RESUMEN
Pluripotent stem cell lines can be derived from blastocyst embryos, which yield embryonic stem cell lines (ES cells), as well as the postimplantation epiblast, which gives rise to epiblast stem cell lines (EpiSCs). Remarkably, ES cells and EpiSCs display profound differences in the combination of growth factors that maintain their pluripotent state. Molecular and functional differences between these two stem cell types demonstrate that the tissue of origin and/or the growth factor milieu may be important determinants of the stem cell identity. We explored how developmental stage of the tissue of origin and culture growth factor conditions affect the stem cell pluripotent state. Our findings indicate that novel stem cell lines, with unique functional and molecular properties, can be generated from murine blastocyst embryos. We demonstrate that the culture growth factor environment and cell-cell interaction play a critical role in defining several unique and stable stem cell ground states.
Asunto(s)
Blastocisto/citología , Línea Celular , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Células Madre Pluripotentes/citología , Animales , Proteína Morfogenética Ósea 4/metabolismo , Cadherinas/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular , Embrión de Mamíferos/citología , Estratos Germinativos/citología , RatonesRESUMEN
Sister chromatid cohesion ensures the faithful segregation of chromosomes in mitosis and in both meiotic divisions. Meiosis-specific components of the cohesin complex, including the recently described SMC1 isoform SMC1 beta, were suggested to be required for meiotic sister chromatid cohesion and DNA recombination. Here we show that SMC1 beta-deficient mice of both sexes are sterile. Male meiosis is blocked in pachytene; female meiosis is highly error-prone but continues until metaphase II. Prophase axial elements (AEs) are markedly shortened, chromatin extends further from the AEs, chromosome synapsis is incomplete, and sister chromatid cohesion in chromosome arms and at centromeres is lost prematurely. In addition, crossover-associated recombination foci are absent or reduced, and meiosis-specific perinuclear telomere arrangements are impaired. Thus, SMC1 beta has a key role in meiotic cohesion, the assembly of AEs, synapsis, recombination, and chromosome movements.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cromátides/genética , Infertilidad/genética , Meiosis/genética , Proteínas Nucleares/metabolismo , Recombinación Genética/genética , Animales , Proteínas de Ciclo Celular/genética , Centrómero/genética , Proteínas Cromosómicas no Histona , Emparejamiento Cromosómico/genética , Segregación Cromosómica/genética , ADN/genética , Femenino , Proteínas Fúngicas , Masculino , Metafase/genética , Ratones , Ratones Noqueados , Proteínas Nucleares/genética , Oogénesis/genética , Espermatogénesis/genética , Telómero/genética , CohesinasRESUMEN
In meiotic prophase, the sister chromatids of each chromosome develop a common axial element (AE) that is integrated into the synaptonemal complex (SC). We analyzed the incorporation of sister chromatid cohesion proteins (cohesins) and other AE components into AEs. Meiotic cohesin REC8 appeared shortly before premeiotic S phase in the nucleus and formed AE-like structures (REC8-AEs) from premeiotic S phase on. Subsequently, meiotic cohesin SMC1beta, cohesin SMC3, and AE proteins SCP2 and SCP3 formed dots along REC8-AEs, which extended and fused until they lined REC8-AEs along their length. In metaphase I, SMC1beta, SMC3, SCP2, and SCP3 disappeared from the chromosome arms and accumulated around the centromeres, where they stayed until anaphase II. In striking contrast, REC8 persisted along the chromosome arms until anaphase I and near the centromeres until anaphase II. We propose that REC8 provides a basis for AE formation and that the first steps in AE assembly do not require SMC1beta, SMC3, SCP2, and SCP3. Furthermore, SMC1beta, SMC3, SCP2, and SCP3 cannot provide arm cohesion during metaphase I. We propose that REC8 then provides cohesion. RAD51 and/or DMC1 coimmunoprecipitates with REC8, suggesting that REC8 may also provide a basis for assembly of recombination complexes.