RESUMO
Decrease in Cdx dosage in an allelic series of mouse Cdx mutants leads to progressively more severe posterior vertebral defects. These defects are corrected by posterior gain of function of the Wnt effector Lef1. Precocious expression of Hox paralogous 13 genes also induces vertebral axis truncation by antagonizing Cdx function. We report here that the phenotypic similarity also applies to patterning of the caudal neural tube and uro-rectal tracts in Cdx and Wnt3a mutants, and in embryos precociously expressing Hox13 genes. Cdx2 inactivation after placentation leads to posterior defects, including incomplete uro-rectal septation. Compound mutants carrying one active Cdx2 allele in the Cdx4-null background (Cdx2/4), transgenic embryos precociously expressing Hox13 genes and a novel Wnt3a hypomorph mutant all manifest a comparable phenotype with similar uro-rectal defects. Phenotype and transcriptome analysis in early Cdx mutants, genetic rescue experiments and gene expression studies lead us to propose that Cdx transcription factors act via Wnt signaling during the laying down of uro-rectal mesoderm, and that they are operative in an early phase of these events, at the site of tissue progenitors in the posterior growth zone of the embryo. Cdx and Wnt mutations and premature Hox13 expression also cause similar neural dysmorphology, including ectopic neural structures that sometimes lead to neural tube splitting at caudal axial levels. These findings involve the Cdx genes, canonical Wnt signaling and the temporal control of posterior Hox gene expression in posterior morphogenesis in the different embryonic germ layers. They shed a new light on the etiology of the caudal dysplasia or caudal regression range of human congenital defects.
Assuntos
Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Tubo Neural/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteínas Wnt/metabolismo , Animais , Fator de Transcrição CDX2 , Forma Celular , Feminino , Proteínas Hedgehog/metabolismo , Proteínas de Homeodomínio/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Tubo Neural/citologia , Fatores de Transcrição/genética , Tretinoína/metabolismo , Proteínas Wnt/genética , Proteína Wnt3 , Proteína Wnt3ARESUMO
The mouse Btaf1 gene, an ortholog of yeast MOT1, encodes a highly conserved general transcription factor. The function of this SNF2-like ATPase has been studied mainly in yeast and human cells, which has revealed that it binds directly to TBP, forming the B-TFIID complex. This complex binds to core promoters of RNA polymerase II-transcribed genes and, of crucial importance, BTAF1-TBP interactions have been shown to affect the kinetics of TBP-promoter interactions. Here we report the isolation of a mouse line carrying a Btaf1 allele containing an ENU-induced point mutation that causes a substitution mutation in the BTAF1 ATPase domain. Embryos homozygous for this loss-of-function mutation appear to be morphologically normal until early somite stages, but die between embryonic days 9 and 10.5 displaying growth arrest and edema. Analyses in vitro suggest that the altered protein is less stable and, independent from this, functionally impaired in releasing of TBP from chromatin, but not in binding to TBP.
Assuntos
Adenosina Trifosfatases/genética , Gastrulação , Genes Letais , Mutação Puntual , Fatores de Transcrição/genética , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Cromatina/metabolismo , Etilnitrosoureia , Feminino , Perfilação da Expressão Gênica , Estudos de Associação Genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Proteínas Mutantes/metabolismo , Fenótipo , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia Estrutural de Proteína , Fatores Associados à Proteína de Ligação a TATA , Proteína de Ligação a TATA-Box/metabolismo , Fator de Transcrição TFIID , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
Directing the orientation of cells in three dimensions is a fundamental aspect of many of the processes underlying the generation of the appropriate shape and function of tissues and organs during embryonic development. In an epithelium, this requires not only the establishment of apicobasal polarity, but also cell arrangement in a specific direction in the plane of the cell sheet. The molecular pathway central to regulating this planar cell polarity (PCP) was originally discovered in the fruit fly Drosophila melanogaster and has more recently been shown to act in a highly analogous way in vertebrates, involving a strongly overlapping set of genes. Mutant studies and molecular analyses have led to insights into the role of ordered planar cell polarity in the development of a wide variety of organs and tissues. In this review, we give an overview of recent developments in the study of planar polarity signaling in vertebrates.
