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1.
PLoS Genet ; 16(12): e1009232, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33347437

RESUMO

Motile cilia can beat with distinct patterns, but how motility variations are regulated remain obscure. Here, we have studied the role of the coiled-coil protein CFAP53 in the motility of different cilia-types in the mouse. While node (9+0) cilia of Cfap53 mutants were immotile, tracheal and ependymal (9+2) cilia retained motility, albeit with an altered beat pattern. In node cilia, CFAP53 mainly localized at the base (centriolar satellites), whereas it was also present along the entire axoneme in tracheal cilia. CFAP53 associated tightly with microtubules and interacted with axonemal dyneins and TTC25, a dynein docking complex component. TTC25 and outer dynein arms (ODAs) were lost from node cilia, but were largely maintained in tracheal cilia of Cfap53-/- mice. Thus, CFAP53 at the base of node cilia facilitates axonemal transport of TTC25 and dyneins, while axonemal CFAP53 in 9+2 cilia stabilizes dynein binding to microtubules. Our study establishes how differential localization and function of CFAP53 contributes to the unique motion patterns of two important mammalian cilia-types.

2.
Nat Commun ; 11(1): 5520, 2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-33139725

RESUMO

Axonemal dynein ATPases direct ciliary and flagellar beating via adenosine triphosphate (ATP) hydrolysis. The modulatory effect of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) on flagellar beating is not fully understood. Here, we describe a deficiency of cilia and flagella associated protein 45 (CFAP45) in humans and mice that presents a motile ciliopathy featuring situs inversus totalis and asthenospermia. CFAP45-deficient cilia and flagella show normal morphology and axonemal ultrastructure. Proteomic profiling links CFAP45 to an axonemal module including dynein ATPases and adenylate kinase as well as CFAP52, whose mutations cause a similar ciliopathy. CFAP45 binds AMP in vitro, consistent with structural modelling that identifies an AMP-binding interface between CFAP45 and AK8. Microtubule sliding of dyskinetic sperm from Cfap45-/- mice is rescued with the addition of either AMP or ADP with ATP, compared to ATP alone. We propose that CFAP45 supports mammalian ciliary and flagellar beating via an adenine nucleotide homeostasis module.


Assuntos
Nucleotídeos de Adenina/metabolismo , Astenozoospermia/genética , Proteínas do Citoesqueleto/deficiência , Situs Inversus/genética , Adolescente , Adulto , Animais , Astenozoospermia/patologia , Axonema/ultraestrutura , Sistemas CRISPR-Cas/genética , Cílios/metabolismo , Cílios/ultraestrutura , Proteínas do Citoesqueleto/genética , Análise Mutacional de DNA , Modelos Animais de Doenças , Epididimo/patologia , Feminino , Flagelos/metabolismo , Flagelos/ultraestrutura , Humanos , Mutação com Perda de Função , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Planárias/citologia , Planárias/genética , Planárias/metabolismo , Mucosa Respiratória/citologia , Mucosa Respiratória/patologia , Situs Inversus/diagnóstico por imagem , Situs Inversus/patologia , Motilidade Espermática/genética , Tomografia Computadorizada por Raios X , Sequenciamento Completo do Exoma
3.
Sci Adv ; 6(30): eaba1195, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32743070

RESUMO

Immotile cilia sense extracellular signals such as fluid flow, but whether Ca2+ plays a role in flow sensing has been unclear. Here, we examined the role of ciliary Ca2+ in the flow sensing that initiates the breaking of left-right (L-R) symmetry in the mouse embryo. Intraciliary and cytoplasmic Ca2+ transients were detected in the crown cells at the node. These Ca2+ transients showed L-R asymmetry, which was lost in the absence of fluid flow or the PKD2 channel. Further characterization allowed classification of the Ca2+ transients into two types: cilium-derived, L-R-asymmetric transients (type 1) and cilium-independent transients without an L-R bias (type 2). Type 1 intraciliary transients occurred preferentially at the left posterior region of the node, where L-R symmetry breaking takes place. Suppression of intraciliary Ca2+ transients delayed L-R symmetry breaking. Our results implicate cilium-derived Ca2+ transients in crown cells in initiation of L-R symmetry breaking in the mouse embryo.

