Jason G Knott

Michigan State University, East Lansing, Michigan, United States

Are you Jason G Knott?

Claim your profile

Publications (28)161.65 Total impact

  • Jason Knott, Soumen Paul
    [Show abstract] [Hide abstract]
    ABSTRACT: Mammalian reproduction is critically dependent on the trophoblast cell lineage, which assures proper establishment of maternal-fetal interactions during pregnancy. Specification of trophoblast cell lineage begins with the development of the trophectoderm (TE) in preimplantation embryos. Subsequently, other trophoblast cell types arise with progression of pregnancy. Studies with transgenic animal models as well as trophoblast stem/progenitor cells have implicated distinct transcriptional and epigenetic regulators in trophoblast lineage development. This review focuses on our current understanding of transcriptional and epigenetic mechanisms regulating specification, determination, maintenance and differentiation of trophoblast cells.
    Reproduction (Cambridge, England) 09/2014; DOI:10.1530/REP-14-0072 · 3.26 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Poor oocyte competence is a major factor limiting efficiency of in vitro embryo production in cattle and women. Despite decades of research, the fundamental questions of what makes an egg good or bad and how to improve egg quality in a clinical setting remain. Previous studies in our laboratory demonstrated the essential requirement of oocyte derived follistatin in early embryonic development with embryotrophic actions of follistatin in promoting early cleavage, increased rates of development to blastocyst stage and increased numbers of trophectoderm cells in bovine blastocyst stage embryos noted. Embryotrophic actions of follistatin on developmental progression of rhesus monkey embryos were also observed. Follistatin is a binding protein for specific members of the transforming growth factor β (TGFβ) superfamily and may exert its embryotrophic effects through modulation of one or more of the SMAD (SMAD2/3, SMAD1/5) or non-SMAD (e.g. AKT, JNK, ERK and P38) signaling pathways regulated by TGFβ superfamily members. The objectives of the present study were to investigate the association of AKT expression with oocyte competence/embryo developmental progression and potential effects of follistatin treatment on AKT phosphorylation in bovine embryos. In experiment 1, bovine oocytes were collected at germinal vesicle (GV) and metaphase II (MII) stages and embryos collected at pronuclear, 2-, 4-, 8- and 16-cell, morula and blastocyst stages and subjected to quantitative RT-PCR analysis of AKT mRNA abundance (n = 4 pools of 10 oocytes/embryos per pool). Results demonstrated increased transcript abundance for AKT in pronuclear through 8-cell embryos relative to GV stage oocytes. AKT transcript abundance was then decreased at 16-cell stage and further decreased in morula and blastocyst stage embryos. In experiment 2, GV stage oocytes were collected following staining with brilliant cresyl blue (BCB; a biochemical indicator of G6PDH activity and oocyte growth linked to egg quality) and differences in AKT and follistatin transcript abundance determined (n = 6 pools of 20 oocytes/group). Abundance of mRNAs for AKT and follistatin was higher in good quality oocytes that stained positive for BCB versus poorer quality oocytes negative for BCB staining. In experiment 3, the effects of exogenous follistatin treatment (0 or 10 ng/ml) of zygote stage embryos on AKT phosphorylation were determined by Western Blot analysis of samples collected at 0, 1, 2, 5 and 10 h after treatment administration (n = 20 embryos per timepoint/experiment; n = 6 replicates). Changes in levels of AKT phosphorylation (phosphorylated AKT/total AKT) in response to follistatin treatment were not detected. Collectively, results demonstrate temporal regulation of AKT mRNA abundance during bovine early embryogenesis and a positive association of AKT transcript abundance with oocyte quality (based on BCB staining) suggestive of a potential functional role in oocyte competence. However, embryotrophic actions of exogenous follistatin do not appear mediated by acute alterations in AKT phosphorylation levels in early bovine embryos. Further studies are required to elucidate the signaling pathways associated with embryotropic actions of follistatin and establish a functional role for maternally derived AKT in oocyte developmental competence and early embryonic development in cattle. (Supported by NIH grant HD072972 to GWS and JK and ARE grant JS2687 to MA)
