Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 19 de 19
Filtrar
1.
Development ; 146(19)2019 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-31488564

RESUMO

Polycomb group (PcG) proteins are transcriptional repressors that are important regulators of cell fate during embryonic development. Among them, Ezh2 is responsible for catalyzing the epigenetic repressive mark H3K27me3 and is essential for animal development. The ability of zebrafish embryos lacking both maternal and zygotic ezh2 to form a normal body plan provides a unique model for comprehensively studying Ezh2 function during early development in vertebrates. By using a multi-omics approach, we found that Ezh2 is required for the deposition of H3K27me3 and is essential for proper recruitment of Polycomb group protein Rnf2. However, despite the complete absence of PcG-associated epigenetic mark and proteins, only minor changes in H3K4me3 deposition and gene and protein expression occur. These changes were mainly due to local dysregulation of transcription factors outside their normal expression boundaries. Altogether, our results in zebrafish show that Polycomb-mediated gene repression is important immediately after the body plan is formed to maintain spatially restricted expression profiles of transcription factors, and we highlight the differences that exist in the timing of PcG protein action between vertebrate species.


Assuntos
Padronização Corporal/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Grupo Polycomb/metabolismo , Proteínas Repressoras/metabolismo , Vertebrados/embriologia , Vertebrados/genética , Animais , Embrião não Mamífero/metabolismo , Epigênese Genética , Histonas/metabolismo , Lisina/metabolismo , Metilação , Mutação/genética , Proteoma/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo , Transcriptoma/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Zigoto/metabolismo
2.
Genes Dev ; 25(6): 529-33, 2011 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-21406552

RESUMO

Heterochromatin formation in fission yeast and the role of RNAi in this process have been intensively studied. So far, however, nothing is known about the regulation of expression of RNAi components during these events. Gullerova and colleagues (pp. 556-568) reveal an autoregulatory loop that regulates the expression of RNAi genes and centromeric heterochromatin formation during the cell cycle. Gene orientation plays a surprising role in this process.


Assuntos
Regulação Fúngica da Expressão Gênica , Interferência de RNA , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Ciclo Celular/genética , Centrômero/metabolismo , Heterocromatina/genética , Heterocromatina/metabolismo
3.
Int J Mol Sci ; 18(4)2017 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-28441764

RESUMO

Early life stage exposure to environmental chemicals may play a role in obesity by altering adipogenesis; however, robust in vivo methods to quantify these effects are lacking. The goal of this study was to analyze the effects of developmental exposure to chemicals on adipogenesis in the zebrafish (Danio rerio). We used label-free Stimulated Raman Scattering (SRS) microscopy for the first time to image zebrafish adipogenesis at 15 days post fertilization (dpf) and compared standard feed conditions (StF) to a high fat diet (HFD) or high glucose diet (HGD). We also exposed zebrafish embryos to a non-toxic concentration of tributyltin (TBT, 1 nM) or Tris(1,3-dichloroisopropyl)phosphate (TDCiPP, 0.5 µM) from 0-6 dpf and reared larvae to 15 dpf under StF. Potential molecular mechanisms of altered adipogenesis were examined by qPCR. Diet-dependent modulation of adipogenesis was observed, with HFD resulting in a threefold increase in larvae with adipocytes, compared to StF and HGD. Developmental exposure to TBT but not TDCiPP significantly increased adipocyte differentiation. The expression of adipogenic genes such as pparda, lxr and lepa was altered in response to HFD or chemicals. This study shows that SRS microscopy can be successfully applied to zebrafish to visualize and quantify adipogenesis, and is a powerful approach for identifying obesogenic chemicals in vivo.


