Germ Cell Nuclear Factor Regulates Gametogenesis in Developing Gonads
2014; 9 (8)
Rapid and Efficient Conversion of Integration-Free Human Induced Pluripotent Stem Cells to GMP-Grade Culture Conditions
2014; 9 (4)
Quantifying Genome-Editing Outcomes at Endogenous Loci with SMRT Sequencing
2014; 7 (1): 293-305
Efficient Endoderm Induction from Human Pluripotent Stem Cells by Logically Directing Signals Controlling Lineage Bifurcations
CELL STEM CELL
2014; 14 (2): 237-252
Targeted genome editing with engineered nucleases has transformed the ability to introduce precise sequence modifications at almost any site within the genome. A major obstacle to probing the efficiency and consequences of genome editing is that no existing method enables the frequency of different editing events to be simultaneously measured across a cell population at any endogenous genomic locus. We have developed a method for quantifying individual genome-editing outcomes at any site of interest with single-molecule real-time (SMRT) DNA sequencing. We show that this approach can be applied at various loci using multiple engineered nuclease platforms, including transcription-activator-like effector nucleases (TALENs), RNA-guided endonucleases (CRISPR/Cas9), and zinc finger nucleases (ZFNs), and in different cell lines to identify conditions and strategies in which the desired engineering outcome has occurred. This approach offers a technique for studying double-strand break repair, facilitates the evaluation of gene-editing technologies, and permits sensitive quantification of editing outcomes in almost every experimental system used.
View details for DOI 10.1016/j.celrep.2014.02.040
View details for Web of Science ID 000334298200028
View details for PubMedID 24685129
Germ cell nuclear factor regulates gametogenesis in developing gonads.
2014; 9 (8)
Human pluripotent stem cell (hPSC) differentiation typically yields heterogeneous populations. Knowledge of signals controlling embryonic lineage bifurcations could efficiently yield desired cell types through exclusion of alternate fates. Therefore, we revisited signals driving induction and anterior-posterior patterning of definitive endoderm to generate a coherent roadmap for endoderm differentiation. With striking temporal dynamics, BMP and Wnt initially specified anterior primitive streak (progenitor to endoderm), yet, 24 hr later, suppressed endoderm and induced mesoderm. At lineage bifurcations, cross-repressive signals separated mutually exclusive fates; TGF-β and BMP/MAPK respectively induced pancreas versus liver from endoderm by suppressing the alternate lineage. We systematically blockaded alternate fates throughout multiple consecutive bifurcations, thereby efficiently differentiating multiple hPSC lines exclusively into endoderm and its derivatives. Comprehensive transcriptional and chromatin mapping of highly pure endodermal populations revealed that endodermal enhancers existed in a surprising diversity of "pre-enhancer" states before activation, reflecting the establishment of a permissive chromatin landscape as a prelude to differentiation.
View details for DOI 10.1016/j.stem.2013.12.007
View details for Web of Science ID 000330835800015
View details for PubMedID 24412311
Characterization of the human ESC transcriptome by hybrid sequencing
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2013; 110 (50): E4821-E4830
Expression of germ cell nuclear factor (GCNF; Nr6a1), an orphan member of the nuclear receptor gene family of transcription factors, during gastrulation and neurulation is critical for normal embryogenesis in mice. Gcnf represses the expression of the POU-domain transcription factor Oct4 (Pou5f1) during mouse post-implantation development. Although Gcnf expression is not critical for the embryonic segregation of the germ cell lineage, we found that sexually dimorphic expression of Gcnf in germ cells correlates with the expression of pluripotency-associated genes, such as Oct4, Sox2, and Nanog, as well as the early meiotic marker gene Stra8. To elucidate the role of Gcnf during mouse germ cell differentiation, we generated an ex vivo Gcnf-knockdown model in combination with a regulated CreLox mutation of Gcnf. Lack of Gcnf impairs normal spermatogenesis and oogenesis in vivo, as well as the derivation of germ cells from embryonic stem cells (ESCs) in vitro. Inactivation of the Gcnf gene in vivo leads to loss of repression of Oct4 expression in both male and female gonads.
View details for DOI 10.1371/journal.pone.0103985
View details for PubMedID 25140725
SHANK3 and IGF1 restore synaptic deficits in neurons from 22q13 deletion syndrome patients.
