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

Jason A. Somarelli, Daneen Schaeffer, Reggie Bosma, Vivian I. Bonano, Jang Wook Sohn, Gabor Kemeny, Abhinav Ettyreddy, Mariano A. Garcia-Blanco

Research output: Contribution to journalArticle

15 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)116-127
Number of pages12
JournalRNA
Volume19
Issue number1
DOIs
StatePublished - Jan 2013

Keywords

  • DsRed
  • Enhanced green fluorescent protein
  • Epithelial-mesenchymal transition
  • In vivo imaging
  • Wound healing
  • pre-mRNA splicing

ASJC Scopus subject areas

  • Molecular Biology

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  • Cite this

    Somarelli, J. A., Schaeffer, D., Bosma, R., Bonano, V. I., Sohn, J. W., Kemeny, G., Ettyreddy, A., & Garcia-Blanco, M. A. (2013). Fluorescence-based alternative splicing reporters for the study of epithelial plasticity in vivo. RNA, 19(1), 116-127. https://doi.org/10.1261/rna.035097.112