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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.
Cell Rep ; 31(10): 107733, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32521258

RESUMO

Endocytosis has been proposed to modulate cell signaling activities. However, the role of endocytosis in embryogenesis, which requires coordination of multiple signaling inputs, has remained less understood. We previously showed that mouse embryos lacking a small guanosine triphosphate (GTP)-binding protein Rab7 implicated in endocytic flow are defective in gastrulation. Here, we investigate how subcellular defects associated with Rab7 deficiency are related to the observed developmental defects. Rab7-deficient embryos fail to organize mesodermal tissues due to defects in Wnt-ß-catenin signaling. Visceral endoderm (VE)-specific ablation of Rab7 results in patterning defects similar to systemic Rab7 deletion. Rab7 mutants accumulate the Wnt antagonist Dkk1 in the extracellular space and in intracellular compartments throughout the VE epithelium. These data indicate that Rab7-dependent endocytosis regulates the concentration and availability of extracellular Dkk1, thereby relieving the epiblast of antagonism. This intercellular mechanism therefore organizes distinct spatiotemporal patterns of canonical Wnt activity during the peri-gastrulation stages of embryonic development.

5.
Genes Cells ; 24(11): 731-745, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31554018

RESUMO

Cluap1/IFT38 is a ciliary protein that belongs to the IFT-B complex and is required for ciliogenesis. In this study, we have examined the behaviors of Cluap1 protein in nonciliated and ciliated cells. In proliferating cells, Cluap1 is located at the distal appendage of the mother centriole. When cells are induced to form cilia, Cluap1 is found in a novel noncentriolar compartment, the cytoplasmic IFT spot, which mainly exists once in a cell. Other IFT-B proteins such as IFT46 and IFT88 are colocalized in this spot. The cytoplasmic IFT spot is present in mouse embryonic fibroblasts (MEFs) but is absent in ciliogenesis-defective MEFs lacking Cluap1, Kif3a or Odf2. The cytoplasmic IFT spot is also found in mouse embryos but is absent in the Cluap1 mutant embryo. When MEFs are induced to form cilia, the cytoplasmic IFT spot appears at an early step of ciliogenesis but starts to disappear when ciliogenesis is mostly completed. These results suggest that IFT-B proteins such as Cluap1 accumulate in a previously undescribed cytoplasmic compartment during ciliogenesis.


Assuntos
Cílios/metabolismo , Citoplasma/metabolismo , Proteínas do Citoesqueleto/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Animais , Cílios/ultraestrutura , Citoplasma/ultraestrutura , Fibroblastos , Proteínas de Choque Térmico , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cinesina , Camundongos , Camundongos Knockout , Proteínas Supressoras de Tumor
6.
Nat Commun ; 8(1): 1492, 2017 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-29138408

RESUMO

Anterior-posterior (A-P) polarity of mouse embryos is established by distal visceral endoderm (DVE) at embryonic day (E) 5.5. Lefty1 is expressed first at E3.5 in a subset of epiblast progenitor cells (L1epi cells) and then in a subset of primitive endoderm cells (L1dve cells) fated to become DVE. Here we studied how prospective DVE cells are selected. Lefty1 expression in L1epi and L1dve cells depends on Nodal signaling. A cell that experiences the highest level of Nodal signaling begins to express Lefty1 and becomes an L1epi cell. Deletion of Lefty1 alone or together with Lefty2 increased the number of prospective DVE cells. Ablation of L1epi or L1dve cells triggered Lefty1 expression in a subset of remaining cells. Our results suggest that selection of prospective DVE cells is both random and regulated, and that a fixed prepattern for the A-P axis does not exist before the blastocyst stage.