2013; 503 (7475): 267-271
Although transcriptional and posttranscriptional events are detected in RNA-Seq data from second-generation sequencing, full-length mRNA isoforms are not captured. On the other hand, third-generation sequencing, which yields much longer reads, has current limitations of lower raw accuracy and throughput. Here, we combine second-generation sequencing and third-generation sequencing with a custom-designed method for isoform identification and quantification to generate a high-confidence isoform dataset for human embryonic stem cells (hESCs). We report 8,084 RefSeq-annotated isoforms detected as full-length and an additional 5,459 isoforms predicted through statistical inference. Over one-third of these are novel isoforms, including 273 RNAs from gene loci that have not previously been identified. Further characterization of the novel loci indicates that a subset is expressed in pluripotent cells but not in diverse fetal and adult tissues; moreover, their reduced expression perturbs the network of pluripotency-associated genes. Results suggest that gene identification, even in well-characterized human cell lines and tissues, is likely far from complete.
View details for DOI 10.1073/pnas.1320101110
View details for Web of Science ID 000328061700004
View details for PubMedID 24282307
SHANK3 and IGF1 restore synaptic deficits in neurons from 22q13 deletion syndrome patients
2013; 503 (7475): 267-?
Generation and characterization of transgene-free human induced pluripotent stem cells and conversion to putative clinical-grade status
STEM CELL RESEARCH & THERAPY
Human amniotic mesenchymal stem cell-derived induced pluripotent stem cells may generate a universal source of cardiac cells.
Stem cells and development
2012; 21 (15): 2798-2808
Phelan-McDermid syndrome (PMDS) is a complex neurodevelopmental disorder characterized by global developmental delay, severely impaired speech, intellectual disability, and an increased risk of autism spectrum disorders (ASDs). PMDS is caused by heterozygous deletions of chromosome 22q13.3. Among the genes in the deleted region is SHANK3, which encodes a protein in the postsynaptic density (PSD). Rare mutations in SHANK3 have been associated with idiopathic ASDs, non-syndromic intellectual disability, and schizophrenia. Although SHANK3 is considered to be the most likely candidate gene for the neurological abnormalities in PMDS patients, the cellular and molecular phenotypes associated with this syndrome in human neurons are unknown. We generated induced pluripotent stem (iPS) cells from individuals with PMDS and autism and used them to produce functional neurons. We show that PMDS neurons have reduced SHANK3 expression and major defects in excitatory, but not inhibitory, synaptic transmission. Excitatory synaptic transmission in PMDS neurons can be corrected by restoring SHANK3 expression or by treating neurons with insulin-like growth factor 1 (IGF1). IGF1 treatment promotes formation of mature excitatory synapses that lack SHANK3 but contain PSD95 and N-methyl-D-aspartate (NMDA) receptors with fast deactivation kinetics. Our findings provide direct evidence for a disruption in the ratio of cellular excitation and inhibition in PMDS neurons, and point to a molecular pathway that can be recruited to restore it.
View details for DOI 10.1038/nature12618
View details for PubMedID 24132240
Ultrastructural Characterization of Mouse Embryonic Stem Cell-Derived Oocytes and Granulosa Cells
STEM CELLS AND DEVELOPMENT
2011; 20 (12): 2205-2215
Human amniotic mesenchymal stem cells (hAMSCs) demonstrated partially pluripotent characteristics with a strong expression of Oct4 and Nanog genes and immunomodulatory properties characterized by the absence of HLA-DR and the presence of HLA-G and CD59. The hAMSCs were reprogrammed into induced pluripotent stem cells (iPSCs) that generate a promising source of universal cardiac cells. The hAMSC-derived iPSCs (MiPSCs) successfully underwent robust cardiac differentiation to generate cardiomyocytes. This study investigated 3 key properties of the hAMSCs and MiPSCs: (1) the reprogramming efficiency of the partially pluripotent hAMSCs to generate MiPSCs; (2) immunomodulatory properties of the hAMSCs and MiPSCs; and (3) the cardiac differentiation potential of the MiPSCs. The characteristic iPSC colony formation was observed within 10 days after the transduction of the hAMSCs with a single integration polycistronic vector containing 4 Yamanaka factors. Immunohistology and reverse transcription-polymerase chain reaction assays revealed that the MiPSCs expressed stem cell surface markers and pluripotency-specific genes. Furthermore, the hAMSCs and MiPSCs demonstrated immunomodulatory properties enabling successful engraftment in the SVJ mice. Finally, the cardiac differentiation of MiPSCs exhibited robust spontaneous contractility, characteristic calcium transience across the membrane, a high expression of cardiac genes and mature cardiac phenotypes, and a contractile force comparable to cardiomyocytes. Our results demonstrated that the hAMSCs are reprogrammed with a high efficiency into MiPSCs, which possess pluripotent, immunomodulatory, and precardiac properties. The MiPSC-derived cardiac cells express a c-kit cell surface marker, which may be employed to purify the cardiac cell population and enable allogeneic cardiac stem cell therapy.
