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1.
Nat Commun ; 15(1): 5090, 2024 Jun 25.
Artículo en Inglés | MEDLINE | ID: mdl-38918373

RESUMEN

The development of haematopoiesis involves the coordinated action of numerous genes, some of which are implicated in haematological malignancies. However, the biological function of many genes remains elusive and unknown functional genes are likely to remain to be uncovered. Here, we report a previously uncharacterised gene in haematopoiesis, identified by screening mutant embryonic stem cells. The gene, 'attenuated haematopoietic development (Ahed)', encodes a nuclear protein. Conditional knockout (cKO) of Ahed results in anaemia from embryonic day 14.5 onward, leading to prenatal demise. Transplantation experiments demonstrate the incapacity of Ahed-deficient haematopoietic cells to reconstitute haematopoiesis in vivo. Employing a tamoxifen-inducible cKO model, we further reveal that Ahed deletion impairs the intrinsic capacity of haematopoietic cells in adult mice. Ahed deletion affects various pathways, and published databases present cancer patients with somatic mutations in Ahed. Collectively, our findings underscore the fundamental roles of Ahed in lifelong haematopoiesis, implicating its association with malignancies.


Asunto(s)
Hematopoyesis , Ratones Noqueados , Animales , Hematopoyesis/genética , Ratones , Humanos , Femenino , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/citología , Ratones Endogámicos C57BL , Mutación , Anemia/genética , Masculino , Células Madre Embrionarias/metabolismo
2.
Nat Cell Biol ; 26(7): 1124-1138, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38902423

RESUMEN

Women are born with all of their oocytes. The oocyte proteome must be maintained with minimal damage throughout the woman's reproductive life, and hence for decades. Here we report that oocyte and ovarian proteostasis involves extreme protein longevity. Mouse ovaries had more extremely long-lived proteins than other tissues, including brain. These long-lived proteins had diverse functions, including in mitochondria, the cytoskeleton, chromatin and proteostasis. The stable proteins resided not only in oocytes but also in long-lived ovarian somatic cells. Our data suggest that mammals increase protein longevity and enhance proteostasis by chaperones and cellular antioxidants to maintain the female germline for long periods. Indeed, protein aggregation in oocytes did not increase with age and proteasome activity did not decay. However, increasing protein longevity cannot fully block female germline senescence. Large-scale proteome profiling of ~8,890 proteins revealed a decline in many long-lived proteins of the proteostasis network in the aging ovary, accompanied by massive proteome remodeling, which eventually leads to female fertility decline.


Asunto(s)
Oocitos , Ovario , Proteoma , Proteostasis , Femenino , Animales , Oocitos/metabolismo , Ovario/metabolismo , Proteoma/metabolismo , Ratones , Ratones Endogámicos C57BL , Envejecimiento/metabolismo , Envejecimiento/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Senescencia Celular , Fertilidad , Proteómica/métodos , Longevidad/fisiología
3.
Artículo en Inglés | MEDLINE | ID: mdl-38625488

RESUMEN

Cancer stem cells (CSCs), which are critical targets for cancer therapy as they are involved in drug resistance to anticancer drugs, and metastasis, are maintained by angiocrine factors produced by particular niches that form within tumor tissue. Secreted frizzled-related protein 1 (Sfrp1) is an extracellular protein that modulates Wnt signaling. However, the cells that produce Sfrp1 in the tumor environment and its function remain unclear. We aimed to elucidate angiocrine factors related to CSC maintenance, focusing on Sfrp1. Although Sfrp1 is a Wnt pathway-related factor, its impact on tumor tissues remains unknown. We investigated the localization of Sfrp1 in tumors and found that it is expressed in some tumor vessels. Analysis of mice lacking Sfrp1 showed that tumor growth was suppressed in Sfrp1-deficient tumor tissues. Flow cytometry analysis indicated that CSCs were maintained in the early tumor growth phase in the Sfrp1 knockout (KO) mouse model of tumor-bearing cancer. However, tumor growth was inhibited in the late tumor growth phase because of the inability to maintain CSCs. Real-time PCR results from tumors of Sfrp1 KO mice showed that the expression of Wnt signaling target genes significantly decreased in the late stage of tumor growth. This suggests that Sfrp1, an angiocrine factor produced by the tumor vascular niche, is involved in Wnt signaling-mediated mechanisms in tumor tissues.