Assuntos
Polaridade Celular/fisiologia , Vertebrados/embriologia , Animais , Polaridade Celular/genética , Drosophila , Humanos , Defeitos do Tubo Neural/metabolismo , Defeitos do Tubo Neural/patologia , Órgão Espiral/citologia , Órgão Espiral/embriologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Vertebrados/crescimento & desenvolvimento , Vertebrados/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMO
Objective Deregulation of the Wnt signalling pathway by mutations in the Apc or ß-catenin genes underlies colorectal carcinogenesis. As a result, ß-catenin stabilises, translocates to the nucleus, and activates gene transcription. Intestinal tumours show a heterogeneous pattern of nuclear ß-catenin, with the highest levels observed at the invasion front. Activation of receptor tyrosine kinases in these tumour areas by growth factors expressed by surrounding stromal cells phosphorylate ß-catenin at tyrosine residues, which is thought to increase ß-catenin nuclear translocation and tumour invasiveness. This study investigates the relevance of ß-catenin tyrosine phosphorylation for Wnt signalling and intestinal tumorigenesis in vivo. Design A conditional knock-in mouse model was generated into which the phospho-mimicking Y654E modification in the endogenous ß-catenin gene was introduced. Results This study provided in vivo evidence that ß-catenin(E654) is characterised by reduced affinity for cadherins, increased signalling and strongly increased phosphorylation at serine 675 by protein kinase A (PKA). In addition, homozygosity for the ß-catenin(E654) targeted allele caused embryonic lethality, whereas heterozygosity predisposed to intestinal tumour development, and strongly enhanced Apc-driven intestinal tumour initiation associated with increased nuclear accumulation of ßcatenin. Surprisingly, the expression of ß-catenin(E654) did not affect histological grade or induce tumour invasiveness. Conclusions A thus far unknown mechanism was uncovered in which Y654 phosphorylation of ß-catenin facilitates additional phosphorylation at serine 675 by PKA. In addition, in contrast to the current belief that ß-catenin Y654 phosphorylation increases tumour progression to a more invasive phenotype, these results show that it rather increases tumour initiation by enhancing Wnt signalling.
Assuntos
Transformação Celular Neoplásica/metabolismo , Neoplasias Colorretais/metabolismo , Proteínas Wnt/fisiologia , beta Catenina/metabolismo , Adenoma/genética , Adenoma/metabolismo , Animais , Células COS , Caderinas/metabolismo , Membrana Celular/metabolismo , Transformação Celular Neoplásica/genética , Chlorocebus aethiops , Neoplasias Colorretais/genética , Proteínas Quinases Dependentes de AMP Cíclico/farmacologia , Perda do Embrião/genética , Técnicas de Introdução de Genes , Genes APC , Genótipo , Heterozigoto , Homozigoto , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologiaRESUMO
BACKGROUND: An omphalocele is one of the major ventral body wall malformations and is characterized by abnormally herniated viscera from the body trunk. It has been frequently found to be associated with other structural malformations, such as genitourinary malformations and digit abnormalities. In spite of its clinical importance, the etiology of omphalocele formation is still controversial. Hedgehog (Hh) signaling is one of the essential growth factor signaling pathways involved in the formation of the limbs and urogenital system. However, the relationship between Hh signaling and ventral body wall formation remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: To gain insight into the roles of Hh signaling in ventral body wall formation and its malformation, we analyzed phenotypes of mouse mutants of Sonic hedgehog (Shh), GLI-Kruppel family member 3 (Gli3) and Aristaless-like homeobox 4 (Alx4). Introduction of additional Alx4(Lst) mutations into the Gli3(Xt/Xt) background resulted in various degrees of severe omphalocele and pubic diastasis. In addition, loss of a single Shh allele restored the omphalocele and pubic symphysis of Gli3(Xt/+); Alx4(Lst/Lst) embryos. We also observed ectopic Hh activity in the ventral body wall region of Gli3(Xt/Xt) embryos. Moreover, tamoxifen-inducible gain-of-function experiments to induce ectopic Hh signaling revealed Hh signal dose-dependent formation of omphaloceles. CONCLUSIONS/SIGNIFICANCE: We suggest that one of the possible causes of omphalocele and pubic diastasis is ectopically-induced Hh signaling. To our knowledge, this would be the first demonstration of the involvement of Hh signaling in ventral body wall malformation and the genetic rescue of omphalocele phenotypes.