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteoglicanos Tipo Condroitín Sulfato , Proteínas Cromosómicas no Histona/metabolismo , Enzimas Reparadoras del ADN , Meiosis/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Espermatocitos/metabolismo , Complejo Sinaptonémico/metabolismo , Ácido Anhídrido Hidrolasas , Anafase/genética , Animales , Proteínas de Ciclo Celular/genética , Células Cultivadas , Proteínas Cromosómicas no Histona/genética , Cromosomas/genética , Cromosomas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Técnica del Anticuerpo Fluorescente , Proteínas Fúngicas , Inmunohistoquímica , Masculino , Modelos Biológicos , Proteínas Nucleares/genética , Fosfoproteínas/genética , Recombinasa Rad51 , Ratas , Fase S/genética , Espermatocitos/citología , Complejo Sinaptonémico/ultraestructura , Testículo/citología , Testículo/metabolismo , CohesinasRESUMEN
BACKGROUND: In the nuclei of most mammalian cells, pericentric heterochromatin is characterized by DNA methylation, histone modifications such as H3K9me3 and H4K20me3, and specific binding proteins like heterochromatin-binding protein 1 isoforms (HP1 isoforms). Maintenance of this specialized chromatin structure is of great importance for genome integrity and for the controlled repression of the repetitive elements within the pericentric DNA sequence. Here we have studied histone modifications at pericentric heterochromatin during primordial germ cell (PGC) development using different fixation conditions and fluorescent immunohistochemical and immunocytochemical protocols. RESULTS: We observed that pericentric heterochromatin marks, such as H3K9me3, H4K20me3, and HP1 isoforms, were retained on pericentric heterochromatin throughout PGC development. However, the observed immunostaining patterns varied, depending on the fixation method, explaining previous findings of a general loss of pericentric heterochromatic features in PGCs. Also, in contrast to the general clustering of multiple pericentric regions and associated centromeres in DAPI-dense regions in somatic cells, the pericentric regions of PGCs were more frequently organized as individual entities. We also observed a transient enrichment of the chromatin remodeler ATRX in pericentric regions in embryonic day 11.5 (E11.5) PGCs. At this stage, a similar and low level of major satellite repeat RNA transcription was detected in both PGCs and somatic cells. CONCLUSIONS: These results indicate that in pericentric heterochromatin of mouse PGCs, only minor reductions in levels of some chromatin-associated proteins occur, in association with a transient increase in ATRX, between E11.5 and E13.5. These pericentric heterochromatin regions more frequently contain only a single centromere in PGCs compared to the surrounding soma, indicating a difference in overall organization, but there is no de-repression of major satellite transcription.
Asunto(s)
Células Germinativas/metabolismo , Heterocromatina/metabolismo , Animales , Centrómero/metabolismo , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Embrión de Mamíferos/metabolismo , Silenciador del Gen , Células Germinativas/citología , Células Germinativas/crecimiento & desarrollo , Histonas/metabolismo , Inmunohistoquímica , Ratones , Microscopía Fluorescente , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMEN
Recent data demonstrates that stem cells can exist in two morphologically, molecularly and functionally distinct pluripotent states; a naïve LIF-dependent pluripotent state which is represented by murine embryonic stem cells (mESCs) and an FGF-dependent primed pluripotent state represented by murine and rat epiblast stem cells (EpiSCs). We find that derivation of induced pluripotent stem cells (iPSCs) under EpiSC culture conditions yields FGF-dependent iPSCs from hereon called FGF-iPSCs) which, unexpectedly, display naïve ES-like/ICM properties. FGF-iPSCs display X-chromosome activation, multi-lineage differentiation, teratoma competence and chimera contribution in vivo. Our findings suggest that in 129 and Bl6 mouse strains, iPSCs can dominantly adopt a naive pluripotent state regardless of culture growth factor conditions. Characterization of the key molecular signalling pathways revealed FGF-iPSCs to depend on the Activin/Nodal and FGF pathways, while signalling through the JAK-STAT pathway is not required for FGF-iPS cell maintenance. Our findings suggest that in 129 and Bl6 mouse strains, iPSCs can dominantly adopt a naive pluripotent state regardless of culture growth factor conditions.