4.
Dev Biol ; 498: 97-105, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-37019373

RESUMEN

Gse1 is a component of the CoREST complex that acts as an H3K4 and H3K9 demethylase and regulates gene expression. Here, we examined the expression and role of Gse1 in mouse development. Gse1 is expressed in male and female germ cells and plays both maternal and zygotic roles. Thus, maternal deletion of Gse1 results in a high incidence of prenatal death, and zygotic deletion leads to embryonic lethality from embryonic day 12.5 (E12.5) and perinatal death. Gse1 is expressed in the junctional zone and the labyrinth of the developing placenta. Gse1 mutant (Gse1Δex3/Δex3) placenta begins to exhibit histological defects from E14.5, being deficient in MCT4+ syncytiotrophoblast II. The number of various cell types was largely maintained in the mutant placenta at E10.5, but several genes were upregulated in giant trophoblasts at E10.5. Placenta-specific deletion of Gse1 with Tat-Cre suggested that defects in Gse1Δex3/Δex3 embryos are due to placental function deficiency. These results suggest that Gse1 is required for placental development in mice, and in turn, is essential for embryonic development.


Asunto(s)
Placenta , Placentación , Ratones , Embarazo , Femenino , Animales , Masculino , Desarrollo Embrionario/genética , Trofoblastos
5.
Development ; 149(9)2022 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-35420656

RESUMEN

For left-right symmetry breaking in the mouse embryo, the basal body must become positioned at the posterior side of node cells, but the precise mechanism for this has remained unknown. Here, we examined the role of microtubules (MTs) and actomyosin in this basal body positioning. Exposure of mouse embryos to agents that stabilize or destabilize MTs or F-actin impaired such positioning. Active myosin II was detected at the anterior side of node cells before the posterior shift of the basal body, and this asymmetric activation was lost in Prickle and dachsous mutant embryos. The organization of basal-body associated MTs (baMTs) was asymmetric between the anterior and posterior sides of node cells, with anterior baMTs extending horizontally and posterior baMTs extending vertically. This asymmetry became evident after polarization of the PCP core protein Vangl1 and before the posterior positioning of the basal body, and it also required the PCP core proteins Prickle and dachsous. Our results suggest that the asymmetry in baMT organization may play a role in correct positioning of the basal body for left-right symmetry breaking.


Asunto(s)
Cuerpos Basales , Polaridad Celular , Actinas/metabolismo , Animales , Polaridad Celular/fisiología , Cilios/metabolismo , Ratones , Microtúbulos/metabolismo
6.
Nat Commun ; 12(1): 4071, 2021 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-34210974

RESUMEN

Molecular left-right (L-R) asymmetry is established at the node of the mouse embryo as a result of the sensing of a leftward fluid flow by immotile cilia of perinodal crown cells and the consequent degradation of Dand5 mRNA on the left side. We here examined how the fluid flow induces Dand5 mRNA decay. We found that the first 200 nucleotides in the 3' untranslated region (3'-UTR) of Dand5 mRNA are necessary and sufficient for the left-sided decay and to mediate the response of a 3'-UTR reporter transgene to Ca2+, the cation channel Pkd2, the RNA-binding protein Bicc1 and their regulation by the flow direction. We show that Bicc1 preferentially recognizes GACR and YGAC sequences, which can explain the specific binding to a conserved GACGUGAC motif located in the proximal Dand5 3'-UTR. The Cnot3 component of the Ccr4-Not deadenylase complex interacts with Bicc1 and is also required for Dand5 mRNA decay at the node. These results suggest that Ca2+ currents induced by leftward fluid flow stimulate Bicc1 and Ccr4-Not to mediate Dand5 mRNA degradation specifically on the left side of the node.