4.
Neuron ; 107(1): 82-94.e6, 2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32330411

RESUMO

Patients lacking PYCR2, a mitochondrial enzyme that synthesizes proline, display postnatal degenerative microcephaly with hypomyelination. Here we report the crystal structure of the PYCR2 apo-enzyme and show that a novel germline p.Gly249Val mutation lies at the dimer interface and lowers its enzymatic activity. We find that knocking out Pycr2 in mice phenocopies the human disorder and depletes PYCR1 levels in neural lineages. In situ quantification of neurotransmitters in the brains of PYCR2 mutant mice and patients revealed a signature of encephalopathy driven by excessive cerebral glycine. Mechanistically, we demonstrate that loss of PYCR2 upregulates SHMT2, which is responsible for glycine synthesis. This hyperglycemia could be partially reversed by SHMT2 knockdown, which rescued the axonal beading and neurite lengths of cultured Pycr2 knockout neurons. Our findings identify the glycine metabolic pathway as a possible intervention point to alleviate the neurological symptoms of PYCR2-mutant patients.


Assuntos
Córtex Cerebral/metabolismo , Glicina Hidroximetiltransferase/metabolismo , Glicina/metabolismo , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/patologia , Pirrolina Carboxilato Redutases/genética , Adolescente , Animais , Córtex Cerebral/patologia , Pré-Escolar , Feminino , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/metabolismo , Humanos , Lactente , Masculino , Camundongos , Camundongos Knockout , Degeneração Neural/genética , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Linhagem , Pirrolina Carboxilato Redutases/deficiência
5.
Elife ; 72018 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-30070635

RESUMO

We have examined the role of Fam60a, a gene highly expressed in embryonic stem cells, in mouse development. Fam60a interacts with components of the Sin3a-Hdac transcriptional corepressor complex, and most Fam60a-/- embryos manifest hypoplasia of visceral organs and die in utero. Fam60a is recruited to the promoter regions of a subset of genes, with the expression of these genes being either up- or down-regulated in Fam60a-/- embryos. The DNA methylation level of the Fam60a target gene Adhfe1 is maintained at embryonic day (E) 7.5 but markedly reduced at E9.5 in Fam60a-/- embryos, suggesting that DNA demethylation is enhanced in the mutant. Examination of genome-wide DNA methylation identified several differentially methylated regions, which were preferentially hypomethylated, in Fam60a-/- embryos. Our data suggest that Fam60a is required for proper embryogenesis, at least in part as a result of its regulation of DNA methylation at specific gene promoters.


Assuntos
Metilação de DNA/genética , Proteínas de Ligação a DNA/genética , Desenvolvimento Embrionário/genética , Animais , Proteínas de Ligação a DNA/química , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Histona Desacetilases/química , Histona Desacetilases/genética , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas , Proteínas Repressoras/química , Proteínas Repressoras/genética , Complexo Correpressor Histona Desacetilase e Sin3
6.
Dev Cell ; 40(5): 439-452.e4, 2017 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-28292423

RESUMO

Polarization of node cells along the anterior-posterior axis of mouse embryos is responsible for left-right symmetry breaking. How node cells become polarized has remained unknown, however. Wnt5a and Wnt5b are expressed posteriorly relative to the node, whereas genes for Sfrp inhibitors of Wnt signaling are expressed anteriorly. Here we show that polarization of node cells is impaired in Wnt5a-/-Wnt5b-/- and Sfrp mutant embryos, and also in the presence of a uniform distribution of Wnt5a or Sfrp1, suggesting that Wnt5 and Sfrp proteins act as instructive signals in this process. The absence of planar cell polarity (PCP) core proteins Prickle1 and Prickle2 in individual cells or local forced expression of Wnt5a perturbed polarization of neighboring wild-type cells. Our results suggest that opposing gradients of Wnt5a and Wnt5b and of their Sfrp inhibitors, together with intercellular signaling via PCP proteins, polarize node cells along the anterior-posterior axis for breaking of left-right symmetry.