View details for DOI 10.1089/scd.2011.0435
View details for PubMedID 22530853
In Situ Genetic Correction of the Sickle Cell Anemia Mutation in Human Induced Pluripotent Stem Cells Using Engineered Zinc Finger Nucleases
2011; 29 (11): 1717-1726
Germ cells are a unique population of cells responsible for transmitting genetic information from one generation to the next. Our understanding of the key mechanisms underlying germ cell development in vivo remains scarce because of insufficient amounts of cell materials available for conducting biological studies. The establishment of in vitro differentiation models that support the generation of germ cells from mouse pluripotent stem cells provides an alternative means for studying reproductive development. The detection and analysis of stem cell-derived germ cells, however, present technical challenges. Methods for determining the developmental stage of germ cells ex vivo, such as gene expression and/or immunochemical analyses are inadequate, frequently necessitating the use of alternative, elaborate methods to prove germ cell identity. We have generated putative oocytes and granulosa cells in vitro from mouse embryonic stem cells and utilized electron microscopy to characterize these cells. Here, we report on the striking ultrastructural similarity of in vitro-generated oocytes and granulosa cells to in vivo oocytes developing within follicles.
View details for DOI 10.1089/scd.2010.0575
View details for Web of Science ID 000297740200017
View details for PubMedID 21244227
Induction of human neuronal cells by defined transcription factors
2011; 476 (7359): 220-U122
The combination of induced pluripotent stem cell (iPSC) technology and targeted gene modification by homologous recombination (HR) represents a promising new approach to generate genetically corrected, patient-derived cells that could be used for autologous transplantation therapies. This strategy has several potential advantages over conventional gene therapy including eliminating the need for immunosuppression, avoiding the risk of insertional mutagenesis by therapeutic vectors, and maintaining expression of the corrected gene by endogenous control elements rather than a constitutive promoter. However, gene targeting in human pluripotent cells has remained challenging and inefficient. Recently, engineered zinc finger nucleases (ZFNs) have been shown to substantially increase HR frequencies in human iPSCs, raising the prospect of using this technology to correct disease causing mutations. Here, we describe the generation of iPSC lines from sickle cell anemia patients and in situ correction of the disease causing mutation using three ZFN pairs made by the publicly available oligomerized pool engineering method (OPEN). Gene-corrected cells retained full pluripotency and a normal karyotype following removal of reprogramming factor and drug-resistance genes. By testing various conditions, we also demonstrated that HR events in human iPSCs can occur as far as 82 bps from a ZFN-induced break. Our approach delineates a roadmap for using ZFNs made by an open-source method to achieve efficient, transgene-free correction of monogenic disease mutations in patient-derived iPSCs. Our results provide an important proof of principle that ZFNs can be used to produce gene-corrected human iPSCs that could be used for therapeutic applications.
View details for DOI 10.1002/stem.718
View details for Web of Science ID 000296565500009
View details for PubMedID 21898685
Telomere shortening and loss of self-renewal in dyskeratosis congenita induced pluripotent stem cells
2011; 474 (7351): 399-?
