RESUMO
Nodal-related signals comprise a subclass of the transforming growth factor-beta (TGF-beta) superfamily and regulate key events in vertebrate embryogenesis, including mesoderm formation, establishment of left-right asymmetry and neural patterning [1-8]. Nodal ligands are thought to act with EGF-CFC protein co-factors to activate activin type I and II or related receptors, which phosphorylate Smad2 and trigger nuclear translocation of a Smad2/4 complex [8-12]. The winged-helix transcription factor forkhead activin signal transducer-1 (Fast-1) acts as a co-factor for Smad2 [12-20]. Xenopus Fast-1 is thought to function as a transcriptional effector of Nodal signals during mesoderm formation [17], but no mutations in the Fast-1 gene have been identified. We report the identification of the zebrafish fast1 gene and show that it is disrupted in schmalspur (sur) mutants, which have defects in the development of dorsal midline cell types and establishment of left-right asymmetry [21-25]. We find that prechordal plate and notochord are strongly reduced in maternal-zygotic sur mutants, whereas other mesendodermal structures are present - a less severe phenotype than that caused by complete loss of Nodal signaling. These results show that fast1 is required for development of dorsal axial structures and left-right asymmetry, and suggest that Nodal signals act through Fast1-dependent and independent pathways.
Assuntos
Padronização Corporal , Proteínas de Ligação a DNA/fisiologia , Fatores de Transcrição/fisiologia , Proteínas de Peixe-Zebra , Peixe-Zebra/embriologia , Sequência de Aminoácidos , Animais , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Desenvolvimento Embrionário , Fatores de Transcrição Forkhead , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Hemizygous interstitial deletions in human chromosome 22q11 are associated with velocardiofacial syndrome and DiGeorge syndrome and lead to multiple congenital abnormalities, including cardiovascular defects. The gene(s) responsible for these disorders is thought to reside in a 1.5-Mb region of 22q11 in which 27 genes have been identified. We have used Cre-mediated recombination of LoxP sites in embryonic stem cells and mice to generate a 550-kb deletion encompassing 16 of these genes in the corresponding region on mouse chromosome 16. Mice heterozygous for this deletion are normal and do not exhibit cardiovascular abnormalities. Because mice with a larger deletion on mouse chromosome 16 do have heart defects, the results allow us to exclude these 16 genes as being solely, or in combination among themselves, responsible for the cardiovascular abnormalities in velocardiofacial/DiGeorge syndrome. We also generated mice with a duplication of the 16 genes that may help dissect the genetic basis of "cat eye" and derivative 22 syndromes that are characterized by extra copies of portions of 22q11, including these 16 genes. We also describe a strategy for selecting cell lines with defined chromosomal rearrangements. The method is based on reconstitution of a dominant selection marker after Cre-mediated recombination of LoxP sites. Therefore it should be widely applicable to many cell lines.
Assuntos
Anormalidades Múltiplas/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 22 , Síndrome de DiGeorge/genética , Deleção de Genes , Animais , Anormalidades Cardiovasculares/genética , Duplicação Gênica , Marcadores Genéticos , Cardiopatias Congênitas/genética , Heterozigoto , Humanos , Camundongos , Camundongos Mutantes , Células-Tronco , TrissomiaRESUMO
In vertebrate embryos, maternal (beta)-catenin protein activates the expression of zygotic genes that establish the dorsal axial structures. Among the zygotically acting genes with key roles in the specification of dorsal axial structures are the homeobox gene bozozok (boz) and the nodal-related (TGF-(beta) family) gene squint (sqt). Both genes are expressed in the dorsal yolk syncytial layer, a source of dorsal mesoderm inducing signals, and mutational analysis has indicated that boz and sqt are required for dorsal mesoderm development. Here we examine the regulatory interactions among boz, sqt and a second nodal-related gene, cyclops (cyc). Three lines of evidence indicate that boz and sqt act in parallel to specify dorsal mesoderm and anterior neuroectoderm. First, boz requires sqt function to induce high levels of ectopic dorsal mesoderm, consistent with sqt acting either downstream or in parallel to boz. Second, sqt mRNA is expressed in blastula stage boz mutants, indicating that boz is not essential for activation of sqt transcription, and conversely, boz mRNA is expressed in blastula stage sqt mutants. Third, boz;sqt double mutants have a much more severe phenotype than boz and sqt single mutants. Double mutants consistently lack the anterior neural tube and axial mesoderm, and ventral fates are markedly expanded. Expression of chordin and noggin1 is greatly reduced in boz;sqt mutants, indicating that the boz and sqt pathways have overlapping roles in activating secreted BMP antagonists. In striking contrast to boz;sqt double mutants, anterior neural fates are specified in boz;sqt;cyc triple mutants. This indicates that cyc represses anterior neural development, and that boz and sqt counteract this repressive function. Our results support a model in which boz and sqt act in parallel to induce dorsalizing BMP-antagonists and to counteract the repressive function of cyc in neural patterning.
