Fluorescence-based alternative splicing reporters for the study of epithelial plasticity in vivo.

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Somarelli, JA; Schaeffer, D; Bosma, R; Bonano, VI; Sohn, JW; Kemeny, G; Ettyreddy, A; Garcia-Blanco, MA

Published Date

  • January 2013

Published In

Volume / Issue

  • 19 / 1

Start / End Page

  • 116 - 127

PubMed ID

  • 23185039

Electronic International Standard Serial Number (EISSN)

  • 1469-9001

International Standard Serial Number (ISSN)

  • 1355-8382

Digital Object Identifier (DOI)

  • 10.1261/rna.035097.112

Language

  • eng