Conserved and non-conserved enhancers direct tissue specific transcription in ancient germ layer specific developmental control genes

Our results show that near four different genes (nkx3.2, pax9, otx1b and foxa2) in zebrafish, only 20-30% of highly conserved DNA sequences can act as developmental enhancers irrespective of the tissue the gene expresses in. We find that some genes also have multiple conserved enhancers expressing in the same tissue at the same or different time points in development. We also located non-conserved enhancers for two of the genes (pax9 and otx1b). Our modified Bacterial artificial chromosome (BACs) studies for these 4 genes revealed that many of these enhancers work in a synergistic fashion, which cannot be captured by individual DNA constructs and are not conserved at the sequence level. Our detailed biochemical and transgenic analysis revealed Foxa1 binds to the otx1b non-conserved enhancer to direct its activity in forebrain and otic vesicle of zebrafish at 24 hpf.Our results clearly indicate that high level of functional conservation of genes is not necessarily associated with sequence conservation of its regulatory elements. Moreover certain non conserved DNA elements might have role in gene regulation. The need is to bring together multiple approaches to bear upon individual genes to decipher all its regulatory elements.One of the paradigms of development is the regulation of the genome in a precise and synchronized manner to form a highly complex embryo with diverse and specialized cell types. Though the major cell types in the embryo contain the same genetic material, they are very different from each other in both morphology as well as function. The generation of this cellular diversity by the genome is controlled by cis-regulatory elements. Cis-regulatory elements are DNA elements that are a key component of the genome's non-coding functional sequences and consist of promoters, enhancers, silencers, insulators and locus control regions (LCR). The idea that animal development is regulated by cis-regulatory DNA elements is well established and has been elegant