Assuntos
Embrião de Mamíferos/metabolismo , Endoderma/metabolismo , Fatores de Determinação Direita-Esquerda/genética , Proteína Nodal/metabolismo , Transdução de Sinais , Vísceras/metabolismo , Animais , Padronização Corporal , Cromossomos Artificiais Bacterianos , Embrião de Mamíferos/citologia , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Camundongos , Camundongos Transgênicos , Processos Estocásticos
7.
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
8.
J Cell Biol ; 215(5): 705-718, 2016 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-27881714

RESUMO

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) can be shed from the cell membrane by GPI cleavage. In this study, we report a novel GPI-processing enzyme, termed post-glycosylphosphatidylinositol attachment to proteins 6 (PGAP6), which is a GPI-specific phospholipase A2 mainly localized at the cell surface. CRIPTO, a GPI-AP, which plays critical roles in early embryonic development by acting as a Nodal coreceptor, is a highly sensitive substrate of PGAP6, whereas CRYPTIC, a close homologue of CRIPTO, is not sensitive. CRIPTO processed by PGAP6 was released as a lysophosphatidylinositol-bearing form, which is further cleaved by phospholipase D. CRIPTO shed by PGAP6 was active as a coreceptor in Nodal signaling, whereas cell-associated CRIPTO activity was reduced when PGAP6 was expressed. Homozygous Pgap6 knockout mice showed defects in early embryonic development, particularly in the formation of the anterior-posterior axis, which are common features with Cripto knockout embryos. These results suggest PGAP6 plays a critical role in Nodal signaling modulation through CRIPTO shedding.


Assuntos
Proteínas Ligadas por GPI/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína Nodal/metabolismo , Fosfolipases A2/metabolismo , Animais , Padronização Corporal , Células CHO , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Células HEK293 , Humanos , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Camundongos Knockout , Modelos Biológicos , Mutação/genética , Monoéster Fosfórico Hidrolases/genética , Fosfotransferases/genética , Transdução de Sinais
9.
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
10.
Elife ; 52016 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-27331609

RESUMO

Transcription factors organize gene expression profiles by regulating promoter activity. However, the role of transcription factors after transcription initiation is poorly understood. Here, we show that the homeoprotein Nkx2-5 and the 5'-3' exonuclease Xrn2 are involved in the regulation of alternative polyadenylation (APA) during mouse heart development. Nkx2-5 occupied not only the transcription start sites (TSSs) but also the downstream regions of genes, serving to connect these regions in primary embryonic cardiomyocytes (eCMs). Nkx2-5 deficiency affected Xrn2 binding to target loci and resulted in increases in RNA polymerase II (RNAPII) occupancy and in the expression of mRNAs with long 3'untranslated regions (3' UTRs) from genes related to heart development. siRNA-mediated suppression of Nkx2-5 and Xrn2 led to heart looping anomaly. Moreover, Nkx2-5 genetically interacts with Xrn2 because Nkx2-5(+/-)Xrn2(+/-), but neither Nkx2-5(+/-)nor Xrn2(+/-), newborns exhibited a defect in ventricular septum formation, suggesting that the association between Nkx2-5 and Xrn2 is essential for heart development. Our results indicate that Nkx2-5 regulates not only the initiation but also the usage of poly(A) sites during heart development. Our findings suggest that tissue-specific transcription factors is involved in the regulation of APA.


Assuntos
Exorribonucleases/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Proteína Homeobox Nkx-2.5/metabolismo , Poliadenilação , Animais , Exorribonucleases/genética , Proteína Homeobox Nkx-2.5/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout
11.
Genes Cells ; 21(7): 728-39, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27353389

RESUMO

Lrrc6 encodes a cytoplasmic protein that is expressed specifically in cells with motile cilia including the node, trachea and testes of the mice. A mutation of Lrrc6 has been identified in human patients with primary ciliary dyskinesia (PCD). Mutant mice lacking Lrrc6 show typical PCD defects such as hydrocephalus and laterality defects. We found that in the absence of Lrrc6, the morphology of motile cilia remained normal, but their motility was completely lost. The 9 + 2 arrangement of microtubules remained normal in Lrrc6(-/-) mice, but the outer dynein arms (ODAs), the structures essential for the ciliary beating, were absent from the cilia. In the absence of Lrrc6, ODA proteins such as DNAH5, DNAH9 and IC2, which are assembled in the cytoplasm and transported to the ciliary axoneme, remained in the cytoplasm and were not transported to the ciliary axoneme. The IC2-IC1 interaction, which is the first step of ODA assembly, was normal in Lrrc6(-/-) mice testes. Our results suggest that ODA proteins may be transported from the cytoplasm to the cilia by an Lrrc6-dependent mechanism.


