Iroquois Homeobox Research Articles

Transforming Properties of 8p11-12 Amplified Genes in Human Breast Cancer

Amplification of the 8p11-12 region has been found in about 15% of human breast cancers and is associated with poor prognosis. Earlier, we used genomic analysis of copy number and gene expression to perform a detailed analysis of the 8p11-12 amplicon to identify candidate oncogenes in breast cancer. We identified 21 candidate genes and provided evidence that three genes, namely, LSM-1, TC-1, and BAG4, have transforming properties when overexpressed. In the present study, we systematically investigated the transforming properties of 13 newly identified 8p11-12 candidate oncogenes in vitro. WHSC1L1, DDHD2, and ERLIN2 were most potently transforming oncogenes based on the number of altered phenotypes expressed by the cells. WHSC1L1 contains a PWWP-domain that is a methyl-lysine recognition motif involved in histone code modification and epigenetic regulation of gene expression. Knockdown of WHSC1L1 in 8p11-12-amplified breast cancer cells resulted in profound loss of growth and survival of these cells. Further, we identified several WHSC1L1 target genes, one of which is iroquois homeobox 3 gene (IRX3), a member of the Iroquois homeobox transcription factor family.

Abstract 3102: Oncogenic PWWP-domain protein WHSC1L1 links the homeobox transcription factor IRX3 in breast cancer

Abstract Amplification of the 8p11-12 region has been found in about 10-15% of human breast cancer and may be associated with poor prognosis. Earlier, we used genomic analysis of copy number and gene expression to perform a detailed analysis of the 8p11-12 amplicon for identifying candidate oncogenes in breast cancer. We identified Wolf-Hirschhorn syndrome candidate 1-like 1 (WHSC1L1) as a candidate oncogene based on statistical analysis of copy number increase and over expression. The WHSC1L1 gene encodes a PWWP domain protein that regulates histone methylation marks, and regulates the transcription profile and the differentiated function of cells. To address the question of whether WHSC1L1 possesses transforming properties, we established lentiviral expression constructs containing the WHSC1L1 as well as other 20 candidate genes of 8p11-12 region. MCF10A cells were infected with these lentiviral vectors and assayed for alterations in growth rates, growth factor-independent proliferation, anchorage-independent growth and for three-dimensional morphogenesis assays in Matrigel culture. From these experiments, we found that the WHSC1L1 is the most potently transforming oncogene we tested from the 8p11-12 region. Furthermore, knockdown of this gene in WHSC1L1 amplified breast cancer cells resulted in profound loss of growth and survival potential in these cells. To identify genes that may be regulated in their expression by the WHSC1L1 when it is amplified and over expressed, we performed expression profiling of MCF10A cells, MCF-10AWHSC1L1 cells, and WHSC1L1 amplified SUM-44 breast cancer cells. Analysis of genes altered by over expression of WHSC1L1 in MCF10A-WHSC1L1 cells and in SUM-44 cells resulted in the identification of 184 WHSC1L1 candidate target genes. Among them was the iroquois homeobox 3 gene (IRX3), a member of the Iroquois homeobox transcription factor family. The regulation of IRX3 gene expression by WHSC1L1 was validated in SUM44 breast cancer cells using WHSC1L1 knock down. IRX3 mRNA was dramatically down-regulated in SUM-44 cells after WHSC1L1 shRNA infection. Intriguingly, in our array CGH study we found that SUM-44 cells also have a gene amplification of IRX3. Thus, our finding indicates that WHSC1L1 is the major driving oncogene in the 8p11-12 amplicon, and provides the first evidence that WHSC1L1 and IRX3 are genetically linked in subset of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3102.

Gene Expression Profiling in Down Syndrome Acute Lymphoblastic Leukemia Identifies Distinct Profiles Associated with CRLF2 Expression Status.

