Recent years advances in high-throughput sequencing have improved our understanding of how transcripts regulate early vertebrate development. Here, we review the transcriptome dynamics and diversity ...during early stages of zebrafish embryogenesis. Transcriptome dynamics is characterized by different patterns of mRNA degradation, activation of dormant transcripts and onset of transcription. Several studies have shown a striking diversity of both coding and non-coding transcripts. However, in the aftermath of this immense increase in data, functional studies of both protein-coding and non-coding transcripts are lagging behind. We anticipate that the forthcoming years will see studies relying on different high-throughput sequencing technologies and genomic tools developed for zebrafish embryos to further pin down yet un-annotated transcript-function relationships.
Procedures to improve nuclear transplantation efficiency in the rabbit were evaluated. We report the influence of recipient oocyte age on the different steps of nuclear transplantation. The effect of ...multiple pulses and the influence of manipulation medium and cytochalasin B in the post-fusion/activation medium on activation and development were studied. Recently ovulated oocytes were enucleated at a higher rate (60%) than aged oocytes (3%, p less than 0.005); they also fused at a higher rate (85% vs. 26%, p less than 0.001). Activation was low with freshly ovulated oocytes compared to aged oocytes (3% vs. 37%, respectively; p less than 0.005), but was increased by using multiple pulses (85% vs. 68%, p less than 0.05). Multiple pulses also improved development to blastocysts (48% vs. 5%, p less than 0.001). Incubation of oocytes in a bicarbonate-buffered medium with 10% fetal calf serum for manipulation also enhanced rates of activation (100% vs. 89%, p less than 0.05) and development of oocytes to blastocysts (77% vs. 26%, p less than 0.001). Furthermore, 7.5 micrograms/ml cytochalasin B in the post-fusion/activation medium increased activation rates (78% vs. 50%, p less than 0.05) and development to blastocysts of manipulated embryos (46% vs. 11%, p less than 0.001). When the above modifications were applied, 10% (23/230) of the total nuclear transplant embryos (8-16-cell-stage donor nuclei) or 21% (23/110) of those transferred to recipients developed to offspring, rates similar to the development of nonmanipulated control embryos (10%, 4/41, p greater than 0.1).
We evaluated the influence of the stage of the cell cycle of the donor nucleus on development in vitro of nuclear transplant rabbit embryos. The developmental potential of nuclei in early, mid-, and ...late stages of the cell cycle was determined. Duration of the G1 phase in early embryos was determined, and a procedure for reversibly synchronizing donor embryos in the G1 phase was developed. In addition, the extent of development in vitro of nuclear transplant embryos with donor nuclei synchronized in the G1 phase was evaluated. Development to blastocysts was greatly affected by the stage of the cycle of the donor nucleus. Use of early-stage nuclei led to 59% nuclear transplant blastocysts, whereas 32% and 3% were obtained with mid- and late-stage nuclear donors, respectively (p 0.001). The short duration of the G1 phase in 16- and 32-cell-stage embryos (approximately 30 min) necessitated a procedure for synchronizing blastomeres in the G1 phase. This entailed, first, a 10-h incubation in 0.5 micrograms/ml colcemid to arrest embryos in metaphase. After release from colcemid, embryos were allowed to cleave in 0.1 microgram/ml of the DNA synthesis inhibitor, aphidicolin, and remained blocked at the G1/S transition. This treatment was reversible, as assessed by the resumption of DNA synthesis, cleavage rate, and development to blastocysts of treated embryos. The beneficial effect of using early-stage donor blastomeres was confirmed by the enhanced rate of development of manipulated embryos to blastocysts with donor nuclei in the G1 phase (71%), as opposed to the late S phase (15%, p 0.001). It is suggested that progression of the nuclear donor in the cell cycle generates chromosome and other cellular defects in nuclear transplant embryos, which are responsible for impaired development
We report the differentiation of human adipose tissue stem cells (ATSCs) to take on cardiomyocyte properties following transient exposure to a rat cardiomyocyte extract. Reversibly permeabilized ...ATSCs were incubated for 1
h in a nuclear and cytoplasmic extract of rat cardiomyocytes, resealed with CaCl
2, and cultured. Three weeks after exposure to extract, ATSCs expressed several cardiomyocyte markers including sarcomeric α-actinin, desmin, and cardiac troponin I, and displayed targeted expression of the gap junction protein connexin 43. Formation of binucleated and striated cells, and spontaneous beating in culture were also observed. A low proportion of intact ATSCs exposed to the extract also showed signs of α-actinin and connexin 43 expression. Additional evidence of differentiation was provided by induction of expression of nuclear lamin A/C, a marker of terminally differentiated cells, and a remarkable increase in cell cycle length. Together with our previous data Nat. Biotechnol 20 (2002) 460, this study suggests that alteration of cell fate using cellular extracts may be applied to multiple cell types. Cell extracts may also prove useful for investigating the molecular mechanisms of stem cell differentiation.