    Annual meeting of the Society for the Study of Reproduction SSR, Michigan, USA; 07/2014
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Transforming growth factor beta (TGFbeta) superfamily signaling controls various aspects of female fertility. However, the functional roles of TGFbeta-superfamily cognate signal transduction pathway components (e.g. SMAD2/3, SMAD4, SMAD1/5/8) in early embryonic development are not completely understood. We have previously demonstrated pronounced embryotropic actions of the TGFbeta superfamily member-binding protein, follistatin, on oocyte competence in cattle. Given SMAD4 is a common "SMAD" required for both SMAD2/3 and SMAD1/5/8 signaling pathways, the objectives of the present studies were to determine the temporal expression and functional role of SMAD4 in bovine early embryogenesis and whether embryotropic actions of follistatin are SMAD4-dependent. SMAD4 mRNA is increased in bovine oocytes during meiotic maturation, is maximal in 2-cell stage embryos, remains elevated through the 8-cell stage and is decreased and remains low through the blastocyst stage. Ablation of SMAD4 via siRNA microinjection of zygotes reduced proportions of embryos cleaving early and development to the 8- to 16-cell and blastocyst stages. Stimulatory effects of follistatin on early cleavage, but not on development to 8- to 16-cell and blastocyst stages were observed in SMAD4-depleted embryos. Therefore, results suggest SMAD4 is obligatory for early embryonic development in cattle and embryotropic actions of follistatin on development to 8- to 16-cell and blastocyst stages are SMAD4 dependent.
    Biology of Reproduction 07/2014; 91(3). DOI:10.1095/biolreprod.114.120105 · 3.45 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: A growing body of evidence suggests oocyte developmental competence is a limiting factor in efficiency of assisted reproductive technologies and pregnancy success in livestock species and humans, but the inherent phenotypic characteristics of competent oocytes are not well understood. Oocytes gradually and sequentially acquire developmental competence (during the course of folliculogenesis) by synthesizing and accumulating transcripts and proteins critical for successful meiotic maturation, fertilization, and early embryogenesis. We have conducted fundamental studies using the bovine model to elucidate differences in oocyte transcriptome associated with poor oocyte competence and the functional and therapeutic relevance of such results. Of particular interest were studies that showed a positive association of follistatin mRNA abundance with oocyte competence in two distinct bovine models. Follistatin treatment of bovine embryos during initial stages of in vitro culture increases proportion of embryos cleaving early, proportion of embryos developing to the blastocyst stage and numbers of blastocyst trophectoderm cells. Comparative studies in the rhesus monkey model demonstrated stimulatory actions of exogenous follistatin on rates of blastocyst development and support potential clinical relevance of results in the bovine model. Complementary loss of function studies in early embryos established a functional role for follistatin in control of bovine blastocyst development and cell allocation. To increase understanding of intrinsic role of TGFβ superfamily in regulation of early embryogenesis and elucidate mechanism of action of follistatin in mediating its embryotropic effects, studies utilizing a combination of pharmacological and siRNA mediated inhibition of TGFβ superfamily signaling pathway components in the presence or absence of follistatin treatment are being conducted. Results to date suggest a functional requirement for the common SMAD (SMAD4) and SMAD2/3 signaling pathways in promoting bovine early embryonic development and indicate stimulatory effects of follistatin on blastocyst development, but not early cleavage are blocked when SMAD signaling is inhibited. Elucidation of the mechanism of action of follistatin in mediating above described embryotropic actions is critical to further understanding of the functional significance of follistatin to regulation of bovine early embryogenesis and the translational relevance of results to improvements in assisted reproductive technologies. (Supported by NIH grant HD072972 to GWS and JK).