Assuntos
Padronização Corporal , Ectoderma/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Mesoderma/fisiologia , Sistema Nervoso/embriologia , Fator de Crescimento Transformador beta/genética , Proteínas de Peixe-Zebra , Peixe-Zebra/embriologia , Animais , Embrião não Mamífero/fisiologia , Genótipo , Proteínas de Homeodomínio/metabolismo , Mutação , Proteína Nodal , Ligantes da Sinalização Nodal , Fator de Crescimento Transformador beta/metabolismoRESUMO
We have generated the first monoclonal antibodies (MAbs) to Armadillo repeat gene deleted in velo-cardiofacial syndrome (ARVCF), a recently identified Armadillo repeat-containing protein closely related to the catenin p120ctn. Six ARVCF-specific MAbs were characterized for isotype, species cross-reactivity, and utility in assays including immunofluorescence, immunoprecipitation, and Western blotting. All six antibodies were isotyped as IgG1 and several cross-reacted with ARVCF from a variety of species including human, rat, dog, and monkey, but not mouse. Importantly, none of the ARVCF MAbs cross-reacted with p120ctn, despite the high homology between these proteins. MAbs 3B2 and 4B1 were consistently the best in all applications and will provide valuable tools for further study of the role of ARVCF in cells.
Assuntos
Anormalidades Múltiplas/genética , Anormalidades Múltiplas/imunologia , Anticorpos Monoclonais/imunologia , Tatus/genética , Tatus/imunologia , Fosfoproteínas/imunologia , Fosfoproteínas/metabolismo , Sequência de Aminoácidos , Animais , Afinidade de Anticorpos , Formação de Anticorpos , Sítios de Ligação , Western Blotting , Caderinas/metabolismo , Cateninas , Moléculas de Adesão Celular/análise , Moléculas de Adesão Celular/imunologia , Moléculas de Adesão Celular/metabolismo , Linhagem Celular/química , Anormalidades Craniofaciais/genética , Anormalidades Craniofaciais/imunologia , Síndrome de DiGeorge/genética , Síndrome de DiGeorge/imunologia , Cães , Imunofluorescência , Deleção de Genes , Haplorrinos , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/imunologia , Humanos , Hibridomas/imunologia , Junções Intercelulares/química , Camundongos , Dados de Sequência Molecular , Testes de Precipitina , Ratos , Sequências Repetitivas de Aminoácidos/genética , Sequências Repetitivas de Aminoácidos/imunologia , Sequências Repetitivas de Ácido Nucleico/genética , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Insuficiência Velofaríngea/genética , Insuficiência Velofaríngea/imunologia , delta CateninaRESUMO
Genetic screens in zebrafish (Danio rerio) have isolated mutations in hundreds of genes with essential functions. To facilitate the identification of candidate genes for these mutations, we have genetically mapped 104 genes and expressed sequence tags by scoring single-strand conformational polymorphisms in a panel of haploid siblings. To integrate this map with existing genetic maps, we also scored 275 previously mapped genes, microsatellites, and sequence-tagged sites in the same haploid panel. Systematic phylogenetic analysis defined likely mammalian orthologs of mapped zebrafish genes, and comparison of map positions in zebrafish and mammals identified significant conservation of synteny. This comparative analysis also identified pairs of zebrafish genes that appear to be orthologous to single mammalian genes, suggesting that these genes arose in a genome duplication that occurred in the teleost lineage after the divergence of fish and mammal ancestors. This comparative map analysis will be useful in predicting the locations of zebrafish genes from mammalian gene maps and in understanding the evolution of the vertebrate genome.
