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1.
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
2.
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.

3.
Nat Ecol Evol ; 4(2): 261-269, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31907383

RESUMO

Unidirectional fluid flow generated by motile cilia at the left-right organizer (LRO) breaks left-right (L-R) symmetry during early embryogenesis in mouse, frog and zebrafish. The chick embryo, however, does not require motile cilia for L-R symmetry breaking. The diversity of mechanisms for L-R symmetry breaking among vertebrates and the trigger for such symmetry breaking in non-mammalian amniotes have remained unknown. Here we examined how L-R asymmetry is established in two reptiles, Madagascar ground gecko and Chinese softshell turtle. Both of these reptiles appear to lack motile cilia at the LRO. The expression of the Nodal gene at the LRO in the reptilian embryos was found to be asymmetric, in contrast to that in vertebrates such as mouse that are dependent on cilia for L-R patterning. Two paralogues of the Nodal gene derived from an ancient gene duplication are retained and expressed differentially in cilia-dependent and cilia-independent vertebrates. The expression of these two Nodal paralogues is similarly controlled in the lateral plate mesoderm but regulated differently at the LRO. Our in-depth analysis of reptilian embryos thus suggests that mammals and non-mammalian amniotes deploy distinct strategies dependent on different Nodal paralogues for rendering Nodal activity asymmetric at the LRO.


Assuntos
Padronização Corporal , Cílios , Animais , Embrião de Galinha , Madagáscar , Camundongos , Répteis , Peixe-Zebra
4.
Biol Open ; 9(2)2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-31988094

RESUMO

In the anterior foregut (AFG) of mouse embryos, the transcription factor SOX2 is expressed in the epithelia of the esophagus and proximal branches of respiratory organs comprising the trachea and bronchi, whereas NKX2.1 is expressed only in the epithelia of respiratory organs. Previous studies using hypomorphic Sox2 alleles have indicated that reduced SOX2 expression causes the esophageal epithelium to display some respiratory organ characteristics. In the present study, we produced mouse embryos with AFG-specific SOX2 deficiency. In the absence of SOX2 expression, a single NKX2.1-expressing epithelial tube connected the pharynx and the stomach, and a pair of bronchi developed in the middle of the tube. Expression patterns of NKX2.1 and SOX9 revealed that the anterior and posterior halves of SOX2-deficient AFG epithelial tubes assumed the characteristics of the trachea and bronchus, respectively. In addition, we found that mesenchymal tissues surrounding the SOX2-deficient NKX2.1-expressing epithelial tube changed to those surrounding the trachea and bronchi in the anterior and posterior halves, as indicated by the arrangement of smooth muscle cells and SOX9-expressing cells and by the expression of Wnt4 (esophagus specific), Tbx4 (respiratory organ specific), and Hoxb6 (distal bronchus specific). The impact of mesenchyme-derived signaling on the early stage of AFG epithelial specification has been indicated. Our study demonstrated an opposite trend where epithelial tissue specification causes concordant changes in mesenchymal tissues, indicating a reciprocity of epithelial-mesenchymal interactions.

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.
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.
Congenit Anom (Kyoto) ; 56(3): 112-8, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26662860

RESUMO

The "Kyoto Collection of Human Embryos" at Kyoto University was begun in 1961. Although morphological analyses of samples in the Kyoto Collection have been performed, these embryos have been considered difficult to genetically analyze because they have been preserved in formalin or Bouin's solution for 20-50 years. Owing to the recent advances in molecular biology, it has become possible to extract DNA from long-term fixed tissues. The purpose of this study was to extract DNA from wet preparations of human embryo samples after long-term preservation in fixing solution. We optimized the DNA extraction protocol to be suitable for tissues that have been damaged by long-term fixation, including DNA-protein crosslinking damage. Diluting Li2 CO3 with 70% ethanol effectively removed picric acid from samples fixed in Bouin's solution. Additionally, 20.0 mg/mL proteinase was valuable to lyse the long-term fixed samples. The extracted DNA was checked with PCR amplification using several sets of primers and sequence analysis. The PCR products included at least 295- and 838-bp amplicons. These results show that the extracted DNA is applicable for genetic analyses, and indicate that old embryos in the Kyoto Collection should be made available for future studies. The protocol described in this study can successfully extract DNA from old specimens and, with improvements, should be applicable in research aiming to understand the molecular mechanisms of human congenital anomalies.


Assuntos
Ácido Acético , DNA/isolamento & purificação , Embrião de Mamíferos , Formaldeído , Picratos , Preservação Biológica , Anormalidades Congênitas/diagnóstico , Anormalidades Congênitas/genética , Humanos , Reação em Cadeia da Polimerase , Preservação Biológica/métodos , Fatores de Tempo
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.
Fetal Diagn Ther ; 29(4): 325-30, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21293111

RESUMO

We here describe a case of congenital leukemia that ended in intrauterine fetal demise at 30 weeks of gestation. Acute enlargement of the fetal trunk, elevated pulsatility index of the umbilical artery with concomitant decline of pulsatility index of the middle cerebral artery, pleural effusion, and polyhydramnios preceded the fetal death. Diagnosis of congenital myeloid leukemia was suggested by microscopic examination of the placental tissue, revealing immature myeloid precursors filling the lumina of fetal vessels in the umbilical cord and chorionic villi. Extensive vascular involvement of the placenta by leukemic cells was considered to be a primary cause of the fetal death.


Assuntos
Doenças Fetais/patologia , Leucemia Mieloide/congênito , Leucemia Mieloide/patologia , Cesárea , Vilosidades Coriônicas/patologia , Evolução Fatal , Feminino , Doenças Fetais/fisiopatologia , Idade Gestacional , Humanos , Leucemia Mieloide/fisiopatologia , Masculino , Derrame Pleural , Gravidez , Fluxo Pulsátil , Natimorto , Artérias Umbilicais/fisiopatologia , Cordão Umbilical/patologia
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