Somatic cell nuclear transfer, cell fusion, or expression of lineage-specific factors have been shown to induce cell-fate changes in diverse somatic cell types. We recently observed that forced expression of a combination of three transcription factors, Brn2 (also known as Pou3f2), Ascl1 and Myt1l, can efficiently convert mouse fibroblasts into functional induced neuronal (iN) cells. Here we show that the same three factors can generate functional neurons from human pluripotent stem cells as early as 6?days after transgene activation. When combined with the basic helix-loop-helix transcription factor NeuroD1, these factors could also convert fetal and postnatal human fibroblasts into iN cells showing typical neuronal morphologies and expressing multiple neuronal markers, even after downregulation of the exogenous transcription factors. Importantly, the vast majority of human iN cells were able to generate action potentials and many matured to receive synaptic contacts when co-cultured with primary mouse cortical neurons. Our data demonstrate that non-neural human somatic cells, as well as pluripotent stem cells, can be converted directly into neurons by lineage-determining transcription factors. These methods may facilitate robust generation of patient-specific human neurons for in vitro disease modelling or future applications in regenerative medicine.
View details for DOI 10.1038/nature10202
View details for Web of Science ID 000293731900039
View details for PubMedID 21617644
Embryonic Stem Cells, Derived Either after In Vitro Fertilization or Nuclear Transfer, Prolong Survival of Semiallogeneic Heart Transplants
JOURNAL OF IMMUNOLOGY
2011; 186 (7): 4164-4174
The differentiation of patient-derived induced pluripotent stem cells (iPSCs) to committed fates such as neurons, muscle and liver is a powerful approach for understanding key parameters of human development and disease. Whether undifferentiated iPSCs themselves can be used to probe disease mechanisms is uncertain. Dyskeratosis congenita is characterized by defective maintenance of blood, pulmonary tissue and epidermal tissues and is caused by mutations in genes controlling telomere homeostasis. Short telomeres, a hallmark of dyskeratosis congenita, impair tissue stem cell function in mouse models, indicating that a tissue stem cell defect may underlie the pathophysiology of dyskeratosis congenita. Here we show that even in the undifferentiated state, iPSCs from dyskeratosis congenita patients harbour the precise biochemical defects characteristic of each form of the disease and that the magnitude of the telomere maintenance defect in iPSCs correlates with clinical severity. In iPSCs from patients with heterozygous mutations in TERT, the telomerase reverse transcriptase, a 50% reduction in telomerase levels blunts the natural telomere elongation that accompanies reprogramming. In contrast, mutation of dyskerin (DKC1) in X-linked dyskeratosis congenita severely impairs telomerase activity by blocking telomerase assembly and disrupts telomere elongation during reprogramming. In iPSCs from a form of dyskeratosis congenita caused by mutations in TCAB1 (also known as WRAP53), telomerase catalytic activity is unperturbed, yet the ability of telomerase to lengthen telomeres is abrogated, because telomerase mislocalizes from Cajal bodies to nucleoli within the iPSCs. Extended culture of DKC1-mutant iPSCs leads to progressive telomere shortening and eventual loss of self-renewal, indicating that a similar process occurs in tissue stem cells in dyskeratosis congenita patients. These findings in iPSCs from dyskeratosis congenita patients reveal that undifferentiated iPSCs accurately recapitulate features of a human stem cell disease and may serve as a cell-culture-based system for the development of targeted therapeutics.
View details for DOI 10.1038/nature10084
View details for Web of Science ID 000291647100050
View details for PubMedID 21602826
Oct1 regulates trophoblast development during early mouse embryogenesis
2010; 137 (21): 3551-3560
Tolerance induction toward allogeneic organ grafts represents one of the major aims of transplantation medicine. Stem cells are promising candidates for promoting donor-specific tolerance. In this study, we investigated the immunomodulatory properties of murine embryonic stem cells (ESCs), obtained either by in vitro fertilization (IVF-ESCs) or by nuclear transfer (NT-ESCs), in heart transplant mouse models. IVF-ESCs did not prolong the survival of fully allogeneic cardiac transplants but significantly prolonged the survival of semiallogeneic hearts from the same ESC donor strain for >100 d in 44% of the animals. However, 28% of transplanted animals infused with IVF-ESCs experienced development of a teratoma. NT-ESCs similarly prolonged semiallogeneic heart graft survival (>100 d in 40% of the animals) but were less teratogenic. By in vitro studies, IVF-ESC and NT-ESC immunoregulation was mediated both by cell contact-dependent mechanisms and by the release of soluble factors. By adding specific inhibitors, we identified PGE(2) as a soluble mediator of ESC immunoregulation. Expansion of regulatory T cells was found in lymphoid organs and in the grafts of IVF-ESC- and NT-ESC-tolerized mice. Our study demonstrates that both IVF-ESCs and NT-ESCs modulate recipient immune response toward tolerance to solid organ transplantation, and that NT-ESCs exhibit a lower tendency for teratoma formation. Because NT-ESCs are obtained by NT of a somatic cell from living individuals into an enucleated oocyte, they could represent a source of donor-derived stem cells to induce tolerance to solid organ allograft.