Assuntos
Padronização Corporal , Polaridade Celular , Transdução de Sinais , Proteínas Wnt/metabolismo , Proteína Wnt-5a/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Comunicação Celular , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas com Domínio LIM/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Mutantes , Modelos Biológicos , Proteínas/metabolismo
7.
Am J Hum Genet ; 99(2): 460-9, 2016 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-27486780

RESUMO

Multiprotein complexes referred to as outer dynein arms (ODAs) develop the main mechanical force to generate the ciliary and flagellar beat. ODA defects are the most common cause of primary ciliary dyskinesia (PCD), a congenital disorder of ciliary beating, characterized by recurrent infections of the upper and lower airways, as well as by progressive lung failure and randomization of left-right body asymmetry. Using a whole-exome sequencing approach, we identified recessive loss-of-function mutations within TTC25 in three individuals from two unrelated families affected by PCD. Mice generated by CRISPR/Cas9 technology and carrying a deletion of exons 2 and 3 in Ttc25 presented with laterality defects. Consistently, we observed immotile nodal cilia and missing leftward flow via particle image velocimetry. Furthermore, transmission electron microscopy (TEM) analysis in TTC25-deficient mice revealed an absence of ODAs. Consistent with our findings in mice, we were able to show loss of the ciliary ODAs in humans via TEM and immunofluorescence (IF) analyses. Additionally, IF analyses revealed an absence of the ODA docking complex (ODA-DC), along with its known components CCDC114, CCDC151, and ARMC4. Co-immunoprecipitation revealed interaction between the ODA-DC component CCDC114 and TTC25. Thus, here we report TTC25 as a new member of the ODA-DC machinery in humans and mice.


Assuntos
Axonema/genética , Axonema/metabolismo , Proteínas de Transporte/genética , Cílios/patologia , Dineínas/química , Dineínas/metabolismo , Síndrome de Kartagener/genética , Síndrome de Kartagener/patologia , Mutação , Animais , Axonema/patologia , Axonema/ultraestrutura , Cílios/metabolismo , Cílios/ultraestrutura , Dineínas/genética , Dineínas/ultraestrutura , Exoma/genética , Éxons/genética , Imunofluorescência , Genes Recessivos , Humanos , Camundongos , Microscopia Eletrônica de Transmissão , Ligação Proteica , Xenopus , Proteínas de Xenopus/deficiência , Proteínas de Xenopus/genética
8.
PLoS One ; 10(10): e0140831, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26469858

RESUMO

In the early vertebrate embryo, cardiac progenitor/precursor cells (CPs) give rise to cardiac structures. Better understanding their biological character is critical to understand the heart development and to apply CPs for the clinical arena. However, our knowledge remains incomplete. With the use of single-cell expression profiling, we have now revealed rapid and dynamic changes in gene expression profiles of the embryonic CPs during the early phase after their segregation from the cardiac mesoderm. Progressively, the nascent mesodermal gene Mesp1 terminated, and Nkx2-5+/Tbx5+ population rapidly replaced the Tbx5low+ population as the expression of the cardiac genes Tbx5 and Nkx2-5 increased. At the Early Headfold stage, Tbx5-expressing CPs gradually showed a unique molecular signature with signs of cardiomyocyte differentiation. Lineage-tracing revealed a developmentally distinct characteristic of this population. They underwent progressive differentiation only towards the cardiomyocyte lineage corresponding to the first heart field rather than being maintained as a progenitor pool. More importantly, Tbx5 likely plays an important role in a transcriptional network to regulate the distinct character of the FHF via a positive feedback loop to activate the robust expression of Tbx5 in CPs. These data expands our knowledge on the behavior of CPs during the early phase of cardiac development, subsequently providing a platform for further study.


Assuntos
Células-Tronco Embrionárias/metabolismo , Perfilação da Expressão Gênica/métodos , Miócitos Cardíacos/metabolismo , Análise de Célula Única/métodos , Animais , Diferenciação Celular/genética , Células Cultivadas , Embrião de Mamíferos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Gravidez , Transcriptoma
9.
Dev Biol ; 395(2): 331-41, 2014 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-25224222

RESUMO

The transcription factor Pitx2c is expressed in primordial visceral organs in a left-right (L-R) asymmetric manner and executes situs-specific morphogenesis. Here we show that Pitx2c is also L-R asymmetrically expressed in the developing mouse limb. Human PITX2c exhibits the same transcriptional activity in the mouse limb. The asymmetric expression of Pitx2c in the limb also exhibits dorsal-ventral and anterior-posterior polarities, being confined to the posterior-dorsal region of the left limb. Left-sided Pitx2c expression in the limb is regulated by Nodal signaling through a Nodal-responsive enhancer. Pitx2c is expressed in lateral plate mesoderm (LPM)-derived cells in the left limb that contribute to various limb connective tissues. The number of Pitx2c(+) cells in the left limb was found to be negatively regulated by Pitx2c itself. Although obvious defects were not apparent in the limb of mice lacking asymmetric Pitx2c expression, Pitx2c may regulate functional L-R asymmetry of the limb.


