Expression of two novel mouse Iroquois homeobox genes during neurogenesis

Members of the Drosophila Iroquois homeobox gene family are implicated in the development of peripheral nervous system and the regionalization of wing and eye imaginal discs. Recent studies suggest that Xenopus Iroquois homeobox (Irx) genes are also involved in neurogenesis. Three mouse Irx genes, Irx1, Irx2 and Irx3, have been previously identified and are expressed with distinct spatio-temporal patterns during neurogenesis. We report here the cloning and expression analysis of two novel mouse Irx genes, Irx5 and Irx6. Although Irx5 and Irx6 proteins are structurally more related to one another, we find that Irx5 displays a developmental expression pattern strikingly similar to that of Irx3, whereas Irx6 expression resembles that of Irx1. Consistent with the notion that Mash1 is a putative target gene of the Irx proteins, all four Irx genes display an overlapping expression pattern with Mash1 in the developing CNS. In contrast, the Irx genes and Mash1 are expressed in complementary domains in the developing eye and olfactory epithelium.     

Iroquois genes influence proximo-distal morphogenesis during rat lung development

Lung development is a highly regulated process directed by mesenchymal-epithelial interactions, which coordinate the temporal and spatial expression of multiple regulatory factors required for proper lung formation. The Iroquois homeobox (Irx) genes have been implicated in the patterning and specification of several Drosophila and vertebrate organs, including the heart. Herein, we investigated whether the Irx genes play a role in lung morphogenesis. We found that Irx1-3 and Irx5 expression was confined to the branching lung epithelium, whereas Irx4 was not expressed in the developing lung. Antisense knockdown of all pulmonary Irx genes together dramatically decreased distal branching morphogenesis and increased distention of the proximal tubules in vitro, which was accompanied by a reduction in surfactant protein C-positive epithelial cells and an increase in beta-tubulin IV and Clara cell secretory protein positive epithelial structures. Transmission electron microscopy confirmed the proximal phenotype of the epithelial structures. Furthermore, antisense Irx knockdown resulted in loss of lung mesenchyme and abnormal smooth muscle cell formation. Expression of fibroblast growth factors (FGF) 1, 7, and 10, FGF receptor 2, bone morphogenetic protein 4, and Sonic hedgehog (Shh) were not altered in lung explants treated with antisense Irx oligonucleotides. All four Irx genes were expressed in Shh- and Gli(2)-deficient murine lungs. Collectively, these results suggest that Irx genes are involved in the regulation of proximo-distal morphogenesis of the developing lung but are likely not linked to the FGF, BMP, or Shh signaling pathways.     

Gene and cluster-specific expression of the Iroquois family members during mouse development

Mammalian homologues of the Drosophila Iroquois homeobox gene complex, involved in patterning and regionalization of differentiation, have recently been identified (Mech. Dev., 69 (1997) 169; Dev. Biol., 217 (2000) 266; Dev. Dyn., 218 (2000) 160; Mech. Dev., 91 (2000) 317; Dev. Biol., 224 (2000) 263; Genome Res., 10 (2000) 1453; Mech. Dev., 103 (2001) 193). The six members of the murine family were found to be organized in two cognate clusters of three genes each, Irx1, -2, -4 and Irx3, -5, -6, respectively (Peters et al., 2000). As a basis for further study of their regulation and function we performed a comparative analysis of the genomic organization and of the expression patterns of all six Irx genes. The genes are expressed in highly specific and regionalized patterns of ectoderm, mesoderm and endoderm derived tissues. In most tissues the pattern of expression of the clustered genes, especially of Irx1 and -2 and of Irx3 and -5, respectively, closely resembled each other while those of Irx4 and -6 were very divergent. Interestingly, the expression of cognate genes was found to be mutually exclusive in adjacent and interacting tissues of limb, heart and the laryncho-pharyncheal region. The results indicate that the Irx genes are coordinately regulated at the level of the cluster.     

