Fluorescence-based alternative splicing reporters for the study of epithelial plasticity in vivo
- Jason A. Somarelli1,2,
- Daneen Schaeffer1,2,
- Reggie Bosma1,2,
- Vivian I. Bonano1,2,3,5,
- Jang Wook Sohn1,2,6,
- Gabor Kemeny1,2,
- Abhinav Ettyreddy1,2 and
- Mariano A. Garcia-Blanco1,2,4,7
- 1Center for RNA Biology,
- 2Department of Molecular Genetics and Microbiology,
- 3University Program in Genetics and Genomics,
- 4Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA
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.
Keywords
- pre-mRNA splicing
- epithelial–mesenchymal transition
- wound healing
- in vivo imaging
- enhanced green fluorescent protein
- DsRed
Footnotes
-
↵7 Corresponding author
E-mail m.garciablanco{at}duke.edu
-
Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.035097.112.
- Received June 21, 2012.
- Accepted October 22, 2012.
- Copyright © 2013 RNA Society
Freely available online through the RNA Open Access option.