Agata Kurowski

University of Cambridge, Cambridge, England, United Kingdom

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Publications (15)29.45 Total impact

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    ABSTRACT: The transcription factor Oct4 is required in vitro for establishment and maintenance of embryonic stem cells and for reprogramming somatic cells to pluripotency. In vivo, it prevents the ectopic differentiation of early embryos into trophoblast. Here, we further explore the role of Oct4 in blastocyst formation and specification of epiblast versus primitive endoderm lineages using conditional genetic deletion. Experiments involving mouse embryos deficient for both maternal and zygotic Oct4 suggest that it is dispensable for zygote formation, early cleavage and activation of Nanog expression. Nanog protein is significantly elevated in the presumptive inner cell mass of Oct4 null embryos, suggesting an unexpected role for Oct4 in attenuating the level of Nanog, which might be significant for priming differentiation during epiblast maturation. Induced deletion of Oct4 during the morula to blastocyst transition disrupts the ability of inner cell mass cells to adopt lineage-specific identity and acquire the molecular profile characteristic of either epiblast or primitive endoderm. Sox17, a marker of primitive endoderm, is not detected following prolonged culture of such embryos, but can be rescued by provision of exogenous FGF4. Interestingly, functional primitive endoderm can be rescued in Oct4-deficient embryos in embryonic stem cell complementation assays, but only if the host embryos are at the pre-blastocyst stage. We conclude that cell fate decisions within the inner cell mass are dependent upon Oct4 and that Oct4 is not cell-autonomously required for the differentiation of primitive endoderm derivatives, as long as an appropriate developmental environment is established.
    No preview · Article · Feb 2014 · Development
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    Dataset: Figure S10
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    ABSTRACT: Entry into anagen is delayed in Misu −/− and K14MisuΔ/Δ mice. (A) Percentage of Misu −/− females and their control littermates represented in Table S1. (B–G) Haematoxylin and Eosin (H&E) staining (B,C) and immunohistochemistry for Ki67 (D,E) of dorsal skin sections of Misu −/− and wild-type hair follicles at P19 (B,C), and P27 (D,E). (F–K) Histology of dorsal skin sections at indicated postnatal days shows delay of entry into anagen in K14MisuΔ/Δ mice compared to controls (Misuf/f). Immunohistochemistry for Ki67 of skin sections at P21 (F,G). Haematoxylin and Eosin staining of skin sections at indicated postnatal days (H–K). (L–S) Misu −/− hair follicles are indistinguishable from their wild-type controls at later stages in anagen at P31. Confocal images of wid-type and Misu −/− hair follicles stained for Lef1 (green) (L,M), hair keratin 72 (green) and Dlx3 (red) (N,O), phospho-Smad1/3/5 (green) (P,Q) and hair keratin 31 (green) and Gata3 (red) (R,S). Nuclei are counterstained with Haematoxylin (B–K) and DAPI (L–S). Scale bar: 200 µm (B–E); 100 µm (F–S). (TIF)
    Preview · Dataset · Dec 2011
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    Dataset: Figure S6
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    ABSTRACT: Cell cycle analysis of stem and progenitor cells using flow cytometry. (A–C) Gating of epidermal cells using flow cytometry to analyse the cell cycle profile of bulge stem cells (A), hair germ cells (B) at P21, and bulge stem cells at P24 (C). (TIF)
    Preview · Dataset · Dec 2011
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    ABSTRACT: Homeostasis of most adult tissues is maintained by balancing stem cell self-renewal and differentiation, but whether post-transcriptional mechanisms can regulate this process is unknown. Here, we identify that an RNA methyltransferase (Misu/Nsun2) is required to balance stem cell self-renewal and differentiation in skin. In the epidermis, this methyltransferase is found in a defined sub-population of hair follicle stem cells poised to undergo lineage commitment, and its depletion results in enhanced quiescence and aberrant stem cell differentiation. Our results reveal that post-transcriptional RNA methylation can play a previously unappreciated role in controlling stem cell fate.
    Full-text · Article · Dec 2011 · PLoS Genetics
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    Dataset: Figure S9
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    ABSTRACT: Lack of Misu impairs migration of LRC from the bulge into the hair germ. (A,B) BrdU (green) incorporation into wild-type (A) and Misu −/− (B) epidermis 2 hours after last BrdU injection. Nuclei are counterstained with DAPI. (C–F) Frequency distributions of intensity of the BrdU-label in the whole hair follicle (bulge plus hair germ) (C), the high bulge (D), the low bulge (E) and the hair germ (F). Error bars indicate SEM (n = 5 hair follicles per mouse, from 4 wt and 4 Misu −/− mice). Scale bar: 100 µm in (A,B). (TIF)
    Preview · Dataset · Dec 2011
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    Dataset: Figure S4