    Annual meeting of the Society for the Study of Reproduction SSR, Michigan, USA; 07/2014
  • [Show abstract] [Hide abstract]
    ABSTRACT: Oocyte-expressed genes regulate key aspects of ovarian follicular development and early embryogenesis. We previously demonstrated a requirement of the oocyte-specific protein JY-1 for bovine embryogenesis. Given JY-1 is present in oocytes throughout folliculogenesis and oocyte-derived JY-1 mRNA is temporally regulated post fertilization, we hypothesized that JY-1 levels in oocytes impact nuclear maturation and subsequent early embryogenesis. A novel model system, whereby JY-1 siRNA was microinjected into cumulus-enclosed germinal vesicle stage oocytes and meiotic arrest maintained for 48 h prior to in vitro maturation, was validated and used to determine the effect of reduced oocyte JY-1 expression on nuclear maturation, cumulus expansion and embryonic development after in vitro fertilization. Depletion of JY-1 protein during in vitro maturation effectively reduced cumulus expansion, percentage of oocytes progressing to metaphase II, proportion of embryos that cleaved early, total cleavage rates and development to 8-16 cell stage and totally blocked development to the blastocyst stage relative to controls. Supplementation with JY-1 protein during oocyte culture rescued effects of JY-1 depletion on meiotic maturation, cumulus expansion and early cleavage, but did not rescue development to 8-16 cell and blastocyst stages. However, effects of JY-1 depletion post fertilization on development to 8- to 16-cell and blastocyst stages were rescued by JY-1 supplementation during embryo culture. In conclusion, these results support an important functional role for oocyte-derived JY-1 protein during meiotic maturation in promoting progression to metaphase II, cumulus expansion and subsequent embryonic development.
    Biology of Reproduction 02/2014; 90(3). DOI:10.1095/biolreprod.113.115071 · 3.45 Impact Factor
  • Soumen Paul, Jason G Knott
    [Show abstract] [Hide abstract]
    ABSTRACT: The first cell-fate decision in mammalian preimplantation embryos is the segregation of the inner cell mass (ICM) and trophectoderm (TE) cell lineages. The ICM develops into the embryo proper, whereas the TE ensures embryo implantation and is the source of the extra-embryonic trophoblast cell lineages, which contribute to the functional components of the placenta. The development of a totipotent zygote into a multi-lineage blastocyst is associated with the generation of distinct transcriptional programs. Several key transcription factors participate in the ICM and TE-specific transcriptional networks, and recent studies indicate that post-translational histone modifications as well as ATP-dependent chromatin remodeling complexes converge with these transcriptional networks to regulate ICM and TE lineage specification. This review will discuss our current understanding and future perspectives related to transcriptional and epigenetic regulatory mechanisms that are implicated in the initial mammalian lineage commitment steps, with a focus on events in mice. Mol. Reprod. Dev. © 2013 Wiley Periodicals, Inc.
    Molecular Reproduction and Development 02/2014; 81(2). DOI:10.1002/mrd.22219 · 2.68 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: In mouse blastocysts CDX2 plays a key role in silencing Oct4 and Nanog expression in the trophectoderm (TE) lineage. However, the underlying transcriptional and chromatin-based changes that are associated with CDX2-mediated repression are poorly understood. To address this a Cdx2-inducible mouse embryonic stem (ES) cell line was utilized as a model system. Induction of Cdx2 expression resulted in a decrease in Oct4/Nanog expression, an increase in TE markers, and differentiation into trophoblast-like stem (TS-like) cells within 48 to 120 hours. Consistent with the downregulation of Oct4 and Nanog transcripts, a time-dependent increase in CDX2 binding and a decrease in RNA polymerase II (RNAPII) and OCT4 binding was observed within 48 hours (P<0.05). To test whether transcriptionally active epigenetic marks were erased during differentiation, histone H3K9/14 acetylation and two of its epigenetic modifiers were evaluated. Accordingly, a significant decrease in histone H3K9/14 acetylation and loss of p300 and HDAC1 binding at the Oct4 and Nanog regulatory elements was observed by 48 hours. Accompanying these changes there was a significant increase in total histone H3 and a loss of chromatin accessibility at both the Oct4 and Nanog regulatory elements (P<0.05), indicative of chromatin remodeling. Lastly, DNA methylation analysis revealed that methylation did not occur at Oct4 and Nanog until 96 to 120 hours after induction of CDX2. In conclusion, our results show that silencing of Oct4 and Nanog is facilitated by sequential changes in transcription factor binding, histone acetylation, chromatin remodeling, and DNA methylation at core regulatory elements.