Assuntos
Cílios/genética , Síndrome de Kartagener/genética , Proteínas/genética , Animais , Dineínas do Axonema/genética , Axonema/genética , Axonema/patologia , Cílios/patologia , Citoplasma/genética , Citoplasma/metabolismo , Proteínas do Citoesqueleto , Modelos Animais de Doenças , Dineínas/genética , Humanos , Síndrome de Kartagener/patologia , Camundongos , Camundongos Transgênicos , Mutação
12.
EMBO J ; 34(1): 97-114, 2015 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-25385835

RESUMO

Formation of a new centriole adjacent to a pre-existing centriole occurs only once per cell cycle. Despite being crucial for genome integrity, the mechanisms controlling centriole biogenesis remain elusive. Here, we identify RBM14 as a novel suppressor of assembly of centriolar protein complexes. Depletion of RBM14 in human cells induces ectopic formation of centriolar protein complexes through function of the STIL/CPAP complex. Intriguingly, the formation of such structures seems not to require the cartwheel structure that normally acts as a scaffold for centriole formation, whereas they can retain pericentriolar material and microtubule nucleation activity. Moreover, we find that, upon RBM14 depletion, a part of the ectopic centriolar protein complexes in turn assemble into structures more akin to centrioles, presumably by incorporating HsSAS-6, a cartwheel component, and cause multipolar spindle formation. We further demonstrate that such structures assemble in the cytoplasm even in the presence of pre-existing centrioles. This study sheds light on the possibility that ectopic formation of aberrant structures related to centrioles may contribute to genome instability and tumorigenesis.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Centríolos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Microtúbulos/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Ciclo Celular/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Instabilidade Genômica/genética , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/genética , Complexos Multiproteicos/genética
13.
Philos Trans R Soc Lond B Biol Sci ; 369(1657)2014 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-25349445

RESUMO

The first cell fate decision during mouse development concerns whether a blastomere will contribute to the inner cell mass (ICM; which gives rise to the embryo proper) or to trophectoderm (TE; which gives rise to the placenta). The position of a cell within an 8- to 16-cell-stage embryo correlates with its future fate, with outer cells contributing to TE and inner cells to the ICM. It remains unknown, however, whether an earlier pre-pattern exists. Here, we propose a hypothesis that could account for generation of such a pre-pattern and which is based on epigenetic asymmetry (such as in histone or DNA methylation) between maternal and paternal genomes in the zygote.


Assuntos
Blastômeros/fisiologia , Padronização Corporal/fisiologia , Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Desenvolvimento Embrionário/fisiologia , Epigênese Genética/fisiologia , Genoma/genética , Animais , Camundongos , Modelos Biológicos
14.
Nat Commun ; 3: 1322, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23271656

RESUMO

Breaking of left-right symmetry in mouse embryos requires fluid flow at the node, but the precise action of the flow has remained unknown. Here we show that the left-right asymmetry of Cerl2 expression around the node, a target of the flow, is determined post-transcriptionally by decay of Cerl2 mRNA in a manner dependent on its 3' untranslated region. Cerl2 mRNA is absent specifically from the apical region of crown cells on the left side of the node. Preferential decay of Cerl2 mRNA on the left is initiated by the leftward flow and further enhanced by the operation of Wnt-Cerl2 interlinked feedback loops, in which Wnt3 upregulates Wnt3 expression and promotes Cerl2 mRNA decay, whereas Cerl2 promotes Wnt degradation. Mathematical modelling and experimental data suggest that these feedback loops behave as a bistable switch that can amplify in a noise-resistant manner a small bias conferred by fluid flow.