Assuntos
Ligação Genética , Mapeamento Físico do Cromossomo/métodos , Peixe-Zebra/genética , Animais , Cromossomos Humanos , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Mutação , FilogeniaRESUMO
The dorsal gastrula organizer plays a fundamental role in establishment of the vertebrate axis. We demonstrate that the zebrafish bozozok (boz) locus is required at the blastula stages for formation of the embryonic shield, the equivalent of the gastrula organizer and expression of multiple organizer-specific genes. Furthermore, boz is essential for specification of dorsoanterior embryonic structures, including notochord, prechordal mesendoderm, floor plate and forebrain. We report that boz mutations disrupt the homeobox gene dharma. Overexpression of boz in the extraembryonic yolk syncytial layer of boz mutant embryos is sufficient for normal development of the overlying blastoderm, revealing an involvement of extraembryonic structures in anterior patterning in fish similarly to murine embryos. Epistatic analyses indicate that boz acts downstream of beta-catenin and upstream to TGF-beta signaling or in a parallel pathway. These studies provide genetic evidence for an essential function of a homeodomain protein in beta-catenin-mediated induction of the dorsal gastrula organizer and place boz at the top of a hierarchy of zygotic genes specifying the dorsal midline of a vertebrate embryo.
Assuntos
Proteínas de Homeodomínio/genética , Transativadores , Proteínas de Peixe-Zebra , Peixe-Zebra/embriologia , Animais , Encéfalo/embriologia , Proteínas do Citoesqueleto/genética , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Hibridização In Situ , Mutação , Notocorda/embriologia , RNA Mensageiro/metabolismo , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/genética , beta CateninaRESUMO
The vertebrate body plan is established during gastrulation, when cells move inwards to form the mesodermal and endodermal germ layers. Signals from a region of dorsal mesoderm, which is termed the organizer, pattern the body axis by specifying the fates of neighbouring cells. The organizer is itself induced by earlier signals. Although members of the transforming growth factor-beta (TGF-beta) and Wnt families have been implicated in the formation of the organizer, no endogenous signalling molecule is known to be required for this process. Here we report that the zebrafish squint (sqt) and cyclops (cyc) genes have essential, although partly redundant, functions in organizer development and also in the formation of mesoderm and endoderm. We show that the sqt gene encodes a member of the TGF-beta superfamily that is related to mouse nodal. cyc encodes another nodal-related proteins, which is consistent with our genetic evidence that sqt and cyc have overlapping functions. The sqt gene is expressed in a dorsal region of the blastula that includes the extraembryonic yolk syncytial layer (YSL). The YSL has been implicated as a source of signals that induce organizer development and mesendoderm formation. Misexpression of sqt RNA within the embryo or specifically in the YSL induces expanded or ectopic dorsal mesoderm. These results establish an essential role for nodal-related signals in organizer development and mesendoderm formation.