    Stem cells and development 09/2013; DOI:10.1089/scd.2013.0328 · 4.15 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: BACKGROUND: During implantation the blastocyst trophectoderm attaches to the endometrial epithelium and continues to differentiate into all trophoblast subtypes, which are the major components of a placenta. Aberrant trophoblast proliferation and differentiation are associated with placental diseases. However, due to ethical and practical issues, there is almost no available cell or tissue source to study the molecular mechanism of human trophoblast differentiation, which further becomes a barrier to the study of the pathogenesis of trophoblast-associated diseases of pregnancy. In this study, our goal was to generate a proof-of-concept model for deriving trophoblast lineage cells from induced pluripotency stem (iPS) cells from human fibroblasts. In future studies the generation of trophoblast lineage cells from iPS cells established from patient's placenta will be extremely useful for studying the pathogenesis of individual trophoblast-associated diseases and for drug testing. METHODS AND RESULTS: Combining iPS cell technology with BMP4 induction, we derived trophoblast lineage cells from human iPS cells. The gene expression profile of these trophoblast lineage cells was distinct from fibroblasts and iPS cells. These cells expressed markers of human trophoblasts. Furthermore, when these cells were differentiated they exhibited invasive capacity and placental hormone secretive capacity, suggesting extravillous trophoblasts and syncytiotrophoblasts. CONCLUSION: Trophoblast lineage cells can be successfully derived from human iPS cells, which provide a proof-of-concept tool to recapitulate pathogenesis of patient placental trophoblasts in vitro.
    Biochemical and Biophysical Research Communications 06/2013; DOI:10.1016/j.bbrc.2013.06.016 · 2.28 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In mouse blastocysts segregation of the inner cell mass (ICM) and the trophectoderm (TE) is regulated by the mutually antagonistic effects of the transcription factors Oct4 and Cdx2 expressed in the ICM and TE, respectively. In contrast, in other species such as bovine and human, Oct4 is not restricted to the ICM and continues to be expressed in the Cdx2-positive TE. A recent comparative study of the bovine and mouse Oct4 promoters revealed that additional mechanisms might act in conjunction with Cdx2 to downregulate Oct4 expression in the mouse TE lineage. For instance, the mouse Oct4 distal enhancer contains an AP-2γ (Tcfap2c) binding motif that is absent in the bovine and human Oct4 distal enhancer. Nonetheless, the functional relevance of Tcfap2c in Oct4 repression during mouse preimplantation development was not tested. To elucidate the role of Tcfap2c in Oct4 expression an RNA interference approach was utilized. Depletion of Tcfap2c triggered a decrease in Oct4 expression at the 8-cell and morula stage. Remarkably, at the blastocyst stage depletion of Tcfap2c and/or its family member Tcfap2a had no effect on Oct4 repression. To test whether Tcfap2c interacts with Oct4 to positively regulate Oct4 expression, chromatin immunoprecipitation and in situ co-immunoprecipitation analyses were performed. These experiments revealed Tcfap2c and Oct4 binding were enriched at the Oct4 distal enhancer in embryonic stem (ES) cells, but were rapidly lost during differentiation into trophoblast-like cells when Oct4 became repressed. Moreover, Tcfap2c and Oct4 interactions were detected at the morula stage, but were lost during blastocyst formation. In summary, these data demonstrate that Tcfap2c is not required for Oct4 silencing in mouse blastocysts, but may be necessary for the maintenance of Oct4 expression during the 8 cell-to-morula transition. These findings support the notion Cdx2 is the predominant negative regulator of Oct4 expression during blastocyst formation in mice.