Convergent evolution of clustering of Iroquois homeobox genes across metazoans

Vertebrate and Drosophila Iroquois genes are organized in clusters of 3 genes sharing blocks of conserved regulatory sequences. Here, we report a 3-gene cluster in the basal, preduplicative chordate amphioxus. Surprisingly, however, the origin of the amphioxus cluster is independent of those in vertebrates and drosophilids. Investigation of genomic organization of Iroquois genes in other 17 metazoan genomes revealed a fourth independent 3-gene cluster organization in polychaetes, as well as additional 2- and 4-gene clusters in other clades, in one of the most striking examples of convergence in genomic organization described so far. The recurrent independent evolution of Iroquois clusters suggests a functional importance of this organization for these genes, perhaps related to the sharing of regulatory elements. Consistent with this, comparative analysis of genomic regions flanking the 3 amphioxus Irx genes revealed several blocks of sequences, conserved for at least 100 Myr. Finally, we discuss the possible causes and implications of the convergent evolution of this genomic and regulatory organization throughout metazoans.     

The homeobox gene irx1a is required for the propagation of the neurogenic waves in the zebrafish retina

Neurogenesis in the compound eyes of Drosophila and the camera eyes of vertebrates spreads in a wave-like fashion. In both phyla, waves of hedgehog expression are known to drive the wave of neuronal differentiation. The mechanism controlling the propagation of hedgehog expression during retinogenesis of the vertebrate eye is poorly understood. The Iroquois homeobox genes play important roles in Drosophila eye development; they are required for the up-regulation of hedgehog expression during propagation of the morphogenetic furrow. Here, we show that the zebrafish Iroquois homolog irx1a is expressed during retinogenesis and knockdown of irx1a results in a retinal phenotype strikingly similar to those of sonic hedgehog (shh) mutants. Analysis of shh-GFP transgene expression in irx1a knockdown retinas revealed that irx1a is required for the propagation of shh expression through the retina. Transplantation experiments illustrated that the effects of irx1a on shh expression are both cell-autonomous and non-cell-autonomous. Our results reveal a role for Iroquois genes in controlling hedgehog expression during vertebrate retinogenesis.     

Defects in brain patterning and head morphogenesis in the mouse mutant Fused toes

During vertebrate development, brain patterning and head morphogenesis are tightly coordinated. In this paper, we study these processes in the mouse mutant Fused toes (Ft), which presents severe head defects at midgestation. The Ft line carries a 1.6-Mb deletion on chromosome 8. This deletion eliminates six genes, three members of the Iroquois gene family, Irx3, Irx5 and Irx6, which form the IrxB cluster, and three other genes of unknown function, Fts, Ftm and Fto. We show that in Ft/Ft embryos, both anteroposterior and dorsoventral patterning of the brain are affected. As soon as the beginning of somitogenesis, the forebrain is expanded caudally and the midbrain is reduced. Within the expanded forebrain, the most dorsomedial (medial pallium) and ventral (hypothalamus) regions are severely reduced or absent. Morphogenesis of the forebrain and optic vesicles is strongly perturbed, leading to reduction of the eyes and delayed or absence of neural tube closure. Finally, facial structures are hypoplastic. Given the diversity, localisation and nature of the defects, we propose that some of them are caused by the elimination of the IrxB cluster, while others result from the loss of one or several of the Fts, Ftm and Fto genes.     