Assuntos
Adipogenia/efeitos dos fármacos , Dieta Hiperlipídica , Microscopia Óptica não Linear/métodos , Compostos Organofosforados/toxicidade , Compostos de Trialquitina/toxicidade , Peixe-Zebra/metabolismo , Animais , Análise por Conglomerados , Poluentes Ambientais/toxicidade , Expressão Gênica/efeitos dos fármacos , Glucose/toxicidade , Larva/química , Larva/efeitos dos fármacos , Larva/metabolismo , PPAR alfa/genética , PPAR alfa/metabolismo , PPAR gama/genética , PPAR gama/metabolismo , Peixe-Zebra/crescimento & desenvolvimento
4.
PLoS Genet ; 8(7): e1002702, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22829772

RESUMO

RNA interference (RNAi)-related pathways affect gene activity by sequence-specific recruitment of Ago proteins to mRNA target molecules. The sequence specificity of this process stems from small RNA (sRNA) co-factors bound by the Ago protein. Stability of sRNA molecules in some pathways is in part regulated by Hen1-mediated methylation of their 3' ends. Here we describe the effects of the Caenorhabditis elegans HEN1 RNA-methyl-transferase homolog, HENN-1, on the different RNAi pathways in this nematode. We reveal differential effects of HENN-1 on the two pathways that are known to employ methylated sRNA molecules: the 26G and 21U pathways. Surprisingly, in the germline, stability of 21U RNAs, the C. elegans piRNAs, is only mildly affected by loss of methylation; and introduction of artificial 21U target RNA does not further destabilize non-methylated 21U RNAs. In contrast, most 26G RNAs display reduced stability and respond to loss of HENN-1 by displaying increased 3'-uridylation frequencies. Within the 26G RNA class, we find that specifically ERGO-1-bound 26G RNAs are modified by HENN-1, while ALG-3/ALG-4-bound 26G RNAs are not. Global gene expression analysis of henn-1 mutants reveals mild effects, including down-regulation of many germline-expressed genes. Our data suggest that, apart from direct effects of reduced 26G RNA levels of henn-1 on gene expression, most effects on global gene expression are indirect. These studies further refine our understanding of endogenous RNAi in C. elegans and the roles for Hen1 like enzymes in these pathways.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans , Células Germinativas/metabolismo , Proteínas do Tecido Nervoso/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Regiões 3' não Traduzidas/genética , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Metilação , Mutação , Proteínas do Tecido Nervoso/metabolismo , Estabilidade de RNA/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Transdução de Sinais
5.
EMBO J ; 29(21): 3688-700, 2010 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-20859253

RESUMO

Piwi-interacting RNAs (piRNAs) are germ line-specific small RNA molecules that have a function in genome defence and germ cell development. They associate with a specific class of Argonaute proteins, named Piwi, and function through an RNA interference-like mechanism. piRNAs carry a 2'-O-methyl modification at their 3' end, which is added by the Hen1 enzyme. We show that zebrafish hen1 is specifically expressed in germ cells and is essential for maintaining a female germ line, whereas it is dispensable in the testis. Hen1 protein localizes to nuage through its C-terminal domain, but is not required for nuage formation. In hen1 mutant testes, piRNAs become uridylated and adenylated. Uridylation frequency is highest on retro-transposon-derived piRNAs and is accompanied by decreased piRNA levels and mild derepression of transposon transcripts. Altogether, our data suggest the existence of a uridylation-mediated 3'-5' exonuclease activity acting on piRNAs in zebrafish germ cells, which is counteracted by nuage-bound Hen1 protein. This system discriminates between piRNA targets and is required for ovary development and fully efficient transposon silencing.