Assuntos
Extremidades/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Homeodomínio/metabolismo , Morfogênese/fisiologia , Fatores de Transcrição/metabolismo , Animais , Primers do DNA/genética , Imunofluorescência , Galactosídeos , Técnicas de Introdução de Genes , Hibridização In Situ , Indóis , Camundongos , Camundongos Transgênicos , Tamoxifeno
10.
Semin Cell Dev Biol ; 32: 80-4, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24704359

RESUMO

Two TGFß-related proteins, Nodal and Lefty, are asymmetrically expressed and play central roles in establishing left-right (L-R) asymmetry of our body. Nodal acts as a left-side determinant whereas Lefty restricts Nodal activity to the left side by acting as a feedback inhibitor of Nodal. While the mechanism for symmetry breaking is variable among animals, the pair of Nodal and Lefty has a conserved role in the L-R asymmetry pathway. Function and regulation of Nodal and Lefty have been revealed in the last decades, but in this review we summarize the role of TGFß-related proteins together with more recent findings. We mainly discuss observations made with mouse embryos, unless indicated otherwise.


Assuntos
Padronização Corporal/genética , Fatores de Determinação Direita-Esquerda/genética , Proteína Nodal/genética , Transdução de Sinais/genética , Animais , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Determinação Direita-Esquerda/metabolismo , Camundongos , Modelos Genéticos , Proteína Nodal/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo
11.
J Cell Biol ; 204(2): 203-13, 2014 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-24421334

RESUMO

Axonemal dynein complexes are preassembled in the cytoplasm before their transport to cilia, but the mechanism of this process remains unclear. We now show that mice lacking Pih1d3, a PIH1 domain-containing protein, develop normally but manifest male sterility. Pih1d3(-/-) sperm were immotile and fragile, with the axoneme of the flagellum lacking outer dynein arms (ODAs) and inner dynein arms (IDAs) and showing a disturbed 9+2 microtubule organization. Pih1d3 was expressed specifically in spermatogenic cells, with the mRNA being most abundant in pachytene spermatocytes. Pih1d3 localized to the cytoplasm of spermatogenic cells but was not detected in spermatids or mature sperm. The levels of ODA and IDA proteins were reduced in the mutant testis and sperm, and Pih1d3 was found to interact with an intermediate chain of ODA as well as with Hsp70 and Hsp90. Our results suggest that Pih1d3 contributes to cytoplasmic preassembly of dynein complexes in spermatogenic cells by stabilizing and promoting complex formation by ODA and IDA proteins.


Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Dineínas do Axonema/metabolismo , Espermatozoides/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Axonema/metabolismo , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Fertilidade/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Camundongos , RNA Mensageiro/metabolismo , Espermatozoides/ultraestrutura , Testículo/metabolismo
12.
Dev Biol ; 381(1): 203-12, 2013 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-23742838

RESUMO

Qilin is one of several genes in zebrafish whose mutation results in cystic kidney. We have now studied the role of its mouse ortholog, Cluap1, in embryonic development by generating Cluap1 knockout (Cluap1-/-) mice. Cluap1-/- embryos died mid-gestation manifesting impairment of ciliogenesis in various regions including the node and neural tube. The basal body was found to be properly docked to the apical membrane of cells in the mutant, but the axoneme failed to grow. Cluap1 is a ciliary protein and is preferentially localized at the base and tip of cilia. Hedgehog signaling, as revealed with a Pacthed1-lacZ reporter gene, was lost in Cluap1-/- embryos at embryonic day (E) 8.5 but was ectopically expanded at E9.0. The Cluap1 knockout embryos also failed to manifest left-right asymmetric expression of Nodal in the lateral plate, most likely as a result of the loss of Hedgehog signaling in node crown cells that in turn leads to pronounced down-regulation of Gdf1 expression in these cells. Crown cell-specific restoration of Cluap1 expression rescued Gdf1 expression in crown cells and left-sided Nodal expression in the lateral plate of mutant embryos. Our results suggest that Cluap1 contributes to ciliogenesis by regulating the intraflagellar transport (IFT) cycle at the base and tip of the cilium.


