with previous localization of the DIFF tumor suppressor locus. Our results indicate that the CDKN2-like sequence we have cloned and mapped is a candidate for the DIFF tumor suppressor gene.Genetic hybrids between species of the genus Xiphophorus (Teleostei: Poeciliidae) exhibit spontaneous melanoma formation in several different cross types and have been used for decades to investigate genetic factors contributing to melanoma formation (1). The most studied and best understood Xiphophorus hybrid melanoma is the spotted dorsal GordonKosswig platyfish-swordtail model (2-5), represented by genetic hybrids derived from crossing F 1 hybrids between the platyfish Xiphophorus maculatus Jp 163 A and the swordtail Xiphophorus helleri back to X. helleri. Melanoma formation in this tumor model is genetically controlled by inheritance of a sex-linked receptor tyrosine kinase gene (Xmrk), associated with the spotted dorsal (Sd) pigment pattern locus, and segregation of an autosomal locus in Xiphophorus linkage group (LG) V, variously referred to in the literature as DIFF, R DIFF , and R (3-6). The DIFF locus is believed to regulate macromelanophore pigment cell differentiation (4, 6), and behaves in the Gordon-Kosswig model as a classical tumor suppressor for which loss of species-specific alleles in pigmented backcross hybrids results in melanoma formation according to simple, Mendelian inheritance (1, 3-5).Elegant experiments have shown that Xmrk is a duplicated gene, which has adventitiously acquired the promoter from another gene (7,8). It has been postulated that expression of the oncogenic Xmrk gene duplicate is re...

The Xiphophorus hybrid melanoma model represents one of the earliest reported cases of genetically regulated tumor susceptibility. Melanoma formation in Xiphophorus hybrids may be explained by the inheritance of two genes: a sex-linked oncogene, Xmrk, and a putative tumor suppressor locus, termed DIFF, located in Linkage Group V (LG V). Several genetic mapping procedures were used to produce a new Xiphophorus LG V map with 20 loci. All markers, particularly a recently cloned Xiphophorus CDKN2 gene family member, called CDKN2X, were tested for associations of genotype with degree of macromelanophore pigment pattern modification and susceptibility to melanoma formation in backcross hybrids of seven genetic types, involving 1,110 fish and three pigment patterns. Highly significant associations of CDKN2X genotypes with such phenotypic effects suggests that this gene is a strong candidate for the classically defined DIFF tumor suppressor gene. Because published results have documented the involvement of the CDKN2A (p16, MTS1, and INK4A) tumor suppressor gene in human melanoma formation, the possibility of CDKN2 genes acting as tumor suppressors in both man and Xiphophorus is likely.

The combined use of the arbitrarily primed polymerase chain reaction [AP-PCR, also known as random amplification of polymorphic DNA (RAPD)] and isozyme mapping resulted in the production of 87 potential marker loci, enabling an overall expansion within the genetic map of the fish genus Xiphophorus. Use of DNA sequencing-style acrylamide gels and carefully controlled conditions of amplification and silver staining allowed exceptional resolution and reproducibility of AP-PCR/RAPD generated markers. Linkage analysis of AP-PCR/RAPD and isozyme markers resulted in the addition of 16 new markers to Xiphophorus linkage groups (LGs) I, II, III, V, IX, X, XII, and XIV. Addition of 5 AP-PCR/RAPD markers to linkage group U6 containing the Tailspot pigment pattern locus (P) and designation of eight new unassigned linkage groups with 22 markers was also accomplished. Genetic linkage data allowed inference of the existence of a novel pigment pattern modifier locus. Expansion of the Xiphophorus gene map by linkage analysis of AP-PCR/RAPD markers in conjunction with isozyme polymorphisms should lead to the rapid saturation of genetic linkage groups such as LG V, which will probably be instrumental to cloning the Diff tumor suppressor gene locus.

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Alterations in gene expression that orchestrate eukaryotic cellular differentiation often require appropriate interactions between differentiating cells and a specialized microenvironment. During T-lymphocyte differentiation, immature thymocytes undergo a stringent intrathymic selection process that requires intimate contact with thymic stromal elements. Since this selection process generates T cells that are self-tolerant and recognize nominal antigen only within the context of self-major histocompatibility antigen complex molecules, it is possible that thymocyte/stromal cell interactions are mediated, in part, by antigen-specific receptors expressed on differentiating thymocytes. However, the developmental stage at which a/fl antigen-specific receptors are expressed during T-cell maturation has been a matter of debate. To address this issue, we have studied a/f8 T-cell antigen receptor gene and protein expression on normal thymocyte subsets of AKR/J mice, as well as on a panel of AKR/J primary thymic lymphomas characterized for CD4 (L3T4) and CD8 (Lyt-2) differentiation antigen expression. The data unequivocally demonstrate that a/fl heterodimers are expressed not only on phenotypically mature thymocytes but also on the majority of CD4+8+ double-positive cells that comprise the predominant nonmature thymocyte subset. Furthermore, a fraction of thymocytes in the CD4-8-double-negative compartment, known to contain progenitor cells, also expresses readily detectable cell-surface a/fl receptors. Therefore, during the process of intrathyinic selection, interactions between nonmature thymocytes and stromal cells via the antigen-receptor complex may play a pivotal role in T-cell differentiation and should be considered in formulating schemes for functional T-cell selection.T-cell differentiation occurs under the influence of the thymic microenvironment within which bone marrow-derived progenitor cells proliferate and differentiate to generate the small fraction of immunocompetent cells that emigrates to peripheral lymphoid tissues. Intrathymic residence is a critical step in the evolution and diversification of a functional T-cell antigen receptor repertoire. Thymocyte maturation involves a stringent selection process that generates mature T cells that are self-tolerant yet able to recognize antigen only within the context of self-major histocompatibility complex (MHC) determinants (1-5). Although the intrathymic selection process is poorly understood, interactions between developing thymocytes and stromal elements in the thymus appear to be essential in establishing both tolerance and MHC restriction (6)(7)(8) MATERIALS AND METHODS Animals and Source of Primary Lymphomas. AKR/J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Spontaneous lymphomas were obtained from 6-mo or older untreated mice. N-methyl-N-nitrosourea (MNU)-induced thymic lymphomas were obtained from 4-to 5-moold mice previously injected with MNU (75 mg/kg) at 7 wk of age. MNU-treated mice were sacrificed before they were 6 ...