Assuntos
Metiltransferases/metabolismo , Oócitos/citologia , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Uridina/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Northern Blotting , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Feminino , Imunoprecipitação , Hibridização In Situ , Masculino , Metiltransferases/fisiologia , Dados de Sequência Molecular , Mutação/genética , Oócitos/metabolismo , Processamento de Terminações 3' de RNA/fisiologia , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Retroelementos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Frações Subcelulares , Testículo/citologia , Testículo/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética
6.
Epigenetics Chromatin ; 13(1): 5, 2020 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-32051014

RESUMO

BACKGROUND: Recent studies indicate that exposure to environmental chemicals may increase susceptibility to developing metabolic diseases. This susceptibility may in part be caused by changes to the epigenetic landscape which consequently affect gene expression and lead to changes in lipid metabolism. The epigenetic modifier enhancer of zeste 2 (Ezh2) is a histone H3K27 methyltransferase implicated to play a role in lipid metabolism and adipogenesis. In this study, we used the zebrafish (Danio rerio) to investigate the role of Ezh2 on lipid metabolism and chromatin status following developmental exposure to the Ezh1/2 inhibitor PF-06726304 acetate. We used the environmental chemical tributyltin (TBT) as a positive control, as this chemical is known to act on lipid metabolism via EZH-mediated pathways in mammals. RESULTS: Zebrafish embryos (0-5 days post-fertilization, dpf) exposed to non-toxic concentrations of PF-06726304 acetate (5 µM) and TBT (1 nM) exhibited increased lipid accumulation. Changes in chromatin were analyzed by the assay for transposase-accessible chromatin sequencing (ATAC-seq) at 50% epiboly (5.5 hpf). We observed 349 altered chromatin regions, predominantly located at H3K27me3 loci and mostly more open chromatin in the exposed samples. Genes associated to these loci were linked to metabolic pathways. In addition, a selection of genes involved in lipid homeostasis, adipogenesis and genes specifically targeted by PF-06726304 acetate via altered chromatin accessibility were differentially expressed after TBT and PF-06726304 acetate exposure at 5 dpf, but not at 50% epiboly stage. One gene, cebpa, did not show a change in chromatin, but did show a change in gene expression at 5 dpf. Interestingly, underlying H3K27me3 marks were significantly decreased at this locus at 50% epiboly. CONCLUSIONS: Here, we show for the first time the applicability of ATAC-seq as a tool to investigate toxicological responses in zebrafish. Our analysis indicates that Ezh2 inhibition leads to a partial primed state of chromatin linked to metabolic pathways which results in gene expression changes later in development, leading to enhanced lipid accumulation. Although ATAC-seq seems promising, our in-depth assessment of the cebpa locus indicates that we need to consider underlying epigenetic marks as well.


Assuntos
Cromatina/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Metabolismo dos Lipídeos , Proteínas de Peixe-Zebra/metabolismo , Adipogenia , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Cromatina/química , Montagem e Desmontagem da Cromatina , Sequenciamento de Cromatina por Imunoprecipitação , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Compostos de Trialquitina/farmacologia , Peixe-Zebra , Proteínas de Peixe-Zebra/antagonistas & inibidores
7.
PLoS One ; 14(1): e0210217, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30677064

RESUMO

Polycomb group (PcG) proteins are essential regulators of epigenetic gene silencing and development. The PcG protein enhancer of zeste homolog 2 (Ezh2) is a key component of the Polycomb Repressive Complex 2 and is responsible for placing the histone H3 lysine 27 trimethylation (H3K27me3) repressive mark on the genome through its methyltransferase domain. Ezh2 is highly conserved in vertebrates. We studied the role of ezh2 during development of zebrafish with the use of a mutant allele (ezh2(sa1199), R18STOP), which has a stop mutation in the second exon of the ezh2 gene. Two versions of the same line were used during this study. The first and original version of zygotic ezh2(sa1199) mutants unexpectedly retained ezh2 expression in brain, gut, branchial arches, and eyes at 3 days post-fertilization (dpf), as revealed by in-situ hybridization. Moreover, the expression pattern in homozygous mutants was identical to that of wild types, indicating that mutant ezh2 mRNA is not subject to nonsense mediated decay (NMD) as predicted. Both wild type and ezh2 mutant embryos presented edemas at 2 and 3 dpf. The line was renewed by selective breeding to counter select the non-specific phenotypes and survival was assessed. In contrast to earlier studies on ezh2 mutant zebrafish, ezh2(sa1199) mutants survived until adulthood. Interestingly, the ezh2 mRNA and Ezh2 protein were present during adulthood (70 dpf) in both wild type and ezh2(sa1199) mutant zebrafish. We conclude that the ezh2(sa1199) allele does not exhibit an ezh2 loss-of-function phenotype.


Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/genética , Epigênese Genética/fisiologia , Proteínas de Peixes/genética , Peixe-Zebra/crescimento & desenvolvimento , Animais , Códon sem Sentido , Metilação de DNA/fisiologia , Embrião não Mamífero , Éxons/genética , Histonas/metabolismo , Homozigoto , Fenótipo , RNA Mensageiro/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra
8.
Sci Rep ; 9(1): 4327, 2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30867528

RESUMO

The Polycomb group (PcG) protein family is a well-known group of epigenetic modifiers. We used zebrafish to investigate the role of Rnf2, the enzymatic subunit of PRC1. We found a positive correlation between loss of Rnf2 and upregulation of genes, especially of those whose promoter is normally bound by Rnf2. The heart of rnf2 mutants shows a tubular shaped morphology and to further understand the underlying mechanism, we studied gene expression of single wildtype and rnf2 mutant hearts. We detected the most pronounced differences at 3 dpf, including upregulation of heart transcription factors, such as tbx2a, tbx2b, and tbx3a. These tbx genes were decorated by broad PcG domains in wildtype whole embryo lysates. Chamber specific genes such as vmhc, myh6, and nppa showed downregulation in rnf2 mutant hearts. The marker of the working myocard, nppa, is negatively regulated by Tbx2 and Tbx3. Based on our findings and literature we postulate that loss of Rnf2-mediated repression results in upregulation and ectopic expression of tbx2/3, whose expression is normally restricted to the cardiac conductive system. This could lead to repression of chamber specific gene expression, a misbalance in cardiac cell types, and thereby to cardiac defects observed in rnf2 mutants.


Assuntos
Desenvolvimento Embrionário/genética , Coração/embriologia , Proteínas com Domínio T/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Animais , Mutação , Ubiquitina-Proteína Ligases/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
9.
PLoS One ; 13(7): e0200316, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29985950

RESUMO

Polycomb Group (PcG) genes are transcriptional repressors that are described to be important during development and differentiation. There is significant interest in PcGs proteins because of their role in stem cell biology and tumorigenesis. In this study we characterize the expression of a selection of PcG genes in the adult germline of zebrafish and during embryogenesis. In adults, expression of selected PcG genes is found to be enriched in germ line over somatic tissues. Therefore, the germ line of adult zebrafish was analyzed for the expression pattern of a selection of PcG genes by whole mount in situ hybridization. We detected presence of the tested PcG gene transcripts at early stages of both oogenesis and spermatogenesis. This enriched expression for early stages of gametogenesis is also observed in developing gonads at 4 and 5 weeks post fertilization. Additionally, zebrafish embryos were used to study the spatio-temporal expression patterns of a selection of PcG genes during development. The PcG genes that we tested are maternally loaded and ubiquitously expressed at early developmental stages, except of ezh1. The expression of the PcG genes that were assessed becomes enriched anteriorly and is more defined during tissue specification. The data shown here is an important resource for functional PcG gene studies in vivo.