Abstract 2389Poster Board II-366Children with Down syndrome (DS) have a 10 to 20-fold increased risk of developing acute lymphoblastic leukemia (ALL), and they have experienced poorer outcomes on recent major protocols worldwide. The cytogenetic abnormalities which are generally common in childhood ALL and contribute to risk-based treatment assignment are markedly less frequent in children with DS-ALL. Recently, activating mutations in Janus kinase 2 (JAK2) have been identified in approximately 20% of DS-ALL, and interstitial deletions involving cytokine receptor-like factor 2 (CRLF2) in approximately 50% of DS-ALL. Global gene expression profiling may provide insights into the biologic consequences of these molecular lesions. We performed microarray analysis of RNA from diagnostic bone marrow samples in 23 DS-ALL and 26 non-DS ALL cases using the Affymetrix Human Genome U133 Plus 2.0 array. CRLF2 expression was high in 10 of the 23 DS-ALL cases, 3 of which also bore JAK2 mutations, and in a single non-DS ALL case. Unsupervised hierarchical clustering analysis demonstrated clustering of non-DS ALL cases belonging to known cytogenetic subgroups such as E2A-PBX1, MLL rearrangement, and high hyperdiploidy. In contrast, neither DS-ALL cases overall nor the JAK2-mutated or high CRLF2 expressing cases formed a cohesive cluster. Supervised analysis identified 43 genes that were differentially expressed between CRLF2 high versus low cases with a false discovery rate <10%. Several of the most highly differentially expressed genes were validated by quantitative real-time PCR. These included three genes with high expression in CRLF2-high cases: chemokine (C-C motif) ligand 17 (CCL17) (p=0.01), V-yes-1 Yamaguchi sarcoma viral oncogene homolog 1 (YES1) (p=0.007), and Iroquois homeobox 2 (IRX2) (p=0.008); and one gene with expression inversely correlated with CRLF2 expression: dual specificity phosphatase 6 (DUSP6) (p=0.0015). Our findings suggest that DS-ALL does not form a single distinct biologic subgroup, but nearly half of DS-ALL cases are defined by high CRLF2 expression, a substantial enrichment for this lesion compared to its prevalence in non-DS ALL. Identification of downstream pathways may identify opportunities for targeted intervention, including interactions with other cytokines and activation of the JAK-STAT pathway. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Identification of Biomarkers in Dupuytren's Disease by Comparative Analysis of Fibroblasts Versus Tissue Biopsies in Disease-Specific Phenotypes

Biomarkers are molecular mediators that can serve as indicators of normal biological processes, pathologic processes, and therapeutic interventions. This study aims to identify potential biomarkers in Dupuytren's disease (DD), a fibroproliferative benign tumor with an unknown etiology and high recurrence after surgery. Bioinformatic analytical techniques were employed to identify candidate genes that may be differentially expressed in DD, which included gene expression analysis of microarray data and thorough literature searches in genetic linkage and other related biomolecular studies. All DD cases were males with advanced DD (n = 5, 66 years +/- 14). RNA was extracted from biopsies and corresponding cultures of normal fascia (unaffected transverse palmar fascia), palmar nodule and cord from each patient. Real-time reverse transcription-polymerase chain reactions were performed to determine the gene expression levels for disease-related transcripts. The bioinformatic analysis revealed 25 candidate genes, which were further short-listed to 6 genes via functional annotation. The 6 selected candidate genes included: A disintegrin and metalloproteinase domain (ADAM12), aldehyde dehydrogenase 1 family member (ALDH1) A1, Iroquois homeobox protein 6 (IRX6), proteoglycan 4 (PRG4), tenascin C (TNC), and periostin (POSTN). The culturing treatments were shown to have significant impact on the gene expression for ALDH1A1, PRG4, and TNC. In tissue biopsies, significant fold changes were observed for ADAM12, POSTN, and TNC in the cord and/or nodule when compared with that of normal fascia. ADAM12 and POSTN are associated with accelerated or abnormal cell growth, whereas TNC has been associated with fibrotic diseases and cell migration. This study demonstrated differential gene expression results in DD tissue biopsies compared with that of their corresponding cultures. ADAM12, POSTN, and TNC were identified from the cord and nodule biopsy samples as potential biomarkers in relation to DD development.