Assuntos
Cílios/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Morfogênese/genética , Animais , Padronização Corporal , Regulação para Baixo , Fibroblastos/metabolismo , Genes Reporter , Genótipo , Proteínas Hedgehog/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Óperon Lac , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mutação , Transdução de Sinais
13.
Dev Biol ; 376(1): 23-30, 2013 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-23357539

RESUMO

In the node of mouse embryo, rotational movements of cilia generate an external liquid flow known as nodal flow, which determines left-right asymmetric gene expression. How nodal flow is converted into asymmetric gene expression is still controversial, but the increase of Ca(2+) levels in endodermal cells to the left of the node has been proposed to play a role. However, Ca(2+) signals inside the node itself have not yet been described. By our optimized Ca(2+) imaging method, we were able to observe dynamic Ca(2+) signals in the node in live mouse embryos. Pharmacological disruption of Ca(2+) signals did not affect ciliary movements or nodal flow, but did alter the expression patterns of the Nodal and Cerl-2 genes. Quantitative analyses of Ca(2+) signal frequencies and distributions showed that during left-right axis establishment, formerly symmetric Ca(2+) signals became biased to the left side. In iv/iv mutant embryos that showed randomized laterality due to ciliary immotility, Ca(2+) signals were found to be variously left-sided, right-sided, or bilateral, and thus symmetric on average. In Pkd2 mutant embryos, which lacked polycystin-2, a Ca(2+)-permeable cation channel necessary for left-right axis formation, the Ca(2+) signal frequency was lower than in wild-type embryos. Our data support a model in which dynamic Ca(2+) signals in the node are involved in left-right patterning.


Assuntos
Padronização Corporal/fisiologia , Sinalização do Cálcio/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Organizadores Embrionários/embriologia , Animais , Cílios/fisiologia , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Camundongos Knockout , Modelos Biológicos , Proteína Nodal/metabolismo , Organizadores Embrionários/metabolismo , Canais de Cátion TRPP/genética
14.
Mol Cell Neurosci ; 52: 128-39, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23147109

RESUMO

Transcriptional regulation of gene expression during development is critical for proper neuronal differentiation and migration. Alternative splicing and differential isoform expression have been demonstrated for most mammalian genes, but their specific contributions to gene function are not well understood. In mice, the transcription factor gene Pitx2 is expressed as three different isoforms (PITX2A, PITX2B, and PITX2C) which have unique amino termini and common DNA binding homeodomains and carboxyl termini. The specific roles of these isoforms in neuronal development are not known. Here we report the onset of Pitx2ab and Pitx2c isoform-specific expression by E9.5 in the developing mouse brain. Using isoform-specific Pitx2 deletion mouse strains, we show that collicular neuron migration requires PITX2AB and that collicular GABAergic differentiation and targeting of hypothalamic projections require unique Pitx2 isoform dosage. These results provide insights into Pitx2 dosage and isoform-specific requirements underlying midbrain and hypothalamic development.


Assuntos
Proteínas de Homeodomínio/metabolismo , Hipotálamo/embriologia , Neurogênese/fisiologia , Neurônios/metabolismo , Colículos Superiores/embriologia , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Hipotálamo/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Neurônios/citologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Colículos Superiores/metabolismo , Fatores de Transcrição/genética
15.
Science ; 338(6104): 226-31, 2012 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-22983710

RESUMO

Unidirectional fluid flow plays an essential role in the breaking of left-right (L-R) symmetry in mouse embryos, but it has remained unclear how the flow is sensed by the embryo. We report that the Ca(2+) channel Polycystin-2 (Pkd2) is required specifically in the perinodal crown cells for sensing the nodal flow. Examination of mutant forms of Pkd2 shows that the ciliary localization of Pkd2 is essential for correct L-R patterning. Whereas Kif3a mutant embryos, which lack all cilia, failed to respond to an artificial flow, restoration of primary cilia in crown cells rescued the response to the flow. Our results thus suggest that nodal flow is sensed in a manner dependent on Pkd2 by the cilia of crown cells located at the edge of the node.