Assuntos
Diferenciação Celular/genética , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Grupo Polycomb/genética , Transcriptoma , Peixe-Zebra/genética , Animais , Embrião não Mamífero , Perfilação da Expressão Gênica , Células Germinativas , Peixe-Zebra/embriologia
10.
Sci Rep ; 8(1): 9675, 2018 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-29946172

RESUMO

Mutations in C2orf71 are causative for autosomal recessive retinitis pigmentosa and occasionally cone-rod dystrophy. We have recently discovered that the protein encoded by this gene is important for modulation of the ciliary membrane through the recruitment of an actin assembly module, and have therefore renamed the gene to PCARE (photoreceptor cilium actin regulator). Here, we report on the identification of two copies of the c2orf71/pcare gene in zebrafish, pcare1 and pcare2. To study the role of the gene most similar to human PCARE, pcare1, we have generated a stable pcare1 mutant zebrafish model (designated pcare1 rmc100/rmc100 ) in which the coding sequence was disrupted using CRISPR/Cas9 technology. Retinas of both embryonic (5 dpf) and adult (6 mpf) pcare1 rmc100/rmc100 zebrafish display a clear disorganization of photoreceptor outer segments, resembling the phenotype observed in Pcare-/- mice. Optokinetic response and visual motor response measurements indicated visual impairment in pcare1 rmc100/rmc100 zebrafish larvae at 5 dpf. In addition, electroretinogram measurements showed decreased b-wave amplitudes in pcare1 rmc100/rmc100 zebrafish as compared to age- and strain-matched wild-type larvae, indicating a defect in the transretinal current. Altogether, our data show that lack of pcare1 causes a retinal phenotype in zebrafish and indicate that the function of the PCARE gene is conserved across species.


Assuntos
Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Eletrorretinografia , Imuno-Histoquímica , Mesotelina , Camundongos , Morfogênese/genética , Morfogênese/fisiologia , Estimulação Luminosa , Proteínas de Peixe-Zebra/genética
11.
Sci Rep ; 6: 24658, 2016 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-27145952

RESUMO

Polycomb group (PcG) proteins are transcriptional repressors of numerous genes, many of which regulate cell cycle progression or developmental processes. We used zebrafish to study Enhancer of zeste homolog 2 (Ezh2), the PcG protein responsible for placing the transcriptional repressive H3K27me3 mark. We identified a nonsense mutant of ezh2 and generated maternal zygotic (MZ) ezh2 mutant embryos. In contrast to knockout mice for PcG proteins, MZezh2 mutant embryos gastrulate seemingly normal, but die around 2 days post fertilization displaying pleiotropic phenotypes. Expression analyses indicated that genes important for early development are not turned off properly, revealing a regulatory role for Ezh2 during zygotic gene expression. In addition, we suggest that Ezh2 regulates maternal mRNA loading of zygotes. Analyses of tissues arising later in development, such as heart, liver, and pancreas, indicated that Ezh2 is required for maintenance of differentiated cell fates. Our data imply that the primary role of Ezh2 is to maintain tissues after tissue specification. Furthermore, our work indicates that Ezh2 is essential to sustain tissue integrity and to set up proper maternal mRNA contribution, and presents a novel and powerful tool to study how PcG proteins contribute to early vertebrate development.


Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteínas de Peixe-Zebra/genética , Animais , Diferenciação Celular , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Desenvolvimento Embrionário/fisiologia , Proteína Potenciadora do Homólogo 2 de Zeste/deficiência , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Trato Gastrointestinal/crescimento & desenvolvimento , Expressão Gênica , Genótipo , Coração/crescimento & desenvolvimento , Histonas/genética , Histonas/metabolismo , Proteína Homeobox Nkx-2.5/genética , Proteína Homeobox Nkx-2.5/metabolismo , Hibridização in Situ Fluorescente , Miocárdio/metabolismo , RNA Mensageiro/metabolismo , Imagem com Lapso de Tempo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/metabolismo , Zigoto/metabolismo
12.
PPAR Res ; 2015: 358029, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26697060