After fertilization, the dormant sperm nucleus undergoes morphological and biochemical transformations leading to the development of a functional nucleus, the male pronucleus. We have investigated ...the formation of the male pronucleus in a cell-free system consisting of permeabilized sea urchin sperm nuclei incubated in fertilized sea urchin egg extract containing membrane vesicles. The first sperm nuclear alteration in vitro is the disassembly of the sperm nuclear lamina as a result of lamin phosphorylation mediated by egg protein kinase C. The conical sperm nucleus decondenses into a spherical pronucleus in an ATP-dependent manner. The new nuclear envelope (NE) forms by ATP-dependent binding of vesicles to chromatin and GTP-dependent fusion of vesicles to each other. Three cytoplasmic membrane vesicle fractions with distinct biochemical, chromatin-binding and fusion properties, are required for pronuclear envelope assembly. Binding of each fraction to chromatin requires two detergent-resistant lipophilic structures at each pole of the sperm nucleus, which are incorporated into the NE by membrane fusion. Targeting of the bulk of NE vesicles to chromatin is mediated by a lamin B receptor (LBR)-like integral membrane protein. The last step of male pronuclear formation involves nuclear swelling. Nuclear swelling is associated with import of soluble lamin B into the nucleus and growth of the nuclear envelope by fusion of additional vesicles. In the nucleus, lamin B associates with LBR, which apparently tethers the NE to the lamina. Thus male pronuclear envelope assembly in vitro involves a highly ordered series of events. These events are similar to those characterizing the remodeling of somatic and embryonic nuclei transplanted into oocytes. The relationship between sperm nuclear remodeling at fertilization and nuclear remodeling after nuclear transplantation is discussed.
Molecular markers of the zebrafish inner nuclear membrane (NEP55) and nuclear lamina (L68) were identified, partially characterized and used to demonstrate that disassembly of the zebrafish nuclear ...envelope requires sequential phosphorylation events by first PKC, then Cdc2 kinase. NEP55 and L68 are immunologically and functionally related to human LAP2beta and lamin B, respectively. Exposure of zebrafish nuclei to meiotic cytosol elicits rapid phosphorylation of NEP55 and L68, and disassembly of both proteins. L68 phosphorylation is completely inhibited by simultaneous inhibition of Cdc2 and PKC and only partially blocked by inhibition of either kinase. NEP55 phosphorylation is completely prevented by inhibition or immunodepletion of cytosolic Cdc2. Inhibition of cAMP-dependent kinase, MEK or CaM kinase II does not affect NEP55 or L68 phosphorylation. In vitro, nuclear envelope disassembly requires phosphorylation of NEP55 and L68 by both mammalian PKC and Cdc2. Inhibition of either kinase is sufficient to abolish NE disassembly. Furthermore, novel two-step phosphorylation assays in cytosol and in vitro indicate that PKC-mediated phosphorylation of L68 prior to Cdc2-mediated phosphorylation of L68 and NEP55 is essential to elicit nuclear envelope breakdown. Phosphorylation elicited by Cdc2 prior to PKC prevents nuclear envelope disassembly even though NEP55 is phosphorylated. The results indicate that sequential phosphorylation events elicited by PKC, followed by Cdc2, are required for zebrafish nuclear disassembly. They also argue that phosphorylation of inner nuclear membrane integral proteins is not sufficient to promote nuclear envelope breakdown, and suggest a multiple-level regulation of disassembly of nuclear envelope components during meiosis and at mitosis.
Nuclear reprogramming in cell–free extracts Collas, Philippe
Philosophical transactions of the Royal Society of London. Series B. Biological sciences,
08/2003, Letnik:
358, Številka:
1436
Journal Article
Recenzirano
Odprti dostop
Methods for directly turning a somatic cell type into another type (a process referred to as transdifferentiation) would be beneficial for producing replacement cells for therapeutic applications. ...Adult stem cells have been shown to display a broader differentiation potential than anticipated and may contribute to tissues other than those in which they reside. In addition, novel transdifferentiation strategies are being developed. I report recent results on the functional reprogramming of a somatic cell using a nuclear and cytoplasmic extract derived from another somatic cell type. The reprogramming of 293T fibroblasts in an extract from T cells is evidenced by nuclear uptake and the assembly of transcription factors, induction of activity of a chromatin remodelling complex, changes in chromatin composition and activation of lymphoid cell-specific genes. The reprogrammed cells express T-cell-specific surface molecules and a complex regulatory function. Reprogramming cells in cell-free extracts may create possibilities for producing replacement cells for therapeutic applications. The system may also constitute a powerful tool to examine the mechanisms of nuclear reprogramming, at least as they occur in vitro.
The 22
nd
International Conference on Aquatic Invasive Species (ICAIS) was held as a hybrid event in Oostende, Belgium from 18–22 April 2022. The conference addressed the theme of “Global Climate ...Change Amplifies Aquatic Invasive Species Impacts” and aimed to expand knowledge on the latest science and policy, inspire cooperation and collaboration on research and management projects at a global scale. Seven renowned international scientists provided keynote presentations on perspectives of climate change within their respective areas of expertise. This special issue of Aquatic Invasions presents nine academic papers addressing a range of aquatic invasive species issues including predation, life history dynamics, ecosystem impacts, and physiological tolerances. The papers highlight the need for regional, national, and international cooperation, collaboration on research and management projects, and targeted, specific, and actionable outreach to combat the growing threat posed by aquatic invasive species.
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