View details for DOI 10.4049/jimmunol.1000654
View details for Web of Science ID 000288751200041
View details for PubMedID 21389254
Dynamic link of DNA demethylation, DNA strand breaks and repair in mouse zygotes
2010; 29 (11): 1877-1888
Oct1 (Pou2f1) is a transcription factor of the POU-homeodomain family that is unique in being ubiquitously expressed in both embryonic and adult mouse tissues. Although its expression profile suggests a crucial role in multiple regions of the developing organism, the only essential function demonstrated so far has been the regulation of cellular response to oxidative and metabolic stress. Here, we describe a loss-of-function mouse model for Oct1 that causes early embryonic lethality, with Oct1-null embryos failing to develop beyond the early streak stage. Molecular and morphological analyses of Oct1 mutant embryos revealed a failure in the establishment of a normal maternal-embryonic interface due to reduced extra-embryonic ectoderm formation and lack of the ectoplacental cone. Oct1(-/-) blastocysts display proper segregation of trophectoderm and inner cell mass lineages. However, Oct1 loss is not compatible with trophoblast stem cell derivation. Importantly, the early gastrulation defect caused by Oct1 disruption can be rescued in a tetraploid complementation assay. Oct1 is therefore primarily required for the maintenance and differentiation of the trophoblast stem cell compartment during early post-implantation development. We present evidence that Cdx2, which is expressed at high levels in trophoblast stem cells, is a direct transcriptional target of Oct1. Our data also suggest that Oct1 is required in the embryo proper from late gastrulation stages onwards.
View details for DOI 10.1242/dev.047027
View details for Web of Science ID 000283669300003
View details for PubMedID 20876643
Induction of Pluripotency in Adult Unipotent Germline Stem Cells
CELL STEM CELL
2009; 5 (1): 87-96
In mammalian zygotes, the 5-methyl-cytosine (5mC) content of paternal chromosomes is rapidly changed by a yet unknown but presumably active enzymatic mechanism. Here, we describe the developmental dynamics and parental asymmetries of DNA methylation in relation to the presence of DNA strand breaks, DNA repair markers and a precise timing of zygotic DNA replication. The analysis shows that distinct pre-replicative (active) and replicative (active and passive) phases of DNA demethylation can be observed. These phases of DNA demethylation are concomitant with the appearance of DNA strand breaks and DNA repair markers such as gammaH2A.X and PARP-1, respectively. The same correlations are found in cloned embryos obtained after somatic cell nuclear transfer. Together, the data suggest that (1) DNA-methylation reprogramming is more complex and extended as anticipated earlier and (2) the DNA demethylation, particularly the rapid loss of 5mC in paternal DNA, is likely to be linked to DNA repair mechanisms.
View details for DOI 10.1038/emboj.2010.80
View details for Web of Science ID 000278235100010
View details for PubMedID 20442707
Oct4-Induced Pluripotency in Adult Neural Stem Cells
2009; 136 (3): 411-419
Mouse and human stem cells with features similar to those of embryonic stem cells have been derived from testicular cells. Although pluripotent stem cells have been obtained from defined germline stem cells (GSCs) of mouse neonatal testis, only multipotent stem cells have been obtained so far from defined cells of mouse adult testis. In this study we describe a robust and reproducible protocol for obtaining germline-derived pluripotent stem (gPS) cells from adult unipotent GSCs. Pluripotency of gPS cells was confirmed by in vitro and in vivo differentiation, including germ cell contribution and transmission. As determined by clonal analyses, gPS cells indeed originate from unipotent GSCs. We propose that the conversion process requires a GSC culture microenvironment that depends on the initial number of plated GSCs and the length of culture time.