Asunto(s)
Embrión de Mamíferos/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Estabilidad del ARN/fisiología , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Receptores CCR4/metabolismo , Regiones no Traducidas 3' , Animales , Regulación del Desarrollo de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones , Ratones Noqueados , Proteínas de Unión al ARN/genética , Receptores CCR4/genética , Canales Catiónicos TRPP/metabolismo , Factores de Transcripción
7.
PLoS Genet ; 16(12): e1009232, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33347437

RESUMEN

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.


Asunto(s)
Dineínas Axonemales/metabolismo , Axonema/metabolismo , Transporte Biológico Activo/genética , Movimiento Celular/genética , Cilios/metabolismo , Embrión de Mamíferos/metabolismo , Microtúbulos/metabolismo , Animales , Dineínas Axonemales/genética , Axonema/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cilios/genética , Embrión de Mamíferos/fisiología , Embrión de Mamíferos/ultraestructura , Epéndimo/embriología , Epéndimo/metabolismo , Epéndimo/fisiología , Técnica del Anticuerpo Fluorescente , Genotipo , Inmunoprecipitación , Ratones , Ratones Noqueados , Microscopía Electrónica de Transmisión , Microtúbulos/genética , Mutación , Fenotipo , Tráquea/embriología , Tráquea/metabolismo , Tráquea/fisiología , Tráquea/ultraestructura
8.
Nat Commun ; 11(1): 5520, 2020 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-33139725

RESUMEN

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.


Asunto(s)
Nucleótidos de Adenina/metabolismo , Astenozoospermia/genética , Proteínas del Citoesqueleto/deficiencia , Situs Inversus/genética , Adolescente , Adulto , Animales , Astenozoospermia/patología , Axonema/ultraestructura , Sistemas CRISPR-Cas/genética , Cilios/metabolismo , Cilios/ultraestructura , Proteínas del Citoesqueleto/genética , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Epidídimo/patología , Femenino , Flagelos/metabolismo , Flagelos/ultraestructura , Humanos , Mutación con Pérdida de Función , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Planarias/citología , Planarias/genética , Planarias/metabolismo , Mucosa Respiratoria/citología , Mucosa Respiratoria/patología , Situs Inversus/diagnóstico por imagen , Situs Inversus/patología , Motilidad Espermática/genética , Tomografía Computarizada por Rayos X , Secuenciación del Exoma
9.
Sci Adv ; 6(30): eaba1195, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32743070

RESUMEN

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.

10.
Cell Rep ; 31(10): 107733, 2020 06 09.
Artículo en Inglés | MEDLINE | ID: mdl-32521258

RESUMEN

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.


Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas Wnt/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Desarrollo Embrionario , Endocitosis , Endodermo/metabolismo , Femenino , Gastrulación , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Embarazo , Proteínas Wnt/antagonistas & inhibidores , Vía de Señalización Wnt , Proteínas de Unión al GTP rab/deficiencia , Proteínas de Unión a GTP rab7
11.
Genes Cells ; 24(11): 731-745, 2019 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-31554018

RESUMEN

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.


Asunto(s)
Cilios/metabolismo , Citoplasma/metabolismo , Proteínas del Citoesqueleto/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Animales , Cilios/ultraestructura , Citoplasma/ultraestructura , Fibroblastos , Proteínas de Choque Térmico , Péptidos y Proteínas de Señalización Intracelular/genética , Cinesinas , Ratones , Ratones Noqueados , Proteínas Supresoras de Tumor
12.
Nat Commun ; 8(1): 1492, 2017 11 14.
Artículo en Inglés | MEDLINE | ID: mdl-29138408

RESUMEN

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.


Asunto(s)
Embrión de Mamíferos/metabolismo , Endodermo/metabolismo , Factores de Determinación Derecha-Izquierda/genética , Proteína Nodal/metabolismo , Transducción de Señal , Vísceras/metabolismo , Animales , Tipificación del Cuerpo , Cromosomas Artificiales Bacterianos , Embrión de Mamíferos/citología , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Ratones , Ratones Transgénicos , Procesos Estocásticos
13.
Dev Cell ; 40(5): 439-452.e4, 2017 03 13.
Artículo en Inglés | MEDLINE | ID: mdl-28292423

RESUMEN

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.


