RESUMO
Embryonic germ-line cells are unipotent cells that give rise to either sperm or oocytes. However, pluripotent stem cells can be derived from primordial germ cells (PGCs) or spermatogonia, suggesting that germ-line cells retain a capacity for pluripotency. Here, we made genome-wide comparisons of the gene expression profiles of freshly isolated PGCs, in vitro-formed PGCs (iPGCs), and other stem cell lines, including embryonic stem cells (ESCs), embryonic germ cells (EGCs) and germ-line stem (GS) cells. Comparing PGC with ESC, 382 genes/transcripts were significantly up-regulated in ESC, while 188 were elevated in PGC. This suggests that PGCs possess transcription program distinct from that of ESC, although both share expression of many pluripotency-associated genes. Our micro-array analysis showed that the analyzed samples could be classified into two groups: one consisting of all the ESCs and most of EGCs, and the other containing PGC samples, iPGC, one type of female EGC and GS cells. We then identified "signature" genes for the two groups, and used them to characterize GS cells, EGC, and iPGCs, and revealed developmental status of each cell type. The relationships between PGCs and stem cells derived from embryos or germ cells are discussed in light of these findings.
Assuntos
Células-Tronco Embrionárias/metabolismo , Perfilação da Expressão Gênica , Células-Tronco Pluripotentes/metabolismo , Animais , Células Cultivadas , Feminino , Células Germinativas , Masculino , Camundongos , Camundongos Endogâmicos , Análise Multivariada , Células-TroncoRESUMO
Intravital imaging of brain and bone marrow cells in the skull with subcellular resolution has revolutionized neurobiology, immunology and hematology. However, the application of this powerful technology in studies of abdominal organs has long been impeded by organ motion caused by breathing and heartbeat. Here we describe for the first time a simple device designated 'microstage' that effectively reduces organ motions without causing tissue lesions. Combining this microstage device with an upright intravital laser scanning microscope equipped with a unique stick-type objective lens, the system enables subcellular-level imaging of abdominal organs in live mice. We demonstrate that this technique allows for the quantitative analysis of subcellular structures and gene expressions in cells, the tracking of intracellular processes in real-time as well as three-dimensional image construction in the pancreas and liver of the live mouse. As the aforementioned analyses based on subcellular imaging could be extended to other intraperitoneal organs, the technique should offer great potential for investigation of physiological and disease-specific events of abdominal organs. The microstage approach adds an exciting new technique to the in vivo imaging toolbox.
Assuntos
Abdome , Microscopia/instrumentação , Microscopia/métodos , Imagem Molecular/instrumentação , Imagem Molecular/métodos , Animais , Imageamento Tridimensional , Fígado/citologia , Camundongos , Camundongos Transgênicos , Pâncreas/citologia , Pâncreas/ultraestrutura , Imagem com Lapso de TempoRESUMO
Changes in nuclear organization and the epigenetic state of the genome are important driving forces for developmental gene expression. However, a strategy that allows simultaneous visualization of the dynamics of the epigenomic state and nuclear structure has been lacking to date. We established an experimental system to observe global DNA methylation in living mouse embryonic stem (ES) cells. The methylated DNA binding domain (MBD) and the nuclear localization signal (nls) sequence coding for human methyl CpG-binding domain protein 1 (MBD1) were fused to the enhanced green fluorescent protein (EGFP) reporter gene, and ES cell lines carrying the construct (EGFP-MBD-nls) were established. The EGFP-MBD-nls protein was used to follow DNA methylation in situ under physiological conditions. We also monitored the formation and rearrangement of methylated heterochromatin using EGFP-MBD-nls. Pluripotent mouse ES cells showed unique nuclear organization in that methylated centromeric heterochromatin coalesced to form large clusters around the nucleoli. Upon differentiation, the organization of these heterochromatin clusters changed dramatically. Time-lapse microscopy successfully captured a moment of dramatic change in chromosome positioning during the transition between two differentiation stages. Thus, this experimental system should facilitate studies focusing on relationships between nuclear organization, epigenetic status and cell differentiation.
Assuntos
Diferenciação Celular/fisiologia , Núcleo Celular , Metilação de DNA , Células-Tronco Embrionárias , Epigênese Genética , Animais , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Células Cultivadas , Cromossomos/metabolismo , Cromossomos/ultraestrutura , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Recuperação de Fluorescência Após Fotodegradação , Genes Reporter , Humanos , Camundongos , Camundongos Knockout , Sinais de Localização Nuclear , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
To elucidate the molecular dynamics of HP1beta in mouse preimplantation embryos, we examined the localization, dynamics, and mobility of HP1beta in the (pro)nucleus by live cell imaging. Time-lapse observation revealed that the chromatin association of HP1beta is regulated in a cell cycle-dependent manner. HP1beta was localized in the interphase nucleus and was dynamically dissociated from the nucleus during the metaphase stage. The HP1beta assembly and clustered heterochromatin structure were both found in the nuclei of 2-cell and later-stage embryos. Moreover, fluorescent recovery after photobleaching analysis implied that HP1beta is more freely mobile in the pronucleus of the 1-cell embryo than in the 4-cell nucleus. These results suggest that the chromatin configuration may be regulated by the stability and mobility of chromatin-associated proteins including HP1beta during early embryonic stages.