RESUMO

The Peroxisome Proliferator-Activated Receptors (PPARs) PPARA and PPARD are regulators of lipid metabolism with important roles in energy release through lipid breakdown, while PPARG plays a key role in lipid storage and adipogenesis. The aim of this review is to describe the role of PPARs in lipid metabolism, adipogenesis, and obesity and evaluate the zebrafish as an emerging vertebrate model to study the function of PPARs. Zebrafish are an appropriate model to study human diseases, including obesity and related metabolic diseases, as pathways important for adipogenesis and lipid metabolism which are conserved between mammals and fish. This review synthesizes knowledge on the role of PPARs in zebrafish and focuses on the putative function of PPARs in zebrafish adipogenesis. Using in silico analysis, we confirm the presence of five PPARs (pparaa, pparab, pparda, ppardb, and pparg) in the zebrafish genome with 67-74% identity to human and mouse PPARs. During development, pparda/b paralogs and pparg show mRNA expression around the swim bladder and pancreas, the region where adipocytes first develop, whereas pparg is detectable in adipocytes at 15 days post fertilization (dpf). This review indicates that the zebrafish is a promising model to investigate the specific functions of PPARs in adipogenesis and obesity.

13.
Stem Cells ; 25(4): 836-43, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17170062

RESUMO

Several studies have suggested that the cyclin-dependent kinase (CDK) inhibitor p21 plays a crucial role in regulating hematopoietic stem and progenitor pool size. To allow assessment of long-term stem cell functioning in vivo, we have backcrossed a p21 null allele to C57BL/6 (B6) mice, the most commonly used mouse strain in hematopoietic stem cell research. In various in vitro assays, the homozygous deletion of the p21 allele did not affect the number of hematopoietic cells in B6 mice. Furthermore, the competitive repopulation ability was not different between p21-deficient and wild-type stem cells from both young and aged (20-month-old) mice. These results show that p21 is not essential for regulation of stem cell number in steady state. When proliferative stress was applied on p21-deficient stem cells by serial transplantation of 1,500 Lin(-)Sca-1(+)c-kit(+) (LSK) cells, again no detrimental effect was observed on cobblestone area-forming cell (CAFC) frequency and competitive repopulating ability. However, when bone marrow cells from mice that received 2 Gy of irradiation were transplanted, p21 deficiency resulted in a more than fourfold reduction in competitive repopulation index. Finally, we did not find major differences in cell cycle status and global gene expression patterns between LSK cells from p21-deficient and wild-type mice. Our findings indicate that the background of mice used for studying the function of a gene by genetic modification may determine the outcome. Cumulatively, our data fail to support the notion that p21 is essential for stem cell function during steady-state hematopoiesis, but may be relatively more important under conditions of cellular stress.


Assuntos
Inibidor de Quinase Dependente de Ciclina p21/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/fisiologia , Células da Medula Óssea/efeitos da radiação , Contagem de Células , Ciclo Celular/efeitos da radiação , Divisão Celular , Cruzamentos Genéticos , Inibidor de Quinase Dependente de Ciclina p21/deficiência , Inibidor de Quinase Dependente de Ciclina p21/genética , Embrião de Mamíferos , Fibroblastos/citologia , Fibroblastos/fisiologia , Citometria de Fluxo , Fluoruracila/farmacologia , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout
14.
Cell ; 129(1): 69-82, 2007 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-17418787

RESUMO

Piwi proteins specify an animal-specific subclass of the Argonaute family that, in vertebrates, is specifically expressed in germ cells. We demonstrate that zebrafish Piwi (Ziwi) is expressed in both the male and the female gonad and is a component of a germline-specifying structure called nuage. Loss of Ziwi function results in a progressive loss of germ cells due to apoptosis during larval development. In animals that have reduced Ziwi function, germ cells are maintained but display abnormal levels of apoptosis in adults. In mammals, Piwi proteins associate with approximately 29-nucleotide-long, testis-specific RNA molecules called piRNAs. Here we show that zebrafish piRNAs are present in both ovary and testis. Many of these are derived from transposons, implicating a role for piRNAs in the silencing of repetitive elements in vertebrates. Furthermore, we show that piRNAs are Dicer independent and that their 3' end likely carries a 2'O-Methyl modification.