Assuntos
Retroalimentação Fisiológica , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Regiões 3' não Traduzidas , Animais , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Camundongos Transgênicos , Conformação de Ácido Nucleico , Ligação Proteica , Estabilidade de RNA , RNA Mensageiro/genética , Transdução de Sinais , Proteína Wnt3/genética , Proteína Wnt3/metabolismo
15.
Development ; 139(1): 3-14, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22147950

RESUMO

The mouse embryo generates multiple cell lineages, as well as its future body axes in the early phase of its development. The early cell fate decisions lead to the generation of three lineages in the pre-implantation embryo: the epiblast, the primitive endoderm and the trophectoderm. Shortly after implantation, the anterior-posterior axis is firmly established. Recent studies have provided a better understanding of how the earliest cell fate decisions are regulated in the pre-implantation embryo, and how and when the body axes are established in the pregastrulation embryo. In this review, we address the timing of the first cell fate decisions and of the establishment of embryonic polarity, and we ask how far back one can trace their origins.


Assuntos
Padronização Corporal/fisiologia , Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Embrião de Mamíferos/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Camadas Germinativas/embriologia , Fatores de Transcrição/metabolismo , Animais , Camundongos , Modelos Biológicos , Fatores de Tempo
16.
Nat Cell Biol ; 13(7): 743-52, 2011 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-21623358

RESUMO

Anterior-posterior polarity of the mouse embryo has been thought to be established when distal visceral endoderm (DVE) at embryonic day (E) 5.5 migrates toward the future anterior side to form anterior visceral endoderm (AVE). Lefty1, a marker of DVE and AVE, is asymmetrically expressed in implanting mouse embryos. We now show that Lefty1 is expressed first in a subset of epiblast progenitor cells and then in a subset of primitive endoderm progenitors. Genetic fate mapping indicated that the latter cells are destined to become DVE. In contrast to the accepted notion, however, AVE is not derived from DVE but is newly formed after E5.5 from Lefty1(-) visceral endoderm cells that move to the distal tip. Concomitant with DVE migration, all visceral endoderm cells in the embryonic region undergo global movement. In embryos subjected to genetic ablation of Lefty1-expressing DVE cells, AVE was formed de novo but the visceral endoderm including the newly formed AVE failed to migrate, indicating that DVE guides the migration of AVE by initiating the global movement of visceral endoderm cells. Future anterior-posterior polarity is thus already determined by Lefty1(+) blastomeres in the implanting blastocyst.


Assuntos
Movimento Celular , Polaridade Celular , Células-Tronco Embrionárias/metabolismo , Endoderma/metabolismo , Fatores de Determinação Direita-Esquerda/metabolismo , Animais , Blastocisto/metabolismo , Blastômeros/metabolismo , Diferenciação Celular , Linhagem da Célula , Movimento Celular/genética , Polaridade Celular/genética , Citocinas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Endoderma/citologia , Regulação da Expressão Gênica no Desenvolvimento , Idade Gestacional , Hibridização In Situ , Fatores de Determinação Direita-Esquerda/genética , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Microscopia de Vídeo , Morfogênese , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo
17.
Genes Dev ; 23(14): 1689-98, 2009 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-19605690

RESUMO

The abundance of retinoic acid (RA) is determined by the balance between its synthesis by retinaldehyde dehydrogenase (RALDH) and its degradation by CYP26. In particular, the dynamic expression of three CYP26 genes controls the regional level of RA within the body. Pregastrulation mouse embryos express CYP26 but not RALDH. We now show that mice lacking all three CYP26 genes manifest duplication of the body axis as a result of expansion of the Nodal expression domain throughout the epiblast. Mouse Nodal was found to contain an RA-responsive element in intron 1 that is highly conserved among mammals. In the absence of CYP26, maternally derived RA activates Nodal expression in the entire epiblast of pregastrulation embryos via this element. These observations suggest that maternal RA must be removed by embryonic CYP26 for correct Nodal expression during embryonic patterning.