Frequently Methylated Tumor Suppressor Genes in Head and Neck Squamous Cell Carcinoma

Head and neck squamous cell carcinoma (HNSCC) is a very aggressive cancer. In advanced stages, the patient has poor chances of receiving effective treatment, and survival rates are low. To facilitate timely diagnosis and improve treatment, elucidation of early detection markers is crucial. DNA methylation markers are particularly advantageous because DNA methylation is an early event in tumorigenesis, and the epigenetic modification, 5-methylcytosine, is a stable mark. A genome-wide screen using Restriction Landmark Genomic Scanning found a set of genes that are most commonly methylated in head and neck cancers. Five candidate genes: septin 9 (SEPT9), sodium-coupled monocarboxylate transporter 1 (SLC5A8), functional smad-suppressing element on chromosome 18 (FUSSEL18), early B-cell factor 3 (EBF3), and iroquois homeobox 1 (IRX1) were methylated in 27% to 67% of the HNSCC patient samples tested. Furthermore, approximately 50% of the methylated tumor samples shared methylation between two of the five genes (most commonly between EBF3 and IRX1), and 15% shared methylation between three of the five genes. Expression analysis revealed candidate gene down-regulation in 25% to 93% of the HNSCC samples, and 5-aza-2'-deoxycytidine treatment was able to restore expression in at least 2 of 5 HNSCC cell lines for all of the genes tested. Overexpression of the three most frequently down-regulated candidates, SLC5A8, IRX1, and EBF3, validated their tumor suppressor potential by growth curve analysis and colony formation assay. Interestingly, all of the candidates identified may be involved in the transforming growth factor beta signaling pathway, which is often disrupted in HNSCC.

The Iroquois Homeobox Gene 5 Is Regulated by 1,25-Dihydroxyvitamin D3 in Human Prostate Cancer and Regulates Apoptosis and the Cell Cycle in LNCaP Prostate Cancer Cells

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the most active metabolite of vitamin D3, has significant antitumor activity in a broad range of preclinical models of cancer. In this study, we show that the Iroquois homeobox gene 5 (Irx5) is down-regulated by 1,25(OH)2D3 in human prostate cancer samples from patients randomly assigned to receive weekly high-dose 1,25(OH)2D3 or placebo before radical prostatectomy. Down-regulation of Irx5 by 1,25(OH)2D3 was also shown in the human androgen-sensitive prostate cancer cell line LNCaP and in estrogen-sensitive MCF-7 breast cancer cells. Knockdown of Irx5 by RNA interference showed a significant reduction in LNCaP cell viability, which was accompanied by an increase in p21 protein expression, G2-M arrest, and an increase in apoptosis. The induced apoptosis was partially mediated by p53, and p53 protein expression was increased as a result of Irx5 knockdown. Cell survival was similarly reduced by Irx5 knockdown in the colon cancer cell line HCT 116 and in MCF-7 breast cancer cells, each being derived from clinical tumor types that seem to be inhibited by 1,25(OH)2D3. Overexpression of Irx5 led to a reduction of p21 and p53 expression. This is the first report that Irx5 is regulated by 1,25(OH)2D3 in humans and the first report to show that Irx5 is involved in the regulation of both the cell cycle and apoptosis in human prostate cancer cells. Irx5 may be a promising new therapeutic target in cancer treatment.

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 effect of environmental salinity on the proteome of the sea bass (Dicentrarchus labrax L.)