    PLoS ONE 05/2013; 8(5):e65771. DOI:10.1371/journal.pone.0065771 · 3.53 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The trophectoderm epithelium is the first differentiated cell layer to arise during mammalian development. Blastocyst formation requires the proper expression and localization of tight junction, polarity, ion gradient and H(2)O channel proteins in the outer cell membranes. However, the underlying transcriptional mechanisms that control their expression are largely unknown. Here, we report that transcription factor AP-2γ (Tcfap2c) is a core regulator of blastocyst formation in mice. Bioinformatics, chromatin immunoprecipitation and transcriptional analysis revealed that Tcfap2c binds and regulates a diverse group of genes expressed during blastocyst formation. RNA interference experiments demonstrated that Tcfap2c regulates genes important for tight junctions, cell polarity and fluid accumulation. Functional and ultrastructural studies revealed that Tcfap2c is necessary for tight junction assembly and paracellular sealing in trophectoderm epithelium. Aggregation of control eight-cell embryos with Tcfap2c knockdown embryos rescued blastocyst formation via direct contribution to the trophectoderm epithelium. Finally, we found that Tcfap2c promotes cellular proliferation via direct repression of p21 transcription during the morula-to-blastocyst transition. We propose a model in which Tcfap2c acts in a hierarchy to facilitate blastocyst formation through transcriptional regulation of core genes involved in tight junction assembly, fluid accumulation and cellular proliferation.
    Development 11/2012; DOI:10.1242/dev.086645 · 6.27 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Parthenogenetic embryonic stem cells (P-ESCs) offer an alternative source of pluripotent cells, which hold great promise for autologous transplantation and regenerative medicine. P-ESCs have been successfully derived from blastocysts of several mammalian species. However, compared with biparental embryonic stem cells (B-ESCs), P-ESCs are limited in their ability to fully differentiate into all 3 germ layers. For example, it has been observed that there is a differentiation bias toward ectoderm derivatives at the expense of endoderm and mesoderm derivatives-muscle in particular-in chimeric embryos, teratomas, and embryoid bodies. In the present study we found that H19 expression was highly upregulated in P-ESCs with more than 6-fold overexpression compared with B-ESCs. Thus, we hypothesized that manipulation of the H19 gene in P-ESCs would alleviate their limitations and allow them to function like B-ESCs. To test this hypothesis we employed a small hairpin RNA approach to reduce the amount of H19 transcripts in mouse P-ESCs. We found that downregulation of H19 led to an increase of mesoderm-derived muscle and endoderm in P-ESCs teratomas similar to that observed in B-ESCs teratomas. This phenomenon coincided with upregulation of mesoderm-specific genes such as Myf5, Myf6, and MyoD. Moreover, H19 downregulated P-ESCs differentiated into a higher percentage of beating cardiomyocytes compared with control P-ESCs. Collectively, these results suggest that P-ESCs are amenable to molecular modifications that bring them functionally closer to true ESCs.
    Stem cells and development 07/2011; 21(7):1134-44. DOI:10.1089/scd.2011.0152 · 4.15 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: For more than thirty years, the dog has been used as a model for human diseases. Despite efforts made to develop canine embryonic stem cells, success has been elusive. Here, we report the generation of canine induced pluripotent stem cells (ciPSCs) from canine adult fibroblasts, which we accomplished by introducing human OCT4, SOX2, c-MYC, and KLF4. The ciPSCs expressed critical pluripotency markers and showed evidence of silencing the viral vectors and normal karyotypes. Microsatellite analysis indicated that the ciPSCs showed the same profile as the donor fibroblasts but differed from cells taken from other dogs. Under culture conditions favoring differentiation, the ciPSCs could form cell derivatives from the ectoderm, mesoderm, and endoderm. Further, the ciPSCs required leukemia inhibitory factor and basic fibroblast growth factor to survive, proliferate, and maintain pluripotency. Our results demonstrate an efficient method for deriving canine pluripotent stem cells, providing a powerful platform for the development of new models for regenerative medicine, as well as for the study of the onset, progression, and treatment of human and canine genetic diseases.