Assuntos
Células Germinativas/citologia , RNA não Traduzido/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Embrião não Mamífero/química , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Feminino , Genoma , Células Germinativas/química , Células Germinativas/metabolismo , Masculino , Ovário/citologia , Interferência de RNA , RNA não Traduzido/genética , Proteínas de Ligação a RNA/metabolismo , Retroelementos , Testículo/citologia , Peixe-Zebra
15.
Stem Cells ; 24(5): 1143-9, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16456126

RESUMO

Hematopoietic stem cells (HSCs) balance self-renewal and differentiation in order to sustain lifelong blood production and simultaneously maintain the HSC pool. However, there is clear evidence that HSCs are subject to quantitative and qualitative exhaustion. In this review, we briefly discuss several known aspects of the stem cell aging process, including DNA damage, telomere shortening, and oxidative stress. Besides these known players, there is increasing evidence that higher order chromatin structure, largely defined by the histone code and affecting transcriptional activity, is important. A model is suggested which describes how epigenetic regulation of gene transcription by modulation of the chromatin structure in stem cells can account for regulation of the aging program.


Assuntos
Senescência Celular/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Animais , Cromatina/genética , Cromatina/fisiologia , Dano ao DNA , Humanos , Modelos Biológicos , Proteínas do Grupo Polycomb , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia
16.
Blood ; 107(5): 2170-9, 2006 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-16293602

RESUMO

The molecular mechanism responsible for a decline of stem cell functioning after replicative stress remains unknown. We used mouse embryonic fibroblasts (MEFs) and hematopoietic stem cells (HSCs) to identify genes involved in the process of cellular aging. In proliferating and senescent MEFs one of the most differentially expressed transcripts was Enhancer of zeste homolog 2 (Ezh2), a Polycomb group protein (PcG) involved in histone methylation and deacetylation. Retroviral overexpression of Ezh2 in MEFs resulted in bypassing of the senescence program. More importantly, whereas normal HSCs were rapidly exhausted after serial transplantations, overexpression of Ezh2 completely conserved long-term repopulating potential. Animals that were reconstituted with 3 times serially transplanted control bone marrow cells all died due to hematopoietic failure. In contrast, similarly transplanted Ezh2-overexpressing stem cells restored stem cell quality to normal levels. In a "genetic genomics" screen, we identified novel putative Ezh2 target or partner stem cell genes that are associated with chromatin modification. Our data suggest that stabilization of the chromatin structure preserves HSC potential after replicative stress.


Assuntos
Senescência Celular/fisiologia , Montagem e Desmontagem da Cromatina/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Proteínas/metabolismo , Animais , Transplante de Medula Óssea , Divisão Celular/fisiologia , Sobrevivência Celular/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/fisiologia , Proteína Potenciadora do Homólogo 2 de Zeste , Feminino , Fibroblastos/citologia , Fibroblastos/fisiologia , Células-Tronco Hematopoéticas/citologia , Histona-Lisina N-Metiltransferase , Histonas/metabolismo , Camundongos , Complexo Repressor Polycomb 2 , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas/genética , Retroviridae , Transdução Genética
17.
Blood ; 105(2): 609-16, 2005 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-15374890

RESUMO

Many current experimental results show the necessity of new conceptual approaches to understand hematopoietic stem cell organization. Recently, we proposed a novel theoretical concept and a corresponding quantitative model based on microenvironment-dependent stem cell plasticity. The objective of our present work is to subject this model to an experimental test for the situation of chimeric hematopoiesis. Investigating clonal competition processes in DBA/2-C57BL/6 mouse chimeras, we observed biphasic chimerism development with initially increasing but long-term declining DBA/2 contribution. These experimental results were used to select the parameters of the mathematical model. To validate the model beyond this specific situation, we fixed the obtained parameter configuration to simulate further experimental settings comprising variations of transplanted DBA/2-C57BL/6 proportions, secondary transplantations, and perturbation of stabilized chimeras by cytokine and cytotoxic treatment. We show that the proposed model is able to consistently describe the situation of chimeric hematopoiesis. Our results strongly support the view that the relative growth advantage of strain-specific stem cells is not a fixed cellular property but is sensitively dependent on the actual state of the entire system. We conclude that hematopoietic stem cell organization should be understood as a flexible, self-organized rather than a fixed, preprogrammed process.