The European sea bass, Dicentrarchus labrax L., tolerates a range of salinities from freshwater to hyper-saline. To study differences in protein expression, fish were reared in both freshwater and seawater. After 3-month acclimation, gill and intestine epithelia were collected and the soluble protein extracted. In all, 362 spots were differentially expressed in the gills and intestines of fishes reared in seawater compared to those from freshwater. Fifty differential protein spots were excised from a colloidal Coomassie-stained gel. Nine separate protein spots were identified unambiguously by mass spectrometry and database searching. Among the six proteins over-expressed in gill cells in seawater, five were cytoskeleton proteins and one was the aromatase cytochrome P450. In gill cells under freshwater conditions, the two over-expressed proteins identified were the prolactin receptor and the major histocompatibility complex class II beta-antigen. In intestinal cells under freshwater conditions, the Iroquois homeobox protein Ziro5 was upregulated over ninefold. The expression of these proteins, their possible direct or indirect roles in the adaptation of D. labrax to salinity, and their correspondences with a previous study are discussed.

Apposition of iroquois expressing and non-expressing cells leads to cell sorting and fold formation in the Drosophilaimaginal wing disc

BackgroundThe organization of the different tissues of an animal requires mechanisms that regulate cell-cell adhesion to promote and maintain the physical separation of adjacent cell populations. In the Drosophila imaginal wing disc the iroquois homeobox genes are expressed in the notum anlage and contribute to the specification of notum identity. These genes are not expressed in the adjacent wing hinge territory. These territories are separated by an approximately straight boundary that in the mature disc is associated with an epithelial fold. The mechanism by which these two cell populations are kept separate is unclear.ResultsHere we show that the Iro-C genes participate in keeping the notum and wing cell populations separate. Indeed, within the notum anlage, cells not expressing Iro-C tend to join together and sort out from their Iro-C expressing neighbours. We also show that apposition of Iro-C expressing and non-expressing cells induces invagination and apico-basal shortening of the Iro-C- cells. This effect probably underlies formation of the fold that separates the notum and wing hinge territories. In addition, cells overexpressing a member of the Iro-C contact one another and become organized in a network of thin strings that surrounds and isolates large groups of non-overexpressing cells. The strings appear to exert a pulling force along their longitudinal axis.ConclusionApposition of cells expressing and non-expressing the Iro-C, as it occurs in the notum-wing hinge border of the Drosophila wing disc, influences cell behaviour. It leads to cell sorting, and cellular invagination and apical-basal shortening. These effects probably account for keeping the prospective notum and wing hinge cell populations separate and underlie epithelial fold formation. Cells that overexpress a member of the Iro-C and that confront non-expressing cells establish contacts between themselves and become organized in a network of thin strings. This is a complex and unique phenotype that might be important for the generation of a straight notum-wing hinge border.