Assuntos
Padronização Corporal , Embrião de Mamíferos/fisiologia , Fatores de Determinação Direita-Esquerda/metabolismo , Organizadores Embrionários/fisiologia , Canais de Cátion TRPP/metabolismo , Animais , Líquidos Corporais/fisiologia , Cálcio/metabolismo , Cílios/metabolismo , Cílios/fisiologia , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/citologia , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Cinesina/genética , Fatores de Determinação Direita-Esquerda/genética , Camundongos , Camundongos Mutantes , Mutação , Organizadores Embrionários/citologia , Transdução de Sinais , Canais de Cátion TRPP/genética
16.
Dev Biol ; 371(2): 136-45, 2012 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-22819673

RESUMO

Tight junctions (TJs) connect epithelial cells and form a semipermeable barrier that only allows selective passage of ions and solutes across epithelia. Here we show that mice lacking EpCAM, a putative cell adhesion protein frequently overexpressed in human cancers, manifest intestinal barrier defects and die shortly after birth as a result of intestinal erosion. EpCAM was found to be highly expressed in the developing intestinal epithelium of wild-type mice and to localize to cell-cell junctions including TJs. Claudin-7 colocalized with EpCAM at cell-cell junctions, and the two proteins were found to associate with each other. Claudins 2, 3, 7, and 15 were down-regulated in the intestine of EpCAM mutant mice, with claudin-7 being reduced to undetectable levels. TJs in the mutant intestinal epithelium were morphologically abnormal with the network of TJ strands scattered and dispersed. Finally, the barrier function of the intestinal epithelium was impaired in the mutant animals. These results suggest that EpCAM contributes to formation of intestinal barrier by recruiting claudins to cell-cell junctions.


Assuntos
Antígenos de Neoplasias/metabolismo , Moléculas de Adesão Celular/metabolismo , Claudinas/metabolismo , Mucosa Intestinal/metabolismo , Junções Íntimas/metabolismo , Animais , Antígenos de Neoplasias/genética , Moléculas de Adesão Celular/genética , Claudina-3/genética , Claudina-3/metabolismo , Claudinas/genética , Regulação para Baixo , Molécula de Adesão da Célula Epitelial , Mucosa Intestinal/embriologia , Camundongos , Camundongos Knockout
17.
Nat Commun ; 3: 622, 2012 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-22233632

RESUMO

Determination of left-right asymmetry in mouse embryos is achieved by a leftward fluid flow (nodal flow) in the node cavity that is generated by clockwise rotational movement of 200-300 cilia in the node. The precise action of nodal flow and how much flow input is required for the robust read-out of left-right determination remains unknown. Here we show that a local leftward flow generated by as few as two rotating cilia is sufficient to break left-right symmetry. Quantitative analysis of fluid flow and ciliary rotation in the node of mouse embryos shows that left-right asymmetry is already established within a few hours after the onset of rotation by a subset of nodal cilia. Examination of various ciliary mutant mice shows that two rotating cilia are sufficient to initiate left-right asymmetric gene expression. Our results suggest the existence of a highly sensitive system in the node that is able to sense an extremely weak unidirectional flow, and may favour a model in which the flow is sensed as a mechanical force.


Assuntos
Padronização Corporal/genética , Cílios/fisiologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/fisiologia , Animais , Biofísica/métodos , Biologia do Desenvolvimento/métodos , Técnicas de Cultura Embrionária , Regulação da Expressão Gênica no Desenvolvimento , Metilcelulose/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Modelos Biológicos , Mutação , Organizadores Embrionários/fisiologia , Fatores de Tempo
18.
Development ; 138(10): 1913-23, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21471156