    Stem cells and development 06/2011; 20(10):1669-78. DOI:10.1089/scd.2011.0127 · 4.15 Impact Factor
  • Source
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: During blastocyst formation the segregation of the inner cell mass (ICM) and trophectoderm is governed by the mutually antagonistic effects of the transcription factors Oct4 and Cdx2. Evidence indicates that suppression of Oct4 expression in the trophectoderm is mediated by Cdx2. Nonetheless, the underlying epigenetic modifiers required for Cdx2-dependent repression of Oct4 are largely unknown. Here we show that the chromatin remodeling protein Brg1 is required for Cdx2-mediated repression of Oct4 expression in mouse blastocysts. By employing a combination of RNA interference (RNAi) and gene expression analysis we found that both Brg1 Knockdown (KD) and Cdx2 KD blastocysts exhibit widespread expression of Oct4 in the trophectoderm. Interestingly, in Brg1 KD blastocysts and Cdx2 KD blastocysts, the expression of Cdx2 and Brg1 is unchanged, respectively. To address whether Brg1 cooperates with Cdx2 to repress Oct4 transcription in the developing trophectoderm, we utilized preimplantation embryos, trophoblast stem (TS) cells and Cdx2-inducible embryonic stem (ES) cells as model systems. We found that: (1) combined knockdown (KD) of Brg1 and Cdx2 levels in blastocysts resulted in increased levels of Oct4 transcripts compared to KD of Brg1 or Cdx2 alone, (2) endogenous Brg1 co-immunoprecipitated with Cdx2 in TS cell extracts, (3) in blastocysts Brg1 and Cdx2 co-localize in trophectoderm nuclei and (4) in Cdx2-induced ES cells Brg1 and Cdx2 are recruited to the Oct4 promoter. Lastly, to determine how Brg1 may induce epigenetic silencing of the Oct4 gene, we evaluated CpG methylation at the Oct4 promoter in the trophectoderm of Brg1 KD blastocysts. This analysis revealed that Brg1-dependent repression of Oct4 expression is independent of DNA methylation at the blastocyst stage. In toto, these results demonstrate that Brg1 cooperates with Cdx2 to repress Oct4 expression in the developing trophectoderm to ensure normal development.
    PLoS ONE 05/2010; 5(5):e10622. DOI:10.1371/journal.pone.0010622 · 3.53 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Oct4 and Nanog are crucial for maintaining pluripotency in embryonic stem (ES) cells and early-stage embryos. In the present study, the status of DNA methylation and of histone modifications in the regulatory regions of Oct4 and Nanog in rhesus nuclear transfer-derived ES (ntES) cells was compared with in vitro fertilized embryo-derived ES (IVFES) cell counterparts. Dynamic changes in DNA methylation during differentiation into neural lineage were also monitored and correlated with mRNA abundance and protein levels of both genes. In ntES cells Oct4 exhibited mono-allelic methylation along with relatively lower mRNA levels, and its transcription was seen predominantly from the unmethylated allele. In contrast, in IVFES cells Oct4 was hypomethylated on both alleles and had relatively higher transcript levels, suggesting incomplete reprogramming of DNA methylation on the Oct4 gene following somatic cell nuclear transfer. During neuronal differentiation, Oct4 underwent biallelic methylation and reduced amounts of Oct4 mRNA were detected in both types of ES cells. Analysis of Nanog regulatory regions revealed that both alleles were hypomethylated and similar levels of Nanog transcripts were expressed in ntES cells and IVFES cells. During neuronal differentiation both alleles were methylated and reduced amounts of Nanog mRNA were detected. Other epigenetic modifications including histone 3 lysine 4, 9, and 27 trimethylation (H3K4me3, H3K9me3, and H3K27me3) showed similar patterns around the regulatory regions of Oct4 and Nanog in both kinds of ES cells. During neural differentiation, dramatic enrichment of H3K27me3 and H3K9me3 (repressive marks) was observed on Oct4 and Nanog regulatory regions. Differentiation of ntES and IVFES cells correlated with the silencing of Oct4 and Nanog, reactivation of the neural marker genes Pax6, N-Oct3, and Olig2, and dynamic changes in histone modifications in the upstream regions of Pax6 and N-Oct3. In short, although ES cells derived from somatic cell nuclear transfer showed a different epigenetic status in the Oct4 regulatory region than the IVF-derived counterparts, based on the parameters tested, the neural differentiation potential of ntES and IVFES cells is equivalent.