ABNORMAL DEVELOPMENT IN FEMALE GONADS OF FUSED TOES MUTANT MICE

The goal of the present study is to investigate the functional significance of Iroquois (Irx) genes in developing gonads. The Iroquois homeobox family has been shown to be critical for axis and pattern formation during the development of several organisms. Our experiment utilizes a mutant mouse model called Fused toes (Ft) which lacks 6 genes including the entire IrxB cluster, Irx3, 5 and 6, and 3 other genes of unknown function, Fto, Fts, and Ftm. Homozygote mutants (Ft–/–) die in development (E10–E12) because of severe malformation of the developing brain and loss of left-right asymmetry. All of these genes are present in developing gonads of both sexes except Irx3, which is female specific. Affymetrix GeneChip and quantitative real-time PCR analysis have demonstrated an 8-fold increase in Irx3 expression that is specific to somatic cells of the female gonad during sex determination. Based on these findings, we hypothesize that the IrxB cluster, and specially Irx3, will play an important role in female gonad development. Fused toes embryos were dissected at embryonic day 11.5 and gonads were cultured in vitro for 3 days or in vivo for 2 weeks under the kidney capsule of a castrated wild type male recipient mouse. Embryonic sex and genotype were distinguished by PCR on genomic DNA isolated from embryo tails (wild type Ft/Ft, heterozygote Ft/–, and Ft null −/−). The cultured gonads were assessed for morphology by histological sections and for apoptosis (Tunel) or proliferation (PCNA staining). Morphology and cell proliferation were similar among gonads of both sexes and of all genotypes after 3 days of in vitro explant culture. However, in female gonads from Ft −/− embryos, we observed a significant decrease in cells positive for Tunel staining compared to wild type or heterozygote samples after 3 days of culture. Embryonic gonads subject to 2 weeks of culture under the kidney capsule of recipient mice approximates development at postnatal day 5. In the male gonads, significant development of testicular cords was noted in both wild type and mutant samples. In contrast, we observed differences between the wild type and mutant female gonads. Preliminary data indicates that the wild type ovary can produce primordial follicles and structures consistent with maturation of a single oocyte. Ovaries derived from the Ft–/– embryo, however, do not appear to form primordial follicles and instead, maintain immature structures. Together, these results suggest that the absence of the IrxB cluster, Fto, Fts, and Ftm induces abnormal gonad development in the female. We are currently pursuing additional studies to address the impact of the Ft mutant on gonad development. Supported by the University of Illinois Campus Research Board and the University of Illinois Veterinary Medical Research Fund. (poster)

Aetiology‐specific patterns in end‐stage heart failure patients identified by functional annotation and classification of microarray data

The objective of the present study was to use gene expression profiling, functional annotations and classification to identify aetiology-specific biological processes and potential molecular markers for different aetiologies of end-stage heart failure. Individual left ventricular myocardial samples from eleven coronary artery disease and nine dilated cardiomyopathy transplant patients were co-hybridized with pooled RNA from four non-failing hearts on custom-made arrays of 7000 human genes. Significance analysis identified differential expression of 153 and 147 genes, respectively, in coronary artery disease or dilated cardiomyopathy versus non-failing hearts. Analysis of Gene Ontology biological process annotations indicated aetiology-specific patterns, primarily related to genes involved in catabolism and in regulation of protein kinase activity. Gene expression classifiers were obtained and used for class prediction of random samples of coronary artery diseased and dilated cardiomyopathic hearts. Best classifiers frequently included matrix metalloproteinase 3, fibulin 1, ATP-binding cassette, sub-family B member 1 and iroquois homeobox protein 5. Combining functional annotation from microarray data and classification analysis constitutes a potent strategy to identify disease-specific biological processes and gene expression markers in e.g. end-stage coronary artery disease and dilated cardiomyopathy.

Irxl1, a divergent Iroquois homeobox family transcription factor gene

Iroquois homeodomain (Irx) transcription factors are encoded by a conserved family of six genes that are found in two clusters of three genes each. Irx proteins are highly conserved, and their expression patterns overlap considerably during embryonic development, suggesting genetically redundant functions. We have identified a highly divergent Irx gene, which we term Iroquois homeobox-like 1 (Irxl1). The chromosomal location of Irxl1 is distinct from the Irx gene clusters. Irxl1 is conserved in most vertebrates, and the deduced amino acid sequence of its protein product predicts a homeodomain that bears significant homology to Irx homeodomains, but is clearly very divergent. We also identified in Irxl1 a divergent Iro box, a motif that is the defining feature of the Irx family. Expression of Irxl1 during mouse embryogenesis was distinct from that of most Irx genes, and was largely restricted to the epaxial and hypaxial components of the somites, limb buds, otic vescicle, craniofacial mesenchyme, retinal ganglion cell layer, and lens. We conclude that Irxl1 is a newly identified highly divergent member of the Irx gene family with specific expression patterns in mouse embryogenesis.

