Alternative splicing multiplies genomic coding capacity and enhances proteome diversity. A well-studied example is the alternative splicing of fibroblast growth factor receptor-2 (FGFR2) which leads to the synthesis of two functionally distinct isoforms of FGFR2 (IIIb and IIIc). The regulation of exon IIIb silencing is one of the events necessary for proper isoform regulation. Exon IIIb silencing is mediated by an exonic splicing silencer (ESS), and the flanking intronic splicing silencers (ISSs). The ESS acts by recruiting the heterogeneous nuclear Ribonucleoprotein A1 (hnRNPA1), while the ISSs recruits the Polypyrimidine Tract Binding Protein (PTB).

To visualize this specific splicing decision in vivo, we developed mice harboring a green fluorescent protein construct that reports on the silencing of exon IIIb. The animals also express a red fluorescent protein reporter of constitutive splicing as an allelic control. This dual reporter system revealed that in various organs and cell types in the adult, the silencing of exon IIIb required the intronic silencers. In neurons, which do not express PTB, we observed robust silencer-dependent repression of exon IIIb, suggesting that most probably the neural paralog, brain PTB, can take over this function. In the epidermis, however, the intronic silencers were not absolutely required for efficient silencing.

We also observed that the regulation of exon IIIb silencing not only differs between tissues, but varies between embryonic and adult stages of the same tissue. Like in the adult, most embryonic and newborn tissues that expressed the iv reporter system, regulated exon IIIb silencing in an ISS-dependent manner. The skin from newborn pups absolutely required the presence of the ISS to repress exon IIIb, which contrasted with the previous findings in adult where it exhibited partial independence to the ISS. The heart from embryos and newborns are capable of silencing exon IIIb in an ISS-independent form, while the adult heart requires the ISSs. These findings suggest that the regulation of this splicing event is determined in a spatio-temporal fashion, where not only different tissues within the organism will recur to different mechanisms to accomplish the same goal, but that the same organ utilizes different mechanisms to silence the exon at different developmental stages.