View details for DOI 10.1016/j.stem.2009.05.025
View details for Web of Science ID 000267879200013
View details for PubMedID 19570517
Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors
2008; 454 (7204): 646-U54
The four transcription factors Oct4, Sox2, Klf4, and c-Myc can induce pluripotency in mouse and human fibroblasts. We previously described direct reprogramming of adult mouse neural stem cells (NSCs) by Oct4 and either Klf4 or c-Myc. NSCs endogenously express Sox2, c-Myc, and Klf4 as well as several intermediate reprogramming markers. Here we report that exogenous expression of the germline-specific transcription factor Oct4 is sufficient to generate pluripotent stem cells from adult mouse NSCs. These one-factor induced pluripotent stem cells (1F iPS) are similar to embryonic stem cells in vitro and in vivo. Not only can these cells can be efficiently differentiated into NSCs, cardiomyocytes, and germ cells in vitro, but they are also capable of teratoma formation and germline transmission in vivo. Our results demonstrate that Oct4 is required and sufficient to directly reprogram NSCs to pluripotency.
View details for DOI 10.1016/j.cell.2009.01.023
View details for Web of Science ID 000263120600012
View details for PubMedID 19203577
Experimental demonstration that mammalian oocytes are not selective towards X- or Y-bearing sperm
MOLECULAR REPRODUCTION AND DEVELOPMENT
2005; 71 (2): 245-246
Reprogramming of somatic cells is a valuable tool to understand the mechanisms of regaining pluripotency and further opens up the possibility of generating patient-specific pluripotent stem cells. Reprogramming of mouse and human somatic cells into pluripotent stem cells, designated as induced pluripotent stem (iPS) cells, has been possible with the expression of the transcription factor quartet Oct4 (also known as Pou5f1), Sox2, c-Myc and Klf4 (refs 1-11). Considering that ectopic expression of c-Myc causes tumorigenicity in offspring and that retroviruses themselves can cause insertional mutagenesis, the generation of iPS cells with a minimal number of factors may hasten the clinical application of this approach. Here we show that adult mouse neural stem cells express higher endogenous levels of Sox2 and c-Myc than embryonic stem cells, and that exogenous Oct4 together with either Klf4 or c-Myc is sufficient to generate iPS cells from neural stem cells. These two-factor iPS cells are similar to embryonic stem cells at the molecular level, contribute to development of the germ line, and form chimaeras. We propose that, in inducing pluripotency, the number of reprogramming factors can be reduced when using somatic cells that endogenously express appropriate levels of complementing factors.
View details for DOI 10.1038/nature07061
View details for Web of Science ID 000258026500049
View details for PubMedID 18594515
Cloned pre-implantation mouse embryos show correct timing but altered levels of gene expression
MOLECULAR REPRODUCTION AND DEVELOPMENT
2005; 70 (2): 146-154
Mammalian oocytes are thought to be neutral as for X- or Y-bearing sperm selection is concerned, and penetration of an oocyte by an X- or a Y-bearing sperm is considered a random event. This assumption is mainly based on a posteriori evidences of a nearly equal sex ratio at birth, but it has never been experimentally demonstrated. We have designed a simple experiment, which allowed the penetration of an oocyte by more than one sperm and the further sexing by PCR of each single pronucleus present within the ooplasm. For the first time, we provide experimental evidence that mammalian oocytes do not play a selecting role since a single oocyte may be simultaneously fertilised by both X- and Y-bearing sperm.
View details for DOI 10.1002/mrd.20252
View details for Web of Science ID 000228966300014
View details for PubMedID 15791593
Three-dimensional localization and dynamics of centromeres in mouse oocytes during folliculogenesis
JOURNAL OF MOLECULAR HISTOLOGY
2004; 35 (6): 631-638
Mammalian embryos obtained by somatic nuclear transfer (NT) struggle to survive throughout development, encountering a number of hurdles leading to wrong functional reprogramming of the donor genome. However, despite these obstacles, some of these embryos continue their development, as if the required transcriptional functions are somehow satisfied. The amount of information gathered on the kinetics and quantitative profile of gene expression in NT pre-implantation embryos is still scarce and limited to a handful of genes described in two species, bovine and mouse. Using a single-cell sensitive semi-quantitative RT-PCR, we have compared the onset and profile of abundance of Hprt, Tsx, Bex1, Bax, Cpt2, and Oct4 genes, in in vitro fertilised and NT-derived mouse 1-cell, 2-cell, 4-cell embryos, morulae, and blastocysts. The genes analysed were activated in NT embryos at approximately the correct time compared to control embryos, indicating that the reprogramming phenomenon is developmentally regulated and that the somatic genome is quickly rearranged towards an embryonic-type of expression during the early stages of segmentation. Despite the right timing of genes onset, the high degree of variability in the number of transcripts found in NT embryos at the latest stages of pre-implantation development, suggests that genome reprogramming is incomplete and inaccurate.