Linkage analysis of genetic loci for kyphoscoliosis on chromosomes 5p13, 13q13.3, and 13q32

Kyphoscoliosis, a three-dimensional deformity of spinal growth, is characterized by a curvature in the coronal plane (scoliosis) in conjunction with thoracic kyphosis in excess of the normal range in the sagittal plane. We identified kyphoscoliosis within members of seven families (53 individuals) originally ascertained as part of a large collaborative study of familial idiopathic scoliosis. Model-independent linkage analysis of a genome-wide microsatellite screen identified areas suggestive of linkage on chromosomes 2q22, 5p13, 13q, and 17q11. Single-point and multipoint analyses of an additional 25 flanking microsatellite markers corroborated linkage to these regions, with areas on chromosomes 5p13, 13q13, and 13q32 being the most significant (P < 0.005). Analyses of single nucleotide polymorphism (SNP) markers in the candidate region on chromosome 5 narrowed the region to approximately 3.5 Mb (P < 0.05), with the most significant P values (P < 0.01) occurring in approximately a 1.3-Mb region. Candidate loci in this region include IRX1, IRX2, and IRX4 of the Iroquois Homeobox protein family. On chromosome 13, single-point and multipoint analyses resulted in multiple SNPs having P values < 0.05 within five candidate genes: Osteoblast-specific factor 2 or periostin, forkhead box O1A, A-kinase anchor protein 11, TBC1 domain family member 4, and glypican 5, thus supporting the potential relevance of this region in the pathogenesis of kyphoscoliosis.

A novel primate specific gene, CEI, is located in the homeobox gene IRXA2 promoter in Homo sapiens

The Iroquois (IRX) homeobox gene family consists of six highly conserved transcription factors that are of importance for normal embryonic development. They are organized in two gene clusters in human, one on 5p15.33 and the other one on 16q12.2, respectively, and both the organization and the structure of the genes are highly conserved. An open reading frame coding for an unknown protein is identified in the promoter of IRXA2 on chromosome 5p. This new gene is composed of four exons and it is orientated in a head-to-head manner to IRXA2. Only a short 851 bp segment separates the two translation start codons and the two genes may share a bi-directional promoter. This bi-directional promoter is embedded in a large CpG-island, that also continues into both genes. RT-PCR analysis of the new gene reveals two alternative mRNA transcripts and a third mRNA transcript can be predicted from EST clones. The expression profile of the gene analysed in 9 different human tissues reveals that it is expressed in a coordinated fashion with IRXA2, which has led to the name CEI (Coordinated Expression to IRXA2). The CEI protein lacks homology to any known protein or protein domain in public databases, and a putative amino terminal signal peptide suggests the protein is secreted or ER compartment located. The gene is only found in the human and the chimpanzee genome, but not in the mouse or the rat genome, which suggests that CEI is unique for higher primates. As the identified bi-directional promoter not being a relic of an ancient compact genome, CEI may play an important role in the evolution of higher primates in coordination with the IRX genes.

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.

DWnt4 regulates the dorsoventral specificity of retinal projections in the Drosophila melanogaster visual system

In Drosophila melanogaster, the axons of retinal photoreceptor cells extend to the first optic ganglion, the lamina, forming a topographic representation. Here we show that DWnt4, a secreted protein of the Wnt family, is the ventral cue for the lamina. In DWnt4 mutants, ventral retinal axons misprojected to the dorsal lamina. DWnt4 was normally expressed in the ventral half of the developing lamina and DWnt4 protein was detected along ventral retinal axons. Dfrizzled2 and dishevelled, respectively, encode a receptor and a signaling molecule required for Wnt signaling. Mutations in both genes caused DWnt4-like defects, and both genes were autonomously required in the retina, suggesting a direct role of DWnt4 in retinal axon guidance. In contrast, iroquois homeobox genes are the dorsal cues for the retina. Dorsal axons accumulated DWnt4 and misprojected to the ventral lamina in iroquois mutants; the phenotype was suppressed in iroquois Dfrizzled2 mutants, suggesting that iroquois may attenuate the competence of Dfrizzled2 to respond to DWnt4.