A dual requirement for Iroquois genes during Xenopus kidney development

The Iroquois (Irx) genes encode evolutionary conserved homeoproteins. We report that Xenopus genes Irx1 and Irx3 are expressed and required during different stages of Xenopus pronephros development. They are initially expressed during mid-neurulation in domains extending over most of the prospective pronephric territory. Expression onset takes place after kidney anlage specification, but before pronephric organogenesis occurs. Later, during nephron segmentation, expression becomes restricted to the intermediate tubule region of the proximal-distal axis. Loss- and gain-of-function analyses, performed with specific morpholinos and inducible wild-type and dominant-negative constructs, reveal a dual requirement for Irx1 and Irx3 during pronephros development. During neurula stages, these genes maintain the specification of the pronephric territory and define its size. This seems to occur, at least in part, through positive regulation of Bmp signalling. Subsequently, Irx genes are required for proper formation of the intermediate tubule. Finally, we find that retinoic acid signalling activates both Irx1 and Irx3 genes in the pronephros.     

An ancient mechanism of hindbrain patterning has been conserved in vertebrate evolution

The hindbrain is a vertebrate-specific embryonic structure of the central nervous system formed by iterative transitory units called rhombomeres (r). Rhombomeric cells are segregated by interhombomeric boundaries which are prefigured by sharp gene expression borders. The positioning of the first molecular boundary within the hindbrain (the prospective r4/r5 boundary) responds to the expression of an Iroquois (Irx) gene in the anterior (r4) and the gene vHnf1 at the posterior (r5). However, while Irx3 is expressed anteriorly in amniotes, a novel Irx gene, iro7, acts in teleosts. To assess the evolutionary history of the genes responsible for the positioning of the r4/r5 boundary in vertebrates, we have stepped outside the gnathostomes to investigate these genes in the agnathans Lethenteron japonicum and Petromyzon marinus. We identified one representative of the Hnf1 family in agnathans. Its expression pattern recapitulates that of vHnf1 and Hnf1 in higher vertebrates. Our phylogenetic analysis places this gene basal to gnathostome Hnf1 and vHnf1 genes. We propose that the duplication of an ancestral hnf1 gene present in the common ancestor of agnathans and gnathostomes gave rise to the two genes found in gnathostomes. We have also amplified 3 Irx genes in L. japonicum: LjIrxA, LjIrxC, LjIrxD. The expression pattern of LjIrxA (the agnathan Irx1/3 ortholog) resembles those of Irx3 or iro7 in gnathostomes. We propose that an Irx/hnf1 pair already present in early vertebrates positioned the r4/r5 boundary and that gene duplications occurred in these gene families after the divergence of the agnathans.     

Identification and expression of zebrafish Iroquois homeobox gene irx1

Iroquois homeoproteins are prepatterning factors that positively regulate proneural genes and control neurogenesis. We have identified a zebrafish Iroquois gene, irx1, which is highly homologous to Xenopus Xiro1, Gallus c-Irx1 and mouse Irx1. Expression of irx1 was initially detected at the bud stage. By 16 h post-fertilization (hpf), irx1 expression was exclusively limited to the prospective midbrain and hindbrain. By 24 hpf, irx1 expression was clearly detected in the acousticovestibual ganglia, tectum, tegmentum, cerebellum and rhombomere 1 but not in rhombomere 2 or mid-hindbrain boundary.     

The Homeodomain Iroquois Proteins Control Cell Cycle Progression and Regulate the Size of Developmental Fields

During development, proper differentiation and final organ size rely on the control of territorial specification and cell proliferation. Although many regulators of these processes have been identified, how both are coordinated remains largely unknown. The homeodomain Iroquois/Irx proteins play a key, evolutionarily conserved, role in territorial specification. Here we show that in the imaginal discs, reduced function of Iroquois genes promotes cell proliferation by accelerating the G1 to S transition. Conversely, their increased expression causes cell-cycle arrest, down-regulating the activity of the Cyclin E/Cdk2 complex. We demonstrate that physical interaction of the Iroquois protein Caupolican with Cyclin E-containing protein complexes, through its IRO box and Cyclin-binding domains, underlies its activity in cell-cycle control. Thus, Drosophila Iroquois proteins are able to regulate cell-autonomously the growth of the territories they specify. Moreover, our results provide a molecular mechanism for a role of Iroquois/Irx genes as tumour suppressors.