Asunto(s)
Tipificación del Cuerpo , Polaridad Celular , Transducción de Señal , Proteínas Wnt/metabolismo , Proteína Wnt-5a/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Comunicación Celular , Péptidos y Proteínas de Señalización Intracelular , Proteínas con Dominio LIM/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Mutantes , Modelos Biológicos , Proteínas/metabolismo
14.
J Cell Biol ; 215(5): 705-718, 2016 Dec 05.
Artículo en Inglés | MEDLINE | ID: mdl-27881714

RESUMEN

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.


Asunto(s)
Proteínas Ligadas a GPI/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína Nodal/metabolismo , Fosfolipasas A2/metabolismo , Animales , Tipificación del Cuerpo , Células CHO , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario , Células HEK293 , Humanos , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/genética , Ratones Noqueados , Modelos Biológicos , Mutación/genética , Monoéster Fosfórico Hidrolasas/genética , Fosfotransferasas/genética , Transducción de Señal
15.
Am J Hum Genet ; 99(2): 460-9, 2016 08 04.
Artículo en Inglés | MEDLINE | ID: mdl-27486780

RESUMEN

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.


Asunto(s)
Axonema/genética , Axonema/metabolismo , Proteínas Portadoras/genética , Cilios/patología , Dineínas/química , Dineínas/metabolismo , Síndrome de Kartagener/genética , Síndrome de Kartagener/patología , Mutación , Animales , Axonema/patología , Axonema/ultraestructura , Cilios/metabolismo , Cilios/ultraestructura , Dineínas/genética , Dineínas/ultraestructura , Exoma/genética , Exones/genética , Técnica del Anticuerpo Fluorescente , Genes Recesivos , Humanos , Ratones , Microscopía Electrónica de Transmisión , Unión Proteica , Xenopus , Proteínas de Xenopus/deficiencia , Proteínas de Xenopus/genética
16.
Genes Cells ; 21(7): 728-39, 2016 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-27353389

RESUMEN

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.


Asunto(s)
Cilios/genética , Síndrome de Kartagener/genética , Proteínas/genética , Animales , Dineínas Axonemales/genética , Axonema/genética , Axonema/patología , Cilios/patología , Citoplasma/genética , Citoplasma/metabolismo , Proteínas del Citoesqueleto , Modelos Animales de Enfermedad , Dineínas/genética , Humanos , Síndrome de Kartagener/patología , Ratones , Ratones Transgénicos , Mutación
17.
Elife ; 52016 06 22.
Artículo en Inglés | MEDLINE | ID: mdl-27331609

RESUMEN

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.


Asunto(s)
Exorribonucleasas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Corazón/embriología , Proteína Homeótica Nkx-2.5/metabolismo , Poliadenilación , Animales , Exorribonucleasas/genética , Proteína Homeótica Nkx-2.5/genética , Ratones Endogámicos C57BL , Ratones Noqueados
18.
EMBO J ; 34(1): 97-114, 2015 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-25385835

RESUMEN

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.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Microtúbulos/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas de Ciclo Celular/genética , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Inestabilidad Genómica/genética , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/genética , Complejos Multiproteicos/genética
19.
Philos Trans R Soc Lond B Biol Sci ; 369(1657)2014 Dec 05.
Artículo en Inglés | MEDLINE | ID: mdl-25349445

RESUMEN

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.


Asunto(s)
Blastómeros/fisiología , Tipificación del Cuerpo/fisiología , Diferenciación Celular/fisiología , Linaje de la Célula/fisiología , Desarrollo Embrionario/fisiología , Epigénesis Genética/fisiología , Genoma/genética , Animales , Ratones , Modelos Biológicos
20.
Nat Commun ; 3: 1322, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23271656

RESUMEN

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.


Asunto(s)
Retroalimentación Fisiológica , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismo , Regiones no Traducidas 3' , Animales , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones , Ratones Transgénicos , Conformación de Ácido Nucleico , Unión Proteica , Estabilidad del ARN , ARN Mensajero/genética , Transducción de Señal , Proteína Wnt3/genética , Proteína Wnt3/metabolismo
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