Final Activity Report Summary - IRXAVNS (Functional comparative Genomics and loss of function study of Vertebrate Iroquois A Genes)
Recent studies of the genome architecture of vertebrates have uncovered two unforeseen aspects of their organisation. First, there are large regions of the genome devoid of protein-coding sequences with no obvious biological role, named gene deserts. Second, comparative genomics has highlighted the existence of an array of highly conserved non-coding regions (HCNRs) present in all vertebrates. Most surprisingly, these structural features are strongly associated with genes with essential functions during development. Among these, the vertebrate Iroquois (Irx) genes stand out on both fronts.
Among others, Irx genes play essential function during anterior-posterior and dorso-ventral subdivisions of the developing nervous system. These genes are organised in two clusters (IrxA and IrxB) containing three genes each. These clusters span more than 1Mb each with no other genes located in between. Recently, our group showed that the two gene deserts in between the three IrxB genes contain numerous HCNRs, and most of them harbour enhancer activity in zebrafish and Xenopus transgenic assays. During this grant, we have examined the cis-regulatory activity of most of the HCNRs present in the two gene deserts between the Irx genes of IrxA cluster. We found that the HCNRs analysed promote expression in sub-territories of those expressing IrxA genes. Therefore, the complex patterns of expression of IrxA genes during development are constructed by the coordinated action of multiple enhancers disseminated all along the IrxA gene deserts. In each gene desert, several HCNCRs activate expression in domains expressing both Irx flanking genes. Shared enhancer may explain why Irx genes have been maintained associated in cluster through the evolution of vertebrates. Several HCNRs are conserved between both Irx clusters. We have also found that these cluster-conserved regions promote expression in identical territories. Therefore, these regions are, at least in part, responsible for the common expression domains for some IrxA and IrxB genes.
The similar expression patterns in many tissues of the different Irx genes from both clusters suggest a functional redundancy of this family of genes during development. This has complicated the loss of function studies of individual genes. Indeed, mouse mutant for Irx2, Irx4 or Irx5 genes show little or no phenotype in the nervous system and only specific defects in other tissues like the heart or the eye. As a consequence, the function of Irx genes during vertebrate neural development have been mostly inferred from gain of function of wild type or dominant negative forms of Irx proteins that may interfere with the function of several Irx proteins. Loss of function studies of combinations of Irx genes are difficult to achieve using mouse genetics. In this grant, we have use specific antisense oligonucleotides (morpholinos, MO) to block the function of individual and combination of Irx genes in Xenopus. In these studies, we have demonstrated that the Irx genes have redundant function during neural development and that they are required for neural specification and patterning. In addition, we have also found and essential requirement of Irx1 and Irx3 genes for renal development.
Among others, Irx genes play essential function during anterior-posterior and dorso-ventral subdivisions of the developing nervous system. These genes are organised in two clusters (IrxA and IrxB) containing three genes each. These clusters span more than 1Mb each with no other genes located in between. Recently, our group showed that the two gene deserts in between the three IrxB genes contain numerous HCNRs, and most of them harbour enhancer activity in zebrafish and Xenopus transgenic assays. During this grant, we have examined the cis-regulatory activity of most of the HCNRs present in the two gene deserts between the Irx genes of IrxA cluster. We found that the HCNRs analysed promote expression in sub-territories of those expressing IrxA genes. Therefore, the complex patterns of expression of IrxA genes during development are constructed by the coordinated action of multiple enhancers disseminated all along the IrxA gene deserts. In each gene desert, several HCNCRs activate expression in domains expressing both Irx flanking genes. Shared enhancer may explain why Irx genes have been maintained associated in cluster through the evolution of vertebrates. Several HCNRs are conserved between both Irx clusters. We have also found that these cluster-conserved regions promote expression in identical territories. Therefore, these regions are, at least in part, responsible for the common expression domains for some IrxA and IrxB genes.
The similar expression patterns in many tissues of the different Irx genes from both clusters suggest a functional redundancy of this family of genes during development. This has complicated the loss of function studies of individual genes. Indeed, mouse mutant for Irx2, Irx4 or Irx5 genes show little or no phenotype in the nervous system and only specific defects in other tissues like the heart or the eye. As a consequence, the function of Irx genes during vertebrate neural development have been mostly inferred from gain of function of wild type or dominant negative forms of Irx proteins that may interfere with the function of several Irx proteins. Loss of function studies of combinations of Irx genes are difficult to achieve using mouse genetics. In this grant, we have use specific antisense oligonucleotides (morpholinos, MO) to block the function of individual and combination of Irx genes in Xenopus. In these studies, we have demonstrated that the Irx genes have redundant function during neural development and that they are required for neural specification and patterning. In addition, we have also found and essential requirement of Irx1 and Irx3 genes for renal development.