Assuntos
Padronização Corporal/genética , Indução Embrionária/genética , Camadas Germinativas/fisiologia , Proteínas Repressoras , Transdução de Sinais , Fatores de Transcrição , Fator de Crescimento Transformador beta/fisiologia , Proteínas de Peixe-Zebra , Sequência de Aminoácidos , Animais , Blastocisto/fisiologia , Mapeamento Cromossômico , Gástrula/fisiologia , Proteína Goosecoid , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Mutação , Ligantes da Sinalização Nodal , Óvulo/metabolismo , Fator de Crescimento Transformador beta/genética , Peixe-ZebraRESUMO
Velo-cardio-facial syndrome (VCFS) is a relatively common developmental disorder characterized by craniofacial anomalies and conotruncal heart defects. Many VCFS patients have hemizygous deletions for a part of 22q11, suggesting that haploinsufficiency in this region is responsible for its etiology. Because most cases of VCFS are sporadic, portions of 22q11 may be prone to rearrangement. To understand the molecular basis for chromosomal deletions, we defined the extent of the deletion, by genotyping 151 VCFS patients and performing haplotype analysis on 105, using 15 consecutive polymorphic markers in 22q11. We found that 83% had a deletion and >90% of these had a similar approximately 3 Mb deletion, suggesting that sequences flanking the common breakpoints are susceptible to rearrangement. We found no correlation between the presence or size of the deletion and the phenotype. To further define the chromosomal breakpoints among the VCFS patients, we developed somatic hybrid cell lines from a set of VCFS patients. An 11-kb resolution physical map of a 1,080-kb region that includes deletion breakpoints was constructed, incorporating genes and expressed sequence tags (ESTs) isolated by the hybridization selection method. The ordered markers were used to examine the two separated copies of chromosome 22 in the somatic hybrid cell lines. In some cases, we were able to map the chromosome breakpoints within a single cosmid. A 480-kb critical region for VCFS has been delineated, including the genes for GSCL, CTP, CLTD, HIRA, and TMVCF, as well as a number of novel ordered ESTs.
Assuntos
Aberrações Cromossômicas/genética , Deleção Cromossômica , Cromossomos Humanos Par 22/genética , Anormalidades Craniofaciais/genética , Cardiopatias Congênitas/genética , Anormalidades Múltiplas/genética , Transtornos Cromossômicos , Mapeamento Cromossômico , Fissura Palatina/genética , Marcadores Genéticos , Genótipo , Humanos , Células Híbridas , Fenótipo , RNA Mensageiro/análise , Sitios de Sequências Rotuladas , SíndromeRESUMO
Velo-cardio-facial syndrome (VCFS) and DiGeorge syndrome (DGS) are characterized by a wide spectrum of phenotypes including cleft palate, conotruncal heart defects, and facial dysmorphology. Hemizygosity for a portion of chromosome 22q11 has been detected in 80-85% of VCFS/DGS patients. Using a cDNA selection protocol, we have identified a new gene, TMVCF (transmembrane protein deleted in VCFS), which maps to the deleted interval. The genomic locus is positioned between polymorphic markers D22S944 and D22S941. TMVCF encodes a small protein of 219 amino acids that is predicted to contain two membrane-spanning domains. TMVCF is expressed abundantly in human adult lung, heart, and skeletal muscle, and transcripts can be detected at least as early as Day 9 of mouse development.
Assuntos
Anormalidades Múltiplas/genética , Cromossomos Humanos Par 22/genética , Proteínas de Membrana/genética , Adulto , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Claudina-5 , Fissura Palatina/genética , Clonagem Molecular , DNA Complementar/genética , Desenvolvimento Embrionário e Fetal/genética , Face/anormalidades , Cardiopatias Congênitas/genética , Humanos , Camundongos , Dados de Sequência Molecular , Deleção de Sequência , Homologia de Sequência de Aminoácidos , SíndromeRESUMO
Velo-cardio-facial syndrome (VCFS) and DiGeorge syndrome (DGS) are characterized by a wide spectrum of phenotypes, including conotruncal heart defects, cleft palate, and facial dysmorphology. Hemizygosity for a portion of chromosome 22q11 has been detected in 80-85% of VCFS/DGS patients. Both syndromes are thought to be the result of a developmental field defect. Using two independent gene-isolation procedures, we isolated a new catenin family member termed ARVCF (armadillo repeat gene deleted in VCFS) from the interval deleted in VCFS. ARVCF encodes a protein of 962 amino acids that contains a coiled coil domain and 10 tandem armadillo repeats. The primary structure of the protein is most closely related to the murine catenin p120CAS, which suggests a role for ARVCF in protein-protein interactions at adherens junctions. ARVCF is expressed ubiquitously in all fetal and adult tissues examined. This gene is hemizygous in all VCFS patients with interstitial deletions. Based on the physical location and potential functions of ARVCF, we suggest that hemizygosity at this locus may play a role in the etiology of some of the phenotypes associated with VCFS.