    Cloning and Stem Cells 12/2009; 11(4):483-96. DOI:10.1089/clo.2009.0019 · 2.66 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Oct4 and Nanog are crucial for maintaining pluripotency in embryonic stem (ES) cells and early-stage embryos. In the present study, the status of DNA methylation and of histone modifications in the regulatory regions of Oct4 and Nanog in rhesus nuclear transfer-derived ES (ntES) cells was compared with in vitro fertilized embryo-derived ES (IVFES) cell counterparts. Dynamic changes in DNA methylation during differentiation into neural lineage were also monitored and correlated with mRNA abundance and protein levels of both genes. In ntES cells Oct4 exhibited mono-allelic methylation along with relatively lower mRNA levels, and its transcription was seen predominantly from the unmethylated allele. In contrast, in IVFES cells Oct4 was hypomethylated on both alleles and had relatively higher transcript levels, suggesting incomplete reprogramming of DNA methylation on the Oct4 gene following somatic cell nuclear transfer. During neuronal differentiation, Oct4 underwent biallelic methylation and reduced amounts of Oct4 mRNA were detected in both types of ES cells. Analysis of Nanog regulatory regions revealed that both alleles were hypomethylated and similar levels of Nanog tran-scripts were expressed in ntES cells and IVFES cells. During neuronal differentiation both alleles were meth-ylated and reduced amounts of Nanog mRNA were detected. Other epigenetic modifications including histone 3 lysine 4, 9, and 27 trimethylation (H3K4me3, H3K9me3, and H3K27me3) showed similar patterns around the regulatory regions of Oct4 and Nanog in both kinds of ES cells. During neural differentiation, dramatic en-richment of H3K27me3 and H3K9me3 (repressive marks) was observed on Oct4 and Nanog regulatory regions. Differentiation of ntES and IVFES cells correlated with the silencing of Oct4 and Nanog, reactivation of the neural marker genes Pax6, N-Oct3, and Olig2, and dynamic changes in histone modifications in the upstream regions of Pax6 and N-Oct3. In short, although ES cells derived from somatic cell nuclear transfer showed a different epigenetic status in the Oct4 regulatory region than the IVF-derived counterparts, based on the pa-rameters tested, the neural differentiation potential of ntES and IVFES cells is equivalent.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The SWI/SNF-Brg1 chromatin remodeling protein plays critical roles in cell-cycle control and differentiation through regulation of gene expression. Loss of Brg1 in mice results in early embryonic lethality, and recent studies have implicated a role for Brg1 in somatic stem cell self-renewal and differentiation. However, little is known about Brg1 function in preimplantation embryos and embryonic stem (ES) cells. Here we report that Brg1 is required for ES cell self-renewal and pluripotency. RNA interference-mediated knockdown of Brg1 in blastocysts caused aberrant expression of Oct4 and Nanog. In ES cells, knockdown of Brg1 resulted in phenotypic changes indicative of differentiation, downregulation of self-renewal and pluripotency genes (e.g., Oct4, Sox2, Sall4, Rest), and upregulation of differentiation genes. Using genome-wide promoter analysis (chromatin immunoprecipitation) we found that Brg1 occupied the promoters of key pluripotency-related genes, including Oct4, Sox2, Nanog, Sall4, Rest, and Polycomb group (PcG) proteins. Moreover, Brg1 co-occupied a subset of Oct4, Sox2, Nanog, and PcG protein target genes. These results demonstrate an important role for Brg1 in regulating self-renewal and pluripotency in ES cells.
    Stem Cells 02/2009; 27(2):317-28. DOI:10.1634/stemcells.2008-0710 · 7.70 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Inositol 1,4,5-trisphosphate generated by the action of a phospholipase C (PLC) mediates release of intracellular Ca2+ that is essential for sperm-induced activation of mammalian eggs. Much attention currently focuses on the role of sperm-derived PLCzeta in generating changes in egg intracellular Ca2+ despite the fact that PLCzeta constitutes a very small fraction of the total amount of PLC in a fertilized egg. Eggs express several isoforms of PLC, but a role for an egg-derived PLC in sperm-induced Ca2+ oscillations has not been examined. Reducing egg PLCbeta1 by a transgenic RNAi approach resulted in a significant decrease in Ca2+ transient amplitude, but not duration or frequency, following insemination. Furthermore, overexpressing PLCbeta1 by microinjecting a Plcb1 cRNA significantly perturbed the duration and frequency of Ca2+ transients and disrupted the characteristic shape of the first transient. These results provide the first evidence for a role of an egg-derived PLC acting in conjunction with a sperm-derived PLCzeta in egg activation.