Assuntos
Hérnia Umbilical/etiologia , Hérnia Umbilical/terapia , Transdução de Sinais , Animais , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Desenvolvimento Embrionário , Terapia Genética/métodos , Proteínas Hedgehog/genética , Proteínas Hedgehog/farmacologia , Proteínas Hedgehog/fisiologia , Hérnia Umbilical/patologia , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Mutantes , Mutação , Proteínas do Tecido Nervoso/genética , Fenótipo , Diástase da Sínfise Pubiana , Proteína Gli3 com Dedos de ZincoRESUMO
Among the cellular properties that are essential for the organization of tissues during animal development, the importance of cell polarity in the plane of epithelial sheets has become increasingly clear in the past decades. Planar cell polarity (PCP) signaling in vertebrates has indispensable roles in many aspects of their development, in particular, controlling alignment of various types of epithelial cells. Disrupted PCP has been linked to developmental defects in animals and to human pathology. Neural tube closure defects (NTD) and disorganization of the mechanosensory cells of the organ of Corti are commonly known consequences of disturbed PCP signaling in mammals. We report here a typical PCP phenotype in a mouse mutant for the Sec24b gene, including the severe NTD craniorachischisis, abnormal arrangement of outflow tract vessels and disturbed development of the cochlea. In addition, we observed genetic interaction between Sec24b and the known PCP gene, scribble. Sec24b is a component of the COPII coat protein complex that is part of the endoplasmic reticulum (ER)-derived transport vesicles. Sec24 isoforms are thought to be directly involved in cargo selection, and we present evidence that Sec24b deficiency specifically affects transport of the PCP core protein Vangl2, based on experiments in embryos and in cultured primary cells.
Assuntos
Polaridade Celular , Mutação , Proteínas do Tecido Nervoso/metabolismo , Defeitos do Tubo Neural/metabolismo , Transdução de Sinais/fisiologia , Proteínas de Transporte Vesicular/metabolismo , Animais , Aorta Torácica/anormalidades , Células Cultivadas , Cóclea/anormalidades , Cóclea/anatomia & histologia , Cóclea/embriologia , Embrião de Mamíferos/anormalidades , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/fisiologia , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Feminino , Fibroblastos/citologia , Fibroblastos/fisiologia , Cardiopatias Congênitas , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Defeitos do Tubo Neural/genética , Defeitos do Tubo Neural/patologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMO
The role of the aristaless-related homeobox gene Alx4 in antero-posterior (AP-) patterning of the developing vertebrate limb has remained somewhat elusive. Polydactyly of Alx4 mutant mice is known to be accompanied by ectopic anterior expression of genes like Shh, Fgf4 and 5'Hoxd. We reported previously that polydactyly in Alx4 mutant mice requires SHH signaling, but we now show that in early Alx4-/- limb buds the anterior ectopic expression of Fgf4 and Hoxd13, and therefore disruption of AP-patterning, occurs independently of SHH signaling. To better understand how Alx4 functions in the pathways that regulate AP-patterning, we also studied genomic regulatory sequences that are capable of directing expression of a reporter gene in a pattern corresponding to endogenous Alx4 expression in anterior limb bud mesenchyme. We observed, as expected for authentic Alx4 expression, expansion of reporter construct expression in a Shh-/- background. Total lack of reporter expression in a Gli3-/- background confirms the existence of Gli3-dependent and -independent Alx4 expression in the limb bud. Apparently, these two modules of Alx4 expression are linked to dissimilar functions.