Assuntos
Anormalidades Múltiplas/genética , Proteínas do Citoesqueleto/genética , Deleção de Genes , Família Multigênica , Adulto , Sequência de Aminoácidos , Animais , Sequência de Bases , Cateninas , Moléculas de Adesão Celular/genética , Fissura Palatina/genética , Sequência Conservada , DNA Complementar/genética , Síndrome de DiGeorge/genética , Face/anormalidades , Feto/metabolismo , Expressão Gênica , Cardiopatias Congênitas/genética , Humanos , Camundongos , Dados de Sequência Molecular , Fenótipo , Fosfoproteínas/genética , Especificidade da Espécie , Síndrome , delta CateninaRESUMO
Velocardiofacial syndrome (VCFS) is a developmental disorder characterized by conotruncal heart defects, craniofacial anomalies, and learning disabilities. VCFS is phenotypically related to DiGeorge syndrome (DGS) and both syndromes are associated with hemizygous 22q11 deletions. Because many of the tissues and structures affected in VCFS/DGS derive from the pharyngeal arches of the developing embryo, it is believed that haploinsufficiency of a gene(s) involved in embryonic development may be responsible for its etiology. A homeodomain-containing gene, Goosecoidlike (GSCL), has been recently described, and it resides in the critical region for VCFS/DGS on 22q11. GSCL is related to the Goosecoid gene (GSC) in both sequence of the homeodomain and genomic organization. Gsc in the mouse is expressed during early and midembryogenesis and is required for craniofacial rib, and limb development. The chick homolog of GSCL, termed GSX, is expressed during early chick embryogenesis. We detected GSCL expression in human embryos and biphasic expression in mouse embryos. It is possible that the vertebrate GSCL gene is also required for embryonic development. Due to its location in the critical region on 22q11, GSCL is an excellent candidate gene for VCFS/DGS. The vertebrate GSC protein has the same DNA binding specificity as the Drosophila morphogen, bicoid. Upon examination of the putative GSCL promoter, we found three sequence elements with an exact match to the reverse complement of the bicoid DNA recognition motif, suggesting that GSC, or possibly GSCL itself, regulates the transcription of GSCL. Sequence analysis of the putative promoter and the coding region of GSCL was performed on the DNA template from 17 VCFS patients who did not have a detectable 22q11 deletion to identify mutations. We did not detect a mutation in this set of VCFS patients. A polymorphism was detected in codon 47 of exon 1.
