Recent studies suggest that spatio-temporal regulation of alternative splicing (AS) plays a critical role in metazoan development. However, little is currently known about the factors and mechanisms regulating AS during development in vivo. Moreover, assigning functional roles to regulated alternative splicing events and understanding their contribution to the development of an organism remains a long-standing goal in biology. C. elegans provides an attractive model system to study AS during development in a living organism. Using AS microarray profiling and RNA-Seq, we have profiled annotated cassette exons and novel splice variants throughout C. elegans developme nt. Our analysis has uncovered many novel AS events and suggests that hundreds of exons are differentially spliced during development. To complement these experiments, bichromatic fluorescent protein-based reporters have been developed that are capable of monitoring spatio-temporal splicing patterns at single cell resolution in vivo. These reporters reveal a remarkable degree of regulatory complexity that exquisitely modulates isoform expression among individual cell types during C. elegans development, particularly in the nervous system. In order to assess the functions of such isoforms during development, a fosmid recombineering-based gene rescue approach has been used to selectively re-introduce individual splice variants into strong loss-of-function mutant strains for a number of genes of interest. These experiments have identified an AS event in the collagen-like gene
cle-1 that plays an important role in proper synapse development. The functions of additional AS even ts that display striking cell type-specific regulatory patterns are currently being investigated. Collectively, the resources described above reveal novel patterns of AS regulation at single cell resolution during C. elegans development, and further facilitate the elucidation of important in vivo functions for such splice variants.