    Developmental Biology 01/2008; 312(1):321-30. DOI:10.1016/j.ydbio.2007.09.028 · 3.64 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Fertilization triggers initiation of development and establishment of blocks on the egg coat and plasma membrane to prevent fertilization by multiple sperm (polyspermy). The mechanism(s) by which mammalian eggs establish the membrane block to polyspermy is largely unknown. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) appears to be the key regulator of several egg activation events (completion of meiosis, progression to embryonic interphase, recruitment of maternal mRNAs). Since sperm-induced increases in cytosolic Ca(2+) play a role in establishment of the membrane block to polyspermy in mouse eggs, we hypothesized that CaMKII was a Ca(2+)-dependent effector leading to this change in egg membrane function. To test this hypothesis, we modulated CaMKII activity in two ways: activating eggs parthenogenetically by introducing constitutively active CaMKIIalpha (CA-CaMKII) into unfertilized eggs, and inhibiting endogenous CaMKII in fertilized eggs with myristoylated autocamtide 2-related inhibitory peptide (myrAIP). We find that eggs treated with myrAIP establish a less effective membrane block to polyspermy than do control eggs, but that CA-CaMKII is not sufficient for membrane block establishment, despite the fact that CA-CaMKII-activated eggs undergo other egg activation events. This suggests that: (1) CaMKII activity contributes to the membrane block, but this not faithfully mimicked by CA-CaMKII and furthermore, other pathways, in addition to those activated by Ca(2+) and CaMKII, also participate in membrane block establishment; (2) CA-CaMKII has a range of effects as a parthenogenetic trigger of egg activation (high levels of cell cycle resumption, modest levels of cortical granule exocytosis, and no membrane block establishment).
    Journal of Cellular Physiology 08/2007; 212(2):275-80. DOI:10.1002/jcp.21046 · 3.87 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Fertilization in mammalian eggs is accompanied by oscillatory changes in intracellular Ca(2+) concentration, which are critical for initiating and completing egg activation events and the developmental program. Ca(2+)/Camodulin-dependent protein kinase II (CaMKII) is a multifunctional enzyme that is postulated to be the downstream transducer of the Ca(2+) signal in many cell types. We tested the hypothesis that CaMKII is the major integrator of Ca(2+)-induced egg activation events and embryo development by microinjecting a cRNA that encodes a constitutively active (Ca(2+)-independent) mutant form of CaMKII (CA-CaMKII) into mouse eggs. Expression of this cRNA, which does not increase intracellular Ca(2+), induced a sustained rise in CaMKII activity and triggered egg activation events, including cell cycle resumption, and degradation and recruitment of maternal mRNAs; cortical granule exocytosis, however, did not occur normally. Furthermore, when mouse eggs were injected with sperm devoid of Ca(2+)-releasing activity and activated with either CA-CaMKII cRNA or by SrCl(2), similar rates and incidence of development to the blastocyst stage were observed. These results strongly suggest that CaMKII is a major integrator of the Ca(2+) changes that occur following fertilization.
    Developmental Biology 09/2006; 296(2):388-95. DOI:10.1016/j.ydbio.2006.06.004 · 3.64 Impact Factor

Publication Stats

837 Citations
161.65 Total Impact Points

Institutions

  • 2009–2014
    • Michigan State University
      • Department of Animal Science
      East Lansing, Michigan, United States
    • EMD Serono
      Rockland, Massachusetts, United States
  • 2005–2008
    • University of Pennsylvania
      • • Center for Research on Reproduction and Women's Health
      • • Department of Biology
      Philadelphia, Pennsylvania, United States
  • 2002–2003
    • University of Massachusetts Amherst
      • Department of Veterinary and Animal Sciences
      Amherst Center, Massachusetts, United States