RESUMO

The essential roles of SHH in anteroposterior (AP) and AER-FGF signalling in proximodistal (PD) limb bud development are well understood. In addition, these morphoregulatory signals are key components of the self-regulatory SHH/GREM1/AER-FGF feedback signalling system that regulates distal progression of limb bud development. This study uncovers an additional signalling module required for coordinated progression of limb bud axis development. Transcriptome analysis using Shh-deficient mouse limb buds revealed that the expression of proximal genes was distally extended from early stages onwards, which pointed to a more prominent involvement of SHH in PD limb axis development. In particular, retinoic acid (RA) target genes were upregulated proximally, while the expression of the RA-inactivating Cyp26b1 enzyme was downregulated distally, pointing to increased RA activity in Shh-deficient mouse limb buds. Further genetic and molecular analysis established that Cyp26b1 expression is regulated by AER-FGF signalling. During initiation of limb bud outgrowth, the activation of Cyp26b1 expression creates a distal 'RA-free' domain, as indicated by complementary downregulation of a transcriptional sensor of RA activity. Subsequently, Cyp26b1 expression increases as a consequence of SHH-dependent upregulation of AER-FGF signalling. To better understand the underlying signalling interactions, computational simulations of the spatiotemporal expression patterns and interactions were generated. These simulations predicted the existence of an antagonistic AER-FGF/CYP26B1/RA signalling module, which was verified experimentally. In summary, SHH promotes distal progression of limb development by enhancing CYP26B1-mediated RA clearance as part of a signalling network linking the SHH/GREM1/AER-FGF feedback loop to the newly identified AER-FGF/CYP26B1/RA module.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas Hedgehog/metabolismo , Botões de Extremidades/embriologia , Botões de Extremidades/metabolismo , Tretinoína/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/genética , Ectoderma/embriologia , Ectoderma/metabolismo , Ativação Enzimática , Retroalimentação Fisiológica , Feminino , Fatores de Crescimento de Fibroblastos/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/deficiência , Proteínas Hedgehog/genética , Mesoderma/embriologia , Mesoderma/metabolismo , Camundongos , Camundongos Knockout , Camundongos Mutantes , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Gravidez , Ácido Retinoico 4 Hidroxilase , Transdução de Sinais
19.
Dev Biol ; 353(2): 321-30, 2011 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-21419113

RESUMO

Left-right (L-R) asymmetry in the mouse embryo is generated in the node and is dependent on cilia-driven fluid flow, but how the initial asymmetry is transmitted from the node to the lateral plate has remained unknown. We have now identified a transcriptional enhancer (ANE) in the human LEFTY1 gene that exhibits marked L>R asymmetric activity in perinodal cells of the mouse embryo. Dissection of ANE revealed that it is activated in the perinodal cells on the left side by Nodal signaling, suggesting that Nodal activity in the node is asymmetric at a time when Nodal expression is symmetric. Phosphorylated Smad2/3 (pSmad2) indeed manifested an L-R asymmetric distribution at the node, being detected in perinodal cells preferentially on the left side. This asymmetry in pSmad2 distribution was found to be generated not by unidirectional transport of Nodal but rather as a result of LR distribution of active Nodal in the node is translated into the asymmetry in LPM.


Assuntos
Padronização Corporal/genética , Padronização Corporal/fisiologia , Mesoderma/embriologia , Proteína Nodal/genética , Proteína Nodal/fisiologia , Animais , Transporte Biológico Ativo , Elementos Facilitadores Genéticos , Feminino , Fatores de Transcrição Forkhead/deficiência , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Humanos , Fatores de Determinação Direita-Esquerda/genética , Masculino , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Camundongos Knockout , Camundongos Mutantes Neurológicos , Camundongos Transgênicos , Fosforilação , Gravidez , Transdução de Sinais , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo
20.
Dev Dyn ; 240(1): 195-203, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21089073

RESUMO

To aid in detection and tracking of cells targeted by the left-right (LR) pathway in the heart throughout morphogenesis, expression from a Pitx2c-lacZ transgene (P2Ztg) was analysed in detail. ß-galactosidase expression from P2Ztg was robust, allowing reliable visualisation of low-level Pitx2c expression, and was virtually entirely dependent upon NODAL signalling in the heart. P2Ztg showed expression in trabecular and septal, as well as non-trabecular, myocardium, and a strong expression bias in myocardium associated with individual endocardial cushions of the atrioventricular canal and outflow tract, which are essential for cardiac septation. Expression on the ventral surface of the outflow tract evolved to a specific stripe that could be used to track the future aorta during outflow tract spiralling and remodelling. Our data show that the P2Ztg transgene is a useful resource for detection of molecular disturbances in the LR cascade, as well as morphogenetic defects associated with other cardiac congenital disorders.


Assuntos
Genes Reporter , Proteínas de Homeodomínio/genética , Miocárdio/metabolismo , Fatores de Transcrição/genética , Transgenes , Animais , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter/fisiologia , Coração/embriologia , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/metabolismo , Proteínas de Homeodomínio/metabolismo , Mesoderma/embriologia , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Morfogênese/genética , Crista Neural/metabolismo , Reprodutibilidade dos Testes , Fatores de Transcrição/metabolismo , Transgenes/genética , Transgenes/fisiologia
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