title: Fluorescence-based alternative splicing reporters for the study of epithelial plasticity in vivo.
abstract: 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.
authors.affiliation: Center for RNA Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Jason A
authors.initials: JA
authors.lastname: Somarelli
authors.forename: Daneen
authors.initials: D
authors.lastname: Schaeffer
authors.forename: Reggie
authors.initials: R
authors.lastname: Bosma
authors.forename: Vivian I
authors.initials: VI
authors.lastname: Bonano
authors.forename: Jang Wook
authors.initials: JW
authors.lastname: Sohn
authors.forename: Gabor
authors.initials: G
authors.lastname: Kemeny
authors.forename: Abhinav
authors.initials: A
authors.lastname: Ettyreddy
authors.forename: Mariano A
authors.initials: MA
authors.lastname: Garcia-Blanco
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D002460:Cell Line; D002465:Cell Movement; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000378:metabolism; D058750:Epithelial-Mesenchymal Transition / Q000235:genetics*; D005091:Exons; D005453:Fluorescence; D015603:Keratinocytes / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D020033:Protein Isoforms / Q000235:genetics; D051497:Receptor, Fibroblast Growth Factor, Type 2 / Q000235:genetics; D051598:Whole Body Imaging / Q000379:methods*; D014945:Wound Healing / Q000235:genetics
chemical_list: D020033:Protein Isoforms; C496354:Fgfr2 protein, mouse; D051497:Receptor, Fibroblast Growth Factor, Type 2