Human intronic enhancers control distinct sub-domains of Gli3 expression during mouse CNS and limb development

Here, we demonstrate in chicken and mouse transgenic embryos that human GLI3-intronic conserved non-coding sequence elements (CNEs) autonomously control individual aspects of Gli3 expression. Their combined action shows many aspects of a Gli3-specific pattern of transcriptional activity. In the mouse limb bud, different CNEs enhance Gli3-specific expression in evolutionary ancient stylopod and zeugopod versus modern skeletal structures of the autopod. Limb bud specificity is also found in chicken but had not been detected in zebrafish embryos. Three of these elements govern central nervous system specific gene expression during mouse embryogenesis, each targeting a subset of endogenous Gli3 transcription sites. Even though fish, birds, and mammals share an ancient repertoire of gene regulatory elements within Gli3, the functions of individual enhancers from this catalog have diverged significantly. During evolution, ancient broad-range regulatory elements within Gli3 attained higher specificity, critical for patterning of more specialized structures, by abolishing the potential for redundant expression control.These results not only demonstrate the high level of complexity in the genetic mechanisms controlling Gli3 expression, but also reveal the evolutionary significance of cis-acting regulatory networks of early developmental regulators in vertebrates.Zinc-finger proteins of the GLI family, GLI1, GLI2, and GLI3 act as transcriptional mediators integrating various upstream patterning signals in a context dependent combinatorial and cooperative fashion to direct a multitude of developmental programs. GLI2 and GLI3 can serve both as transcriptional activators or repressors, whereas GLI1, whose expression is transcriptionally regulated by GLI2 and GLI3, appears to play a secondary role, e.g. in potentiating response to the secreted protein sonic hedgehog (SHH) [1].Mutations in the human GLI3 gene cause a variety of dominant developmental syndromes subsumed as "GLI3 mo