Assuntos
Padronização Corporal/fisiologia , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteína Nodal/metabolismo , Tretinoína/metabolismo , Animais , Sequência de Bases , Sequência Conservada , Sistema Enzimático do Citocromo P-450/genética , Feminino , Camadas Germinativas/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Camundongos Knockout , Dados de Sequência Molecular , Proteína Nodal/genética , Filogenia , Ácido Retinoico 4 Hidroxilase , Alinhamento de Sequência , Transdução de Sinais , Fatores de Tempo
18.
J Cell Biol ; 184(2): 323-34, 2009 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-19153222

RESUMO

The anterior-posterior axis of the mouse embryo is established by formation of distal visceral endoderm (DVE) and its subsequent migration. The precise mechanism of DVE formation has remained unknown, however. Here we show that bone morphogenetic protein (BMP) signaling plays dual roles in DVE formation. BMP signaling is required at an early stage for differentiation of the primitive endoderm into the embryonic visceral endoderm (VE), whereas it inhibits DVE formation, restricting it to the distal region, at a later stage. A Smad2-activating factor such as Activin also contributes to DVE formation by generating a region of VE positive for the Smad2 signal and negative for Smad1 signal. DVE is thus formed at the distal end of the embryo, the only region of VE negative for the Smad1 signal and positive for Smad2 signal. An inverse relation between the level of phosphorylated Smad1 and that of phosphorylated Smad2 in VE suggests an involvement of antagonism between Smad1- and Smad2-mediated signaling.


Assuntos
Embrião de Mamíferos/metabolismo , Endoderma/metabolismo , Transdução de Sinais/genética , Proteína Smad1/metabolismo , Proteína Smad2/metabolismo , Animais , Padronização Corporal/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Transgênicos , Gravidez
19.
Curr Opin Genet Dev ; 17(4): 344-50, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17646095

RESUMO

How and at what stage of development are the axes of the body determined? The left-right axis of the mouse embryo is generated de novo at embryonic day (E) 8.0 in a manner dependent on pre-existing positional cues. The anterior-posterior (A-P) axis becomes apparent earlier when distally located visceral endoderm migrates toward the future anterior side at E5.5. The direction of this migration is predetermined by asymmetric expression of Lefty1 and Cerl1(Cerberus-like 1). Asymmetric expression of Lefty1 takes place even earlier, in the primitive endoderm of the implanting blastocyst, pushing back the origin of the A-P axis to the peri-implantation stage. Although its functional significance remains to be seen, studies on how this molecular asymmetry emerges may provide insight into the origin of A-P polarity. The first cell fate decision occurs by the morula stage. Although blastomeres at the two-cell or four-cell stage may have biased fates, it is currently unknown whether this bias has any causal relation to later fate.


Assuntos
Padronização Corporal/genética , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Animais , Padronização Corporal/fisiologia , Linhagem da Célula , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/fisiologia , Fatores de Determinação Direita-Esquerda , Camundongos , Modelos Biológicos , Fatores de Tempo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/fisiologia
20.
Dev Cell ; 10(4): 451-9, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16580991

RESUMO

The earliest recognizable sign of patterning of the mouse embryo along the anteroposterior (A-P) axis is the migration of the distal visceral endoderm (DVE) toward the future anterior side. Here we report an asymmetry in the mouse embryo at an unexpectedly early stage. The gene for Lefty1, a Nodal antagonist that influences the direction of DVE migration, was found to be asymmetrically expressed in the primitive endoderm of the implanting blastocyst. Lefty1 expression begins randomly in the inner cell mass (ICM) of the blastocyst but is regionalized to one side of the tilted ICM shortly after implantation. Asymmetric expression of Lefty1 can be established by in vitro culture, indicating that it does not require interaction with the uterus. The asymmetric Lefty1 expression is induced by Nodal signaling, although Nodal and genes for its effectors are expressed symmetrically. This asymmetry in molecular patterning of the mouse embryo pushes back the origin of the A-P body axis to the peri-implantation stage.


Assuntos
Padronização Corporal/fisiologia , Polaridade Celular/fisiologia , Implantação do Embrião/fisiologia , Endoderma/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Animais , Sequência de Bases , Blastômeros/fisiologia , Padronização Corporal/genética , Polaridade Celular/genética , Células Cultivadas , Implantação do Embrião/genética , Técnicas In Vitro , Fatores de Determinação Direita-Esquerda , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Proteína Nodal , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/genética
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