Cracking the genome's second code: enhancer detection by combined phylogenetic footprinting and transgenic fish and frog embryos.

Genes involved in vertebrate development are unusually enriched for highly conserved non-coding sequence elements. These regions are readily detected in silico, by genome-wide sequence comparisons between different vertebrates, from mammals to fish (phylogenetic footprinting). It follows that sequence conservation must be the result of positive selection for an essential physiological role. An obvious possibility is that these conserved sequences possess regulatory or structural functions important for gene expression and, thus, an in vivo assay becomes necessary. We have developed a rapid testing system using zebrafish and Xenopus laevis embryos that allows us to assign transcriptional regulatory functions to conserved non-coding sequence elements. The sequences are cloned into a vector containing a minimal promoter and the GFP reporter, and are assayed for their putative cis-regulatory activity in zebrafish or Xenopus transgenic experiments. Vectors used include plasmid DNA and the Tol2 transposon system in fish and X. laevis. We have followed this logic to detect and analyze conserved elements in an intergenic region present in the Iroquois (Irx) gene clusters of zebrafish, Xenopus tropicalis, Fugu rubripes and mouse. We have assayed approximately 50 of these conserved elements and shown that the majority behave as modular positive regulatory elements (enhancers) that contribute to specific temporal and spatial domains that are part of the endogenous gene expression pattern. Moreover, comparison of the activity of cognate Irx enhancers from different organisms demonstrates that conservation of sequence is accompanied by in vivo functional conservation across species. Finally, for some of the most conserved elements, we have been able to identify a critical core sequence, essential for correct enhancer function.

The Irx gene family in zebrafish: genomic structure, evolution and initial characterization of irx5b.

Genes of the iroquois ( Iro/Irx) family are highly conserved from Drosophila to mammals and they have been implicated in a number of developmental processes. In flies, the Iro genes participate in patterning events in the early larva and in imaginal disk specification. In vertebrates, the Irx genes regulate developmental events during gastrulation, nervous system regionalization, activation of proneural genes and organ patterning. The Iro genes in Drosophila and the Irx genes of mammals show a clustered organization in the genome. Flies have a single cluster comprising three genes while mammals have two clusters also having three genes each. Moreover, experimental evidence in flies shows that transcriptional regulatory elements are shared among genes within the Iro cluster, suggesting that the same may be true in vertebrates. To date, the genomic organization of the Irx genes in non-mammalian species has not been studied. In this work, we have isolated the irx5b gene from zebrafish, Danio rerio, and have characterized its expression pattern. Furthermore, we have identified the complete set of Irx genes in two fish species, the zebrafish and pufferfish, Takifugu rubripes, and have determined the genomic organization of these genes. Our analysis indicates that early in fish evolutionary history, the Irx gene clusters have been duplicated and that subsequent events have maintained the clustered organization for some of the genes, while others have been lost. In total there are 11 existing Irx genes in zebrafish and 10 in pufferfish. We propose a new nomenclature for the zebrafish Irx genes based on the analysis of their sequences and their genomic relationships.