View details for DOI 10.1002/mrd.20144
View details for Web of Science ID 000226154600004
View details for PubMedID 15570622
Single-cell quantitative RT-PCR analysis of Cpt1b and Cpt2 gene expression in mouse antral oocytes and in preimplantation embryos
CYTOGENETIC AND GENOME RESEARCH
2004; 105 (2-4): 215-221
Very little is known about oocyte nuclear architecture during folliculogenesis. Using antibodies to reveal centromeres, Hoechst-staining to detect the AT-rich pericentromeric heterochromatin (chromocenters), combined with confocal microscopy for the three-dimensional analysis of the nucleus, we demonstrate that during mouse folliculogenesis the oocyte nuclear architecture undergoes dynamic changes. In oocytes isolated from primordial and primary follicles, centromeres and chromocenters were preferentially located at the periphery of the nucleus. During oocyte growth, centromeres and chromocenters were initially found spread within the nucleus and then progressively clustered around the periphery of the nucleolus. Our results indicate that the oocyte nuclear achitecture is developmentally regulated and they contribute to a further understanding of the role of nuclear organization in the regulation of genome functioning during differentiation and development.
View details for Web of Science ID 000225903600011
View details for PubMedID 15614617
Fatty acids represent an important energy source for preimplantation embryos. Fatty acids oxidation is correlated with the embryo oxygen consumption which remains relatively constant up to the 8-cell stage, but suddenly increases between the 8-cell and morula stages. The degradation of fatty acids occurs in mitochondria and is catalyzed by several carnitine acyl transferases, including two carnitine palmitoyl transferases, CPT-I and CPT-II. We have carried out a study to determine the relative number of transcripts of Cpt1b and Cpt2 genes encoding for m-CPT-I and CPT-II enzymes, during mouse preimplantation development. Here we show that Cpt1b transcripts are first and temporally detected at the 2-cell stage and reappear at the morula and blastocyst stage. Cpt2 transcripts decrease following fertilization to undetectable levels and are present again later at the morula stage. These results show that transcription of both Cpt1b and Cpt2 is triggered at the morula stage, concomitantly with known increasing profiles of oxygen uptake and fatty acids oxidation. Based on the number of Cpt2 transcripts detected, we could discriminate the presence of two groups of embryos with high and low number of transcripts, from the zygote throughout preimplantation development. To further investigate if the establishment of these two groups of embryos occurs prior to fertilization, we have analyzed the relative number of transcripts of both genes in antral and ovulated MII oocytes. As for preimplantation embryos, MII oocytes show two groups of Cpt2 expression. Antral oocytes, classified according to their chromatin configuration in SN (surrounded nucleolus, in which the nucleolus is surrounded by a rim of Hoechst-positive chromatin) and NSN (not surrounded nucleolus, in which this rim is absent), show three groups with different numbers of Cpt2 transcripts. All NSN oocytes have a number of Cpt2 transcripts doubled compared to that of the group of MII oocytes with high expression. Instead, SN oocytes could be singled out into two groups with high and low numbers of Cpt2 transcripts, similar to those found in MII oocytes. The results of this study point out a correlation between the timing of fatty acids oxidation during preimplantation development and the expression of two genes encoding two enzymes involved in the oxidative pathway. Furthermore, although the biological meaning for the presence of two groups of oocytes/embryos with different levels of Cpt2 transcripts remains unclear, the data obtained suggest a possible correlation between the levels of Cpt2 expression and embryo developmental competence.
View details for DOI 10.1159/000078191
View details for Web of Science ID 000224158000007
View details for PubMedID 15237209