INACTIVATION OF THE FGF-4 GENE IN EMBRYONIC STEM-CELLS ALTERS THE GROWTH AND OR THE SURVIVAL OF THEIR EARLY DIFFERENTIATED PROGENY/

Previous studies have shown that early mouse embryos with both FGF-4 a lleles inactivated are developmentally arrested shortly after implanta tion. To understand the roles of FGF-4 during early development, we pr epared genetically engineered embryonic stem (ES) cells, which are una ble to produce FGF-4. Specifically, we describe the isolation and char acterization of ES cells with both FGF-4 alleles inactivated. The FGF- 4(-/-) ES cells do not require FGF-4 to proliferate in vitro, and addi tion of FGF-4 to the medium has little or no effect on their growth. T hus, FGF-4 does not appear to act as an autocrine growth factor for cu ltured ES cells. We also demonstrate that FGF-4(-/-) ES cells, like th eir unmodified counterparts, are capable of forming highly complex tum ors in syngeneic mice composed of a wide range of differentiated cells types, including neural tissue, glandular epithelium, and muscle. In addition, we demonstrate that the FGF-4(-/-) ES cells can differentiat e in vitro after exposure to retinoic acid; however, the growth and/or survival of the differentiated cells is severely compromised. Importa ntly, addition of FGF-4 to the culture medium dramatically increases t he number of differentiated cells derived from the FGF-4(-/-) ES cells , in particular cells with many of the properties of parietal extraemb ryonic endoderm. Finally, we demonstrate that there are differences in the RNA profiles expressed by the differentiated progeny formed in vi tro from FGF-4(-/-) ES cells and FGF-4(+/+) ES cells when they are cul tured with FGF-4. Taken together, the studies described in this report indicate that certain lineages formed in vitro are affected by the in activation of the FGF-4 gene, in particular specific cells that form d uring the initial stage of ES cell differentiation. Thus, ES cells wit h both FGF-4 alleles inactivated should shed light on the important ro les of FGF-4 during the early stages of mammalian development and help determine why FGF-4(-/-) embryos die shortly after implantation. (C) 1997 Academic Press.