Assuntos
Hematopoese/fisiologia , Transplante de Células-Tronco Hematopoéticas , Modelos Biológicos , Quimera por Radiação , Quimeras de Transplante , Animais , Antineoplásicos/farmacologia , Citocinas/farmacologia , Feminino , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Gravidez , Processos Estocásticos
18.
Stem Cells ; 23(1): 82-92, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15625125

RESUMO

Adult somatic stem cells possess extensive self-renewal capacity, as their primary role is to replenish aged and functionally impaired tissues. We have previously shown that the stem cell pool in short-lived DBA/2 (D2) mice is reduced during aging, in contrast to long-lived C57BL/6 (B6) mice. This suggests the existence of a genetically determined mitotic clock operating in stem cells, which possibly limits organismal aging. In the study reported here, unfractionated bone marrow (BM) cells or highly purified Lin(-)Sca-1(+)c-kit(+) (LSK) cells were serially transplanted in lethally irradiated D2 and B6 mice. In both strains, serial transplantation resulted in a substantial loss of stem cell activity. However, as we estimate that in B6 mice, the maximum number of population doublings of primitive cells is approximately 30, in D2 mice this is only approximately 20, resulting in a 1,000-fold difference in expansion potential, irrespective of whether whole bone marrow or purified hematopoietic stem cells (HSCs) were transplanted. Interestingly, recipients reconstituted with serially transplanted BM cells were able to accept a freshly isolated graft without any further conditioning. Finally, we show that whereas transplantation of BM cells into healthy, nonconditioned, young B6 recipients does not lead to engraftment, young BM cells do engraft and provide multilineage reconstitution in nonirradiated aged mice. Our data clearly establish the relevance of an intrinsic, genetically controlled program associated with impaired stem cell functioning during aging.


Assuntos
Senescência Celular/fisiologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/fisiologia , Animais , Contagem de Células , Estudos de Avaliação como Assunto , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Fatores de Tempo
19.
Stem Cells ; 22(7): 1181-90, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15579638

RESUMO

Numerous assays exist that measure the function of stem cells. In this article, we review in detail the history and future of existing stem cell assays. Hematopoietic stem cells (HSCs) are historically the most well studied, but new developments in stem cell research, including the claim of stem cell plasticity, have caused controversies related to technical issues, as well as to semantics. Stem cell research requires proper definitions, and utilization of stem cell assays, especially since research on non-HSCs that lack solid stem cell assays, is rapidly evolving. These emerging fields may benefit from what has been learned from HSC assays: most important, that the true potential of stem cells can only be assessed retrospectively. This also relates to new developments in HSC research, when limiting numbers of in vitro-manipulated stem cells are transplanted. The most conflicting results arise when cells express stem cell characteristics in one assay but not in another. Should we adjust our definition of a stem cell? If so, when do we decide a claim of stem cell activity to be justified? We therefore recommend using multiple stem cell assays, preferably at least one in vivo assay. These assays should measure functionality of the putative stem cell population.


Assuntos
Biologia Celular , Separação Celular/métodos , Transplante de Células/métodos , Hematologia/métodos , Células-Tronco/citologia , Animais , Ligação Competitiva , Citometria de Fluxo/métodos , Células-Tronco Hematopoéticas , Humanos , Fenótipo , Pesquisa/tendências , Projetos de Pesquisa
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA