Interaction between IRF6 and TGFA Genes Contribute to the Risk of Nonsyndromic Cleft Lip/Palate

Previous evidence from tooth agenesis studies suggested IRF6 and TGFA interact. Since tooth agenesis is commonly found in individuals with cleft lip/palate (CL/P), we used four large cohorts to evaluate if IRF6 and TGFA interaction contributes to CL/P. Markers within and flanking IRF6 and TGFA genes were tested using Taqman or SYBR green chemistries for case-control analyses in 1,000 Brazilian individuals. We looked for evidence of gene-gene interaction between IRF6 and TGFA by testing if markers associated with CL/P were overtransmitted together in the case-control Brazilian dataset and in the additional family datasets. Genotypes for an additional 142 case-parent trios from South America drawn from the Latin American Collaborative Study of Congenital Malformations (ECLAMC), 154 cases from Latvia, and 8,717 individuals from several cohorts were available for replication of tests for interaction. Tgfa and Irf6 expression at critical stages during palatogenesis was analyzed in wild type and Irf6 knockout mice. Markers in and near IRF6 and TGFA were associated with CL/P in the Brazilian cohort (p<10⁻⁶). IRF6 was also associated with cleft palate (CP) with impaction of permanent teeth (p<10⁻⁶). Statistical evidence of interaction between IRF6 and TGFA was found in all data sets (p = 0.013 for Brazilians; p = 0.046 for ECLAMC; p = 10⁻⁶ for Latvians, and p = 0.003 for the 8,717 individuals). Tgfa was not expressed in the palatal tissues of Irf6 knockout mice. IRF6 and TGFA contribute to subsets of CL/P with specific dental anomalies. Moreover, this potential IRF6-TGFA interaction may account for as much as 1% to 10% of CL/P cases. The Irf6-knockout model further supports the evidence of IRF6-TGFA interaction found in humans.     

Cell-Autonomous and Non-cell-autonomous Function of Hox Genes Specify Segmental Neuroblast Identity in the Gnathal Region of the Embryonic CNS in Drosophila

During central nervous system (CNS) development neural stem cells (Neuroblasts, NBs) have to acquire an identity appropriate to their location. In thoracic and abdominal segments of Drosophila, the expression pattern of Bithorax-Complex Hox genes is known to specify the segmental identity of NBs prior to their delamination from the neuroectoderm. Compared to the thoracic, ground state segmental units in the head region are derived to different degrees, and the precise mechanism of segmental specification of NBs in this region is still unclear. We identified and characterized a set of serially homologous NB-lineages in the gnathal segments and used one of them (NB6-4 lineage) as a model to investigate the mechanism conferring segment-specific identities to gnathal NBs. We show that NB6-4 is primarily determined by the cell-autonomous function of the Hox gene Deformed (Dfd). Interestingly, however, it also requires a non-cell-autonomous function of labial and Antennapedia that are expressed in adjacent anterior or posterior compartments. We identify the secreted molecule Amalgam (Ama) as a downstream target of the Antennapedia-Complex Hox genes labial, Dfd, Sex combs reduced and Antennapedia. In conjunction with its receptor Neurotactin (Nrt) and the effector kinase Abelson tyrosine kinase (Abl), Ama is necessary in parallel to the cell-autonomous Dfd pathway for the correct specification of the maxillary identity of NB6-4. Both pathways repress CyclinE (CycE) and loss of function of either of these pathways leads to a partial transformation (40%), whereas simultaneous mutation of both pathways leads to a complete transformation (100%) of NB6-4 segmental identity. Finally, we provide genetic evidences, that the Ama-Nrt-Abl-pathway regulates CycE expression by altering the function of the Hippo effector Yorkie in embryonic NBs. The disclosure of a non-cell-autonomous influence of Hox genes on neural stem cells provides new insight into the process of segmental patterning in the developing CNS.     

Dominant Mutations in GRHL3 Cause Van der Woude Syndrome and Disrupt Oral Periderm Development

Mutations in interferon regulatory factor 6 (IRF6) account for ∼70% of cases of Van der Woude syndrome (VWS), the most common syndromic form of cleft lip and palate. In 8 of 45 VWS-affected families lacking a mutation in IRF6, we found coding mutations in grainyhead-like 3 (GRHL3). According to a zebrafish-based assay, the disease-associated GRHL3 mutations abrogated periderm development and were consistent with a dominant-negative effect, in contrast to haploinsufficiency seen in most VWS cases caused by IRF6 mutations. In mouse, all embryos lacking Grhl3 exhibited abnormal oral periderm and 17% developed a cleft palate. Analysis of the oral phenotype of double heterozygote (Irf6^+/−;Grhl3^+/−) murine embryos failed to detect epistasis between the two genes, suggesting that they function in separate but convergent pathways during palatogenesis. Taken together, our data demonstrated that mutations in two genes, IRF6 and GRHL3, can lead to nearly identical phenotypes of orofacial cleft. They supported the hypotheses that both genes are essential for the presence of a functional oral periderm and that failure of this process contributes to VWS.     

Interferon Regulatory Factor 6 Has a Protective Role in the Host Response to Endotoxic Shock

Interferon Regulatory Factor (IRF) 6, a member of the IRF family, is essential for epidermal and orofacial embryonic development. Irf6 is strongly expressed in keratinocytes, in which it regulates epidermal proliferation, differentiation, and migration. A recent role for Irf6 in Toll-like receptor 2-dependent chemokine gene expression was also reported in an epithelial cell line. However, a function for Irf6 in innate immune cells was not previously reported. In the present study, we investigated the expression and function of Irf6 in bone marrow-derived neutrophils and macrophages. We show here, using a conditional knockout of Irf6 in lysosymeM expressing cells, that Irf6 is required for resistance to LPS-induced endotoxic shock. In addition, Irf6-deficient bone marrow-derived neutrophils exhibited increased chemotactic index and velocity compared with wild-type cells in vitro. TLR4-specific KC and IL6 secretions were upregulated in Irf6-deficient bone marrow-derived macrophages in vitro. These cells also exhibited an increased level of phosphorylated IkBa. Collectively, our findings suggest a role for Irf6 in the resistance to endotoxic shock due to NFk-B-mediated alteration of cytokine production.     

Identification of a Van der Woude syndrome mutation in the cleft palate 1 mutant mouse

Mutations in Interferon Regulatory Factor 6 (IRF6) have been identified in two human allelic syndromes with cleft lip and/or palate: Van der Woude (VWS) and Popliteal Pterygium syndromes (PPS). Furthermore, common IRF6 haplotypes and single nucleotide polymorphisms (SNP) alleles are strongly associated with nonsyndromic clefting defects in multiple ethnic populations. Mutations in the mouse often provide good models for the study of human diseases and developmental processes. We identified the cleft palate 1 (clft1) mouse mutant in a forward genetic screen for phenotypes modeling human congenital disease. In the clft1 mutant, we have identified a novel missense point mutation in the mouse Irf6 gene, which confers an amino acid alteration that has been found in a VWS family. Phenotypic comparison of clft1 mutants to previously reported Irf6 mutant alleles demonstrates the Irf6^clft1 allele is a hypomorphic allele. The cleft palate seen in these mutants appears to be due to abnormal adhesion between the palate and tongue. The Irf6^clft1 allele provides the first mouse model for the study of an etiologic IRF6 missense mutation observed in a human VWS family. genesis 48:303–308, 2010. © 2010 Wiley‐Liss, Inc.     

Generation and characterization of a conditional allele of Interferon Regulatory Factor 6

Interferon Regulatory Factor 6 (IRF6) is a critical regulator of differentiation, proliferation, and migration of keratinocytes. Mutations in IRF6 cause two autosomal dominant disorders characterized by cleft lip with or without cleft palate. In addition, DNA variation in IRF6 confers significant risk for non‐syndromic cleft lip and palate. IRF6 is also implicated in adult onset development and disease processes, including mammary gland development and squamous cell carcinoma. Mice homozygous for a null allele of Irf6 die shortly after birth due to severe skin, limb, and craniofacial defects, thus impeding the study of gene function after birth. To circumvent this, a conditional allele of Irf6 was generated. To validate the functionality of the conditional allele, we used three “deleter” Cre strains: Gdf9‐Cre, CAG‐Cre, and Ella‐Cre. When Cre expression was driven by the Gdf9‐Cre or CAG‐Cre transgenes, 100% recombination was observed as indicated by DNA genotyping and phenotyping. In contrast, use of the Ella‐Cre transgenic line resulted in incomplete recombination, despite expression at the one‐cell stage. In sum, we generated a novel tool to delete Irf6 in a tissue specific fashion, allowing for study of gene function past perinatal stages. However, recombination efficiency of this allele was dictated by the Cre‐driver used.     

Mesoderm progenitor cells of common origin contribute to the head musculature and the cardiac outflow tract

During early embryogenesis, heart and skeletal muscle progenitor cells are thought to derive from distinct regions of the mesoderm (i.e. the lateral plate mesoderm and paraxial mesoderm, respectively). In the present study, we have employed both in vitro and in vivo experimental systems in the avian embryo to explore how mesoderm progenitors in the head differentiate into both heart and skeletal muscles. Using fate-mapping studies, gene expression analyses, and manipulation of signaling pathways in the chick embryo, we demonstrate that cells from the cranial paraxial mesoderm contribute to both myocardial and endocardial cell populations within the cardiac outflow tract. We further show that Bmp signaling affects the specification of mesoderm cells in the head: application of Bmp4, both in vitro and in vivo, induces cardiac differentiation in the cranial paraxial mesoderm and blocks the differentiation of skeletal muscle precursors in these cells. Our results demonstrate that cells within the cranial paraxial mesoderm play a vital role in cardiogenesis, as a new source of cardiac progenitors that populate the cardiac outflow tract in vivo. A deeper understanding of mesodermal lineage specification in the vertebrate head is expected to provide insights into the normal, as well as pathological, aspects of heart and craniofacial development.     

Bmp signaling promotes intermediate mesoderm gene expression in a dose-dependent, cell-autonomous and translation-dependent manner

The intermediate mesoderm lies between the somites and the lateral plate and is the source of all kidney tissue in the developing vertebrate embryo. While bone morphogenetic protein (Bmp) signaling is known to regulate mesodermal cell type determination along the medio-lateral axis, its role in intermediate mesoderm formation has not been well characterized. The current study finds that low and high levels of Bmp ligand are both necessary and sufficient to activate intermediate and lateral mesodermal gene expression, respectively, both in vivo and in vitro. Dose-dependent activation of intermediate and lateral mesodermal genes by Bmp signaling is cell-autonomous, as demonstrated by electroporation of the avian embryo with constitutively active Bmp receptors driven by promoters of varying strengths. In explant cultures, Bmp activation of Odd-skipped related 1 (Odd-1), the earliest known gene expressed in the intermediate mesoderm, is blocked by cyclohexamide, indicating that the activation of Odd-1 by Bmp signaling is translation-dependent. The data from this study are integrated with that of other studies to generate a model for the role of Bmp signaling in trunk mesodermal patterning in which low levels of Bmp activate intermediate mesoderm gene expression by inhibition of repressors present in medial mesoderm, whereas high levels of Bmp repress both medial and intermediate mesoderm gene expression and activate lateral plate genes.     

Direct and indirect requirements of Shh/Gli signaling in early pituitary development

Induction of early pituitary progenitors is achieved through combined activities of signals from adjacent embryonic tissues. Previous studies have identified a requirement for oral ectoderm derived Sonic Hedgehog (Shh) in specification and/or proliferation of early pituitary progenitors, however how different Gli genes mediate Shh signaling to control pituitary progenitor development has not yet been determined. Here we show that Gli2, which encodes a major Gli activator, is required for proliferation of specific groups of pituitary progenitors but not for initial dorsoventral patterning. We further show that the action of Gli2 occurs prior to the closure of Rathke' pouch. Lastly, we show that Shh/Gli2 signaling controls the diencephalic expression of Bone morphogenetic protein 4 (Bmp4) and Fibroblast growth factor 8 (Fgf8), two genes that are known to play critical roles in patterning and growth of Rathke's pouch. Our results therefore suggest both cell-autonomous and non-cell-autonomous requirements for Gli2 in regulation of pituitary progenitor specification, proliferation and differentiation.     

Chiari Malformation Type I: A Case-Control Association Study of 58 Developmental Genes

Chiari malformation type I (CMI) is a disorder characterized by hindbrain overcrowding into an underdeveloped posterior cranial fossa (PCF), often causing progressive neurological symptoms. The etiology of CMI remains unclear and is most likely multifactorial. A putative genetic contribution to CMI is suggested by familial aggregation and twin studies. Experimental models and human morphometric studies have suggested an underlying paraxial mesoderm insufficiency. We performed a case-control association study of 303 tag single nucleotide polymorphisms (SNP) across 58 candidate genes involved in early paraxial mesoderm development in a sample of 415 CMI patients and 524 sex-matched controls. A subgroup of patients diagnosed with classical, small-PCF CMI by means of MRI-based PCF morphometry (n = 186), underwent additional analysis. The genes selected are involved in signalling gradients occurring during segmental patterning of the occipital somites (FGF8, Wnt, and retinoic acid pathways and from bone morphogenetic proteins or BMP, Notch, Cdx and Hox pathways) or in placental angiogenesis, sclerotome development or CMI-associated syndromes. Single-marker analysis identified nominal associations with 18 SNPs in 14 genes (CDX1, FLT1, RARG, NKD2, MSGN1, RBPJ1, FGFR1, RDH10, NOG, RARA, LFNG, KDR, ALDH1A2, BMPR1A) considering the whole CMI sample. None of these overcame corrections for multiple comparisons, in contrast with four SNPs in CDX1, FLT1 and ALDH1A2 in the classical CMI group. Multiple marker analysis identified a risk haplotype for classical CMI in ALDH1A2 and CDX1. Furthermore, we analyzed the possible contributions of the most significantly associated SNPs to different PCF morphometric traits. These findings suggest that common variants in genes involved in somitogenesis and fetal vascular development may confer susceptibility to CMI.