In epithelial cells, alternative splicing of fibroblast growth factor receptor 2 (FGFR2) transcripts leads to the expression of the FGFR2(IIIb) isoform, whereas in mesenchymal cells, the same process results in the synthesis of FGFR2(IIIc). Expression of the FGFR2(IIIc) isoform during prostate tumor progression suggests a disruption of the epithelial character of these tumors. To visualize the use of FGFR2 exon IIIc in prostate AT3 tumors in syngeneic rats, we constructed minigene constructs that report on alternative splicing. Imaging these alternative splicing decisions revealed unexpected mesenchymal-epithelial transitions in these primary tumors. These transitions were observed more frequently where tumor cells were in contact with stroma. Indeed, these transitions were frequently observed among lung micrometastases in the organ parenchyma and immediately adjacent to blood vessels. Our data suggest an unforeseen relationship between epithelial mesenchymal plasticity and malignant fitness.alternative splicing ͉ mesenchymal-epithelial transitions ͉ tumor plasticity R egulation of alternative splicing is essential for normal gene expression (1), and alterations of this regulation are linked to disease (2), as illustrated by the association between cancer and splicing defects (3-7). An elegant example of this is provided by the splicing of transcripts encoding fibroblast growth factor receptor 2 (FGFR2). The status of FGFR2 alternative splicing depends on the interplay between several cis-acting elements in the FGFR2 premRNA and transacting factors, some of which are cell-type-specific (8). In mesenchymal cells, exon IIIb is silenced by the action of an exonic splicing silencer (9) and two flanking intronic splicing silencers (10-12). This silencing is mediated by Polypyrimidine tract-binding protein (PTB), hnRNP A1, and heretofore unknown factors. In epithelial cells, exon IIIb silencing is countered by several intronic elements. The best characterized are the intronic activating sequence 2, the intronic splicing activator and repressor (ISAR, also known as IAS3), and several GCAUG repeats (13-18). These elements have a dual function in epithelial cells, because they are also involved in silencing exon IIIc (14-18).FGFR2 splicing has been studied in tumors derived from the R-3327 Dunning rat prostate tumor, which arose spontaneously from the dorsal lobe of the prostate in a Copenhagen rat (19). Some R-3327-derived tumors (DT or DT3) express FGFR2(IIIb), which is consistent with their epithelial phenotype (20), whereas AT tumors (e.g., AT3), which have lost epithelial markers and display many mesenchymal indicators (21), express FGFR2(IIIc) (20). The significance of these alternative decisions for tumor behavior is underscored by the fact that forced expression of FGFR2(IIIb) suppresses tumor progression of AT3 tumors (22). Most importantly, however, the differential splicing of FGFR2 transcripts in these two cell types highlights broad differences in gene expression programs. Arguably, monitoring alternative sp...

Alternative splicing generates a vast diversity of protein isoforms from a limited number of protein-coding genes, with many of the isoforms possessing unique, and even contrasting, functions. Fluorescence-based splicing reporters have the potential to facilitate studies of alternative splicing at the single-cell level and can provide valuable information on phenotypic transitions in almost real time. Fibroblast growth factor receptor 2 (FGFR2) pre-mRNA is alternatively spliced to form the epithelial-specific and mesenchymal-specific IIIb and IIIc isoforms, respectively, which are useful markers of epithelial-mesenchymal transitions (EMT). We have used our knowledge of FGFR2 splicing regulation to develop a fluorescence-based reporter system to visualize exon IIIc regulation in vitro and in vivo. Here we show the application of this reporter system to the study of EMT in vitro in cell culture and in vivo in transgenic mice harboring these splicing constructs. In explant studies, the reporters revealed that FGFR2 isoform switching is not required for keratinocyte migration during cutaneous wound closure. Our results demonstrate the value of the splicing reporters as tools to study phenotypic transitions and cell fates at single cell resolution. Moreover, our data suggest that keratinocytes migrate efficiently in the absence of a complete EMT.