Assuntos
Anormalidades Múltiplas/genética , Cromossomos Humanos Par 22 , Análise Mutacional de DNA , Proteínas de Ligação a DNA/genética , Síndrome de DiGeorge/genética , Proteínas de Homeodomínio/genética , Proteínas Repressoras , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , DNA Complementar , Embrião de Mamíferos , Proteína Goosecoid , Humanos , Células Híbridas , Camundongos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
The region of human chromosome 22q11 is prone to rearrangements. The resulting chromosomal abnormalities are involved in Velo-cardio-facial and DiGeorge syndromes (VCFS and DGS) (deletions), "cat eye" syndrome (duplications), and certain types of tumors (translocations). As a prelude to the development of mouse models for VCFS/DGS by generating targeted deletions in the mouse genome, we examined the organization of genes from human chromosome 22q11 in the mouse. Using genetic linkage analysis and detailed physical mapping, we show that genes from a relatively small region of human 22q11 are distributed on three mouse chromosomes (MMU6, MMU10, and MMU16). Furthermore, although the region corresponding to about 2.5 megabases of the VCFS/DGS critical region is located on mouse chromosome 16, the relative organization of the region is quite different from that in humans. Our results show that the instability of the 22q11 region is not restricted to humans but may have been present throughout evolution. The results also underscore the importance of detailed comparative mapping of genes in mice and humans as a prerequisite for the development of mouse models of human diseases involving chromosomal rearrangements.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 22 , Rearranjo Gênico , Animais , Evolução Biológica , Ligação Genética , Humanos , Camundongos , Especificidade da EspécieRESUMO
Velo-cardio-facial syndrome (VCFS) and DiGeorge syndrome (DGS) are developmental disorders characterized by a spectrum of phenotypes including velopharyngeal insufficiency, conotruncal heart defects and facial dysmorphology among others. Eighty to eighty-five percent of VCFS/DGS patients are hemizygous for a portion of chromosome 22. It is likely that the genes encoded by this region play a role in the etiology of the phenotypes associated with the disorders. Using a cDNA selection protocol, we isolated a novel clathrin heavy chain cDNA (CLTD) from the VCFS/DGS minimally deleted interval. The cDNA encodes a protein of 1638 amino acids. CLTD shares significant homology, but is not identical to the ubiquitously expressed clathrin heavy chain gene. The CLTD gene also shows a unique pattern of expression, having its maximal level of expression in skeletal muscle. Velopharyngeal insufficiency and muscle weakness are common features of VCFS patients. Based on the location and expression pattern of CLTD, we suggest hemizygosity at this locus may play a role in the etiology of one of the VCFS-associated phenotypes.
Assuntos
Anormalidades Múltiplas/genética , Clatrina/genética , Músculos/metabolismo , Anormalidades Múltiplas/metabolismo , Sequência de Aminoácidos , Autorradiografia , Sequência de Bases , Northern Blotting , Southern Blotting , Mapeamento Cromossômico , Cromossomos Humanos Par 22 , Cadeias Pesadas de Clatrina , DNA Complementar/análise , Face/anormalidades , Regulação da Expressão Gênica no Desenvolvimento , Cardiopatias Congênitas/genética , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Faringe/anormalidades , Reação em Cadeia da PolimeraseRESUMO
Velo-cardio-facial syndrome (VCFS) is a common genetic disorder among individuals with cleft palate and is associated with hemizygous deletions in human chromosome 22q11. Toward the molecular definition of the deletions, we constructed a physical map of 22q11 in the form of overlapping YACs. The physical map covers > 9 cM of genetic distance, estimated to span 5 Mb of DNA, and contains a total of 64 markers. Eleven highly polymorphic short tandem-repeat polymorphic (STRP) markers were placed on the physical map, and 10 of these were unambiguously ordered. The 11 polymorphic markers were used to type the DNA from a total of 61 VCFS patients and 49 unaffected relatives. Comparison of levels of heterozygosity of these markers in VCFS patients and their unaffected relatives revealed that four of these markers are commonly hemizygous among VCFS patients. To confirm these results and to define further the breakpoints in VCFS patients, 15 VCFS individuals and their unaffected parents were genotyped for the 11 STRP markers. Haplotypes generated from this study revealed that 82% of the patients have deletions that can be defined by the STRP markers. The results revealed that all patients who have a deletion share a common proximal breakpoint, while there are two distinct distal breakpoints. Markers D22S941 and D22S944 appear to be consistently hemizygous in patients with deletions. Both of these markers are located on a single nonchimeric YAC that is 400 kb long. The results also show that the parental origin of the deleted chromosome does not have any effect on the phenotypic manifestation.