Assuntos
Padronização Corporal/genética , Extremidades/embriologia , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais/genética , Transativadores/metabolismo , Animais , Primers do DNA , Genômica/métodos , Proteínas Hedgehog , Hibridização In Situ , Camundongos , Camundongos Knockout , Plasmídeos/genética , Proteína Gli3 com Dedos de Zinco , beta-GalactosidaseRESUMO
The diverse cellular contributions to the skeletal elements of the vertebrate shoulder and pelvic girdles during embryonic development complicate the study of their patterning. Research in avian embryos has recently clarified part of the embryological basis of shoulder formation. Although dermomyotomal cells provide the progenitors of the scapular blade, local signals appear to have an essential guiding role in this process. These signals differ from those that are known to pattern the more distal appendicular skeleton. We have studied the impact of Tbx15, Gli3, Alx4 and related genes on formation of the skeletal elements of the mouse shoulder and pelvic girdles. We observed severe reduction of the scapula in double and triple mutants of these genes. Analyses of a range of complex genotypes revealed aspects of their genetic relationship, as well as functions that had been previously masked due to functional redundancy. Tbx15 and Gli3 appear to have synergistic functions in formation of the scapular blade. Scapular truncation in triple mutants of Tbx15, Alx4 and Cart1 indicates essential functions for Alx4 and Cart1 in the anterior part of the scapula, as opposed to Gli3 function being linked to the posterior part. Especially in Alx4/Cart1 mutants, the expression of markers such as Pax1, Pax3 and Scleraxis is altered prior to stages when anatomical aberrations are visible in the shoulder region. This suggests a disorganization of the proximal limb bud and adjacent flank mesoderm, and is likely to reflect the disruption of a mechanism providing positional cues to guide progenitor cells to their destination in the pectoral girdle.
Assuntos
Osso e Ossos/anormalidades , Osso e Ossos/embriologia , Proteínas de Homeodomínio/genética , Ombro/embriologia , Proteínas com Domínio T/genética , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição Kruppel-Like , Camundongos , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Gli3 com Dedos de ZincoRESUMO
During embryogenesis, target genes of retinoid signaling are able to respond differently to identical concentrations of retinoids. Small differences in the retinoic acid response elements (RARE) may be essential for these distinct responses. Recently, we identified a RARE in a Hox enhancer (dubbed distal element) that is active relatively late during mouse development. We now show that the RARE motif in the distal element is necessary and sufficient for the induction of gene expression by retinoic acid (RA) in P19 embryonic carcinoma cells. Furthermore, the significance of these results was established by RA treatment of transgenic mouse lines carrying the distal element containing the wild-type or a mutated RARE. We compared the in vitro activity of the distal element-RARE with that of the direct repeat with 5-bp spacer RARE of the RARbeta2 gene, which is active during early during mouse development. We found that these RAREs, despite their similarity, responded differently to RA. By making single point mutations we show that the specificity resides in their retinoid X receptor-binding sites and is determined by base pairs located just outside the RARE consensus sequence. We suggest that the context of RARE motifs is important for the distinct transcriptional activities of genes under control of retinoid signaling.
Assuntos
Regulação da Expressão Gênica/genética , Genes Homeobox , Elementos de Resposta/fisiologia , Tretinoína/fisiologia , Animais , Embrião de Mamíferos , Genes Reporter , Camundongos , Mutação , Regiões Promotoras Genéticas , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/fisiologia , Elementos de Resposta/genética , Receptores X de Retinoides , Fatores de TranscriçãoRESUMO
Distal limb development and specification of digit identities in tetrapods are under the control of a mesenchymal organizer called the polarizing region. Sonic Hedgehog (SHH) is the morphogenetic signal produced by the polarizing region in the posterior limb bud. Ectopic anterior SHH signaling induces digit duplications and has been suspected as a major cause underlying congenital malformations that result in digit polydactyly. Here, we report that the polydactyly of Gli3-deficient mice arises independently of SHH signaling. Disruption of one or both Gli3 alleles in mouse embryos lacking Shh progressively restores limb distal development and digit formation. Our genetic analysis indicates that SHH signaling counteracts GLI3-mediated repression of key regulator genes, cell survival, and distal progression of limb bud development.