Iroquoisgenes 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.

The Iroquois homeobox gene, Irx5, is required for retinal cone bipolar cell development

In the mouse retina, at least ten distinct types of bipolar interneurons are involved in the transmission of visual signals from photoreceptors to ganglion cells. How bipolar interneuron diversity is generated during retinal development is poorly understood. Here, we show that Irx5, a member of the Iroquois homeobox gene family, is expressed in developing bipolar cells starting at postnatal day 5 and is localized to a subset of cone bipolar cells in the mature mouse retina. In Irx5-deficient mice, defects were observed in the expression of some, but not all, immunohistological markers that define mature Type 2 and Type 3 OFF cone bipolar cells, indicating a role for Irx5 in bipolar cell differentiation. The differentiation of these two bipolar cell types has previously been shown to require the homeodomain-CVC transcription factor, Vsx1. However, the defects observed in Irx5-deficient retinas do not coincide with a reduction of Vsx1 expression, and conversely, the expression of Irx5 in cone bipolar cells does not require the presence of a functional Vsx1 allele. These results indicate that there are at least two distinct genetic pathways ( Irx5-dependent and Vsx1-dependent) regulating the development of Type 2 and Type 3 cone bipolar cells.

Irx3 is differentially up-regulated in female gonads during sex determination

Irx3 is a member of the Iroquois homeobox gene family that encodes a protein known for its essential role in spinal cord development. Transcript screening of male and female gonads during the critical period of sex determination (E12–13.5) revealed a sexually dimorphic expression pattern for Irx3 with female gonads exhibiting a sixfold increase in expression over time. Whole mount in situ hybridization confirmed the sexually dimorphic nature of Irx3 expression and immunohistochemical analysis of gonads at E13.5 determined that IRX3 and GATA4 proteins co-localized to somatic cells of XX gonads. The Irx3 signal persisted in germ cell-depleted XX gonads resulting from Busulfan treatment suggesting that its expression was independent of germ cell regulation. Quantitative real-time PCR analysis over an extended time course determined that Irx3 message was low initially and then increased in XX gonads until E13.5, remained elevated until birth, diminished shortly after birth, and remained low in the adult ovary. In contrast, Irx3 message was 50% lower in male compared to female gonads at the initial time point, and continued to decrease over time. Further analysis of adult ovaries suggested that IRX3 expression is not present in any subpopulations of cells of the differentiated ovary. Together, these results suggest that the Irx3 signal is restricted to the somatic cell component of XX gonads and is present at a discreet period of ovarian development that ends abruptly at birth. This timing coincides with the transition of female primordial germ cells from mitotic proliferation to meiotic division, and the organization of germ cell cysts prior to primordial follicle development at birth.

Xiro homeoproteins coordinate cell cycle exit and primary neuron formation by upregulating neuronal-fate repressors and downregulating the cell-cycle inhibitor XGadd45-γ

The iroquois (iro) homeobox genes participate in many developmental processes both in vertebrates and invertebrates, among them are neural plate formation and neural patterning. In this work, we study in detail Xenopus Iro (Xiro) function in primary neurogenesis. We show that misexpression of Xiro genes promotes the activation of the proneural gene Xngnr1 but suppresses neuronal differentiation. This is probably due to upregulation of at least two neuronal-fate repressors: XHairy2A and XZic2. Accordingly, primary neurons arise at the border of the Xiro expression domains. In addition, we identify XGadd45-γ as a new gene repressed by Xiro. XGadd45-γ encodes a cell-cycle inhibitor and is expressed in territories where cells will exit mitosis, such as those where primary neurons arise. Indeed, XGadd45-γ misexpression causes cell cycle arrest. We conclude that, during Xenopus primary neuron formation, in Xiro expressing territories neuronal differentiation is impaired, while in adjacent cells, XGadd45-γ may help cells stop dividing and differentiate as neurons.