Cell, Vol. 53, 847-856, June 17, 1988, Copyright © 1988 by Cell Press c-los Expression Interferes with Thymus Development in Transgenic Mice Ulrich RLither,* Werner MLiller, t Takayuki Sumida,t Takeshi Tokuhisa,t Klaus Rajewsky, t and Erwin F. Wagner* * European Molecular Biology Laboratory Postfach 10.2209 D-6900 Heidelberg, Federal Republic of Germany t Instltut f0r Genetik, Umversit&t zu K61n Im Weyertal 121 D-5000 KSIn, Federal Republic of Germany Summary To study the function of the proto-oncogene c-fos in hematopoietic tissues, transgenic mice were gener- ated that express c-los from the H2-K b promoter in several organs. These H2-c-fos mice have enlarged spleens and hyperplastic thymuses containing an in- creased number of thymic epithelial cells. The exoge- nous c-fos expression specifically affects T cell devel- opment in the thymus, thereby increasing the fraction of mature thymocytes. Results obtained with bone marrow radiation chimeras suggest that the altered distribution of T cell subsets is not a direct effect of c-los expression within the T cell lineage. No changes in the proportion of hematopoietic cell lineages are seen in the spleen, and these mice do not develop lymphoid malignancies. B and T cell function, how- ever, is impaired, and H2-c-fos mice are immune defi- cient. It appears that c-fos specifically stimulates the proliferation of thymic epithelial cells, and may thus indirectly affect T cell development. Introduction The expression of proto-oncogenes is thought to be im- portant in various differentiation processes as well as in development (Varmus, 1984; MSIler, 1986b). The proto- oncogene c-fos, which encodes a nuclear protein, has been extensively analyzed, and the results of many studies strongly suggest multifunctional properties for the c-fos gene product (MI311er, 1986a; Verma, 1986). Expression during development has been demonstrated in extraem- bryonic tissues in mice and humans. Recently, in situ hy- bridization experiments with mouse embryos have shown a stage-specific expression of c-fos RNA in the growth regions of fetal bone as well as in mesodermal web tissue (Dony and Gruss, 1987). Furthermore, high fos expression occurs in several tissues immediately following birth (Ka- sik et al., 1987). In adults, only certain hematopoietic cell lineages, such as macrophages and blood neutrophils, express c-fos mRNA at high levels (Gonda and Metcalf, 1984; Kreipe et al., 1987). In mitogen-stimulated thyme- cytes and peripheral blood mononuclear cells, which comprise 75% of T cells, c-fos is transiently highly ex- ~:Present address: ICMR Kobe Un=verslty, Kobe, Japan pressed (Moore et al., 1986; Reed et al., 1986). Although a substantial number of in vitro studies have indicated that c-fos expression can be altered by various growth factors and other proliferative stimuli (ML~ller, 1986a; Verma, 1986; Verma and Sassone-Corsi, 1987; Treisman, 1986, 1987), the function of this proto-oncogene is still unknown. Since the fos protein is localized in the nucleus, it has been proposed that fos might be involved in the transduc- t=on of signals generated by growth or differentiation fac- tors (M011er, 1986a). The interaction of fos with a transcrip- tion complex has indeed been shown in the adipocyte differentiation system (Distel et al., 1987). However, no sequence-specific DNA binding of fos has yet been demonstrated. Currently, the role of c-fos in biological processes can best be explained by assuming that c-los can modulate the expression of different sets of genes. To examine the biological potential of c-fos in various or- gans and cell lineages during development in vivo, we have chosen to express c-fos genes in transgenic mice (Rether et al., 1987). In earlier studies we introduced the mouse c-fos gene under the control of the human metal- Iothionein (MT) promoter. High MT-c-fos expression could be demonstrated only in certain tissues such as pancreas, heart, bone, muscle, and brain when a 3' modification removing sequences responsible for mRNA stability was introduced into the DNA construct. The deregulated c-fos expression affected the bone tissue exclusively, leading to characteristic swellings on the long bones (RL~ther et al., 1987). No stable expression of the transgene was detected in hematopoietic tissues, which express the endogenous c-fos gene. To obtain c-fos expression in a broad range of cell types, including the hematopoietic cells, we fused the gene to the munne major histocompatibility complex pro- moter H2-K b, which is known to be active in a broad range of cell types (Morello et al., 1986). In the present study, we asked whether c-fos expressed from a promoter with a different specificity can exert an ef- fect on cell lineages other than bone tissue. We were par- ticularly interested in determining whether constitutive expression of the c-fos gene in hematopoietic cells predi- sposes them to malignant transformation. Here we report the consequences of H2-c-fos expression in hematopoi- etic tissues in transgenic mice. These mice have enlarged spleens and abnormally expanded thymic epithelial cells, most likely influencing the differentiation of thymocytes and affecting the function of T and B cells. Results Generation of Transgenic Mice The complete murine c-los gene (with its own translational start and stop codons and the polyadenylation signal), fused to the H2-K b promoter (H2-c-fos), was injected as a purified fragment into fertilized F2 (C57BL/6 x SJL) eggs. About 15% of injected and transferred eggs developed to term, and of those, 19% contained variable numbers of ex- ogenous DNA copies (data not shown). A total of four