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    ABSTRACT: Expression of bulge and hair germ markers. (A–C) Whole mount labelling for Itgα6 (red), P-cadherin (P-cadh) (green) and DAPI (blue) in tail skin. Scale bar: 100 µm. (TIF)
    Preview · Dataset · Dec 2011
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    Dataset: Figure S2
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    ABSTRACT: Misu is expressed in committed progenitor cells. (A–H) Immunohistochemistry of LacZ (blue) of hair follicles of adult mice in anagen at P31 co-stained for the proliferation marker Ki67 (A), markers for the cortex and pre-cortex; Dlx3 (B) and keratin 31 (C), markers for the inner root sheath; Gata3 (D) and keratin 71 and 72 (E,F), a marker for the companion layer; keratin 6 (G) and a marker for the outer root sheath; keratin 14 (H). Scale bars: 50 µm. (TIF)
    Preview · Dataset · Dec 2011
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    Dataset: Figure S5
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    ABSTRACT: Expression profile of FDG+ve cells at the onset of anagen. (A–B) Gating for FDG+ve and FDG−ve epidermal cells from Misu +/− mice at P21 using flow cytometry. Cells were gated by F2Log (autofluorescence) versus F1Log (FDG). FDG+ve cells were sorted as indicated by the blue square and FDG−ve cells were sorted as indicated by the black square. Epidermal cells from wild-type mice are negative for FDG (A). (C–G) QPCR for the indicated genes of total RNA isolated from FDG+ve and FDG−ve sorted epidermal cells. Error bars indicate SEM (n = 3). (TIF)
    Preview · Dataset · Dec 2011
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    Dataset: Table S1
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    ABSTRACT: Hair cycle staging of Misu −/− mice and their control littermates. For each postnatal day (P), mice are grouped by genotype and gender. The total number of mice is indicated with N. Each mouse is classified into specific hair cycle phases based on established morphological guidelines. Anagen I–IIIa is defined as period starting from the onset of mitotic activity in the hair germ (I) to cells show differentiation into all follicular components (IIIa). Anagen IIIb–VI includes the stage of melanocyte activation (IIIb) to a new hair shaft emerges from skin surface (VI). (DOCX)
    Preview · Dataset · Dec 2011
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    Dataset: Figure S8
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    ABSTRACT: Conditional deletion of Misu in skin increases dormancy of bulge stem cells. (A) Schematic overview of MISUf/f allele. Exon-intron organization of murine MISU gene, showing the SUN domain (red box) and LoxP sites flanking exon 6 (blue triangles). (B–C) Validation of conditional deletion of Misu in the epidermis in K14MisuΔ/Δ mice. QPCR using total back skin shows a decrease of Misu-mRNA levels in K14MisuΔ/Δ mice, especially in anagen (P27), when Misu-mRNA levels are the highest in the control Misuf/f mice (B). QPCR using total back skin and epidermis of mice in telogen (P49) shows a specific reduction in Misu-mRNA levels in K14MisuΔ/Δ epidermis compared to total skin (C). (D) Epidermis in telogen (P49) analysed by flow cytometry for expression of Itgα6 and CD34. The percentage of Itgα6low/CD34+ve and Itgα6high/CD34+ve cells is shown ± SEM (n = 3). The red bars in (D) indicate a cell population enriched in K14MisuΔ/Δ epidermis. The grey box indicates cells sorted as population L (Itgα6low/CD34+ve) and the black box cells sorted as population H (Itgα6high/CD34+ve). (E) QPCR for the sorted cell populations indicated in (D) from K14MisuΔ/Δ and Misuf/f mice. RNA levels were normalized to GAPDH and values for K14MisuΔ/Δ versus controls (Misuf/f) measured. Error bars represent SEM (n = 4). (TIF)
    Preview · Dataset · Dec 2011
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    Dataset: Figure S7
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    ABSTRACT: Flow cytometry analysis for bulge stem cells and progenitor cells in anagen and telogen. (A) Flow cytometry analysis for expression of Itgα6 and CD34 in epidermis at anagen (P30) and (B) Itgα6 and P-Cadherin at telogen (P49) in wild-type and Misu −/− mice. (TIF)
    Preview · Dataset · Dec 2011
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    Dataset: Figure S1
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    ABSTRACT: Validation of Misu −/− mice. (A) Exon-intron organization of murine MISU gene, showing the SUN domain (red box) and the Gene Trap insertion (filled blue box). (B) Gene-specific PCR to detect MISU (upper panel) and LacZ alleles (lower panel). (C) QPCR using total back skin shows lack of Misu-mRNA in Misu −/− mice. (D) Western blot confirms deletion of Misu-protein. Tubulin (Tub) was used as loading control. (TIF)
    Preview · Dataset · Dec 2011
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    Dataset: Figure S3
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    ABSTRACT: LacZ-expression and Misu protein are absent in controls. (A,B) LacZ staining of wild-type skin at telogen (A) and anagen (B). Arrows indicate unspecific staining in sebaceous glands. Sections are counterstained with Eosin. (C) Confocal image of Misu −/− hair follicle in anagen co-stained for K15 (red), Misu (green) and nulei (DAPI, in blue). Scale bars: 50 µm. (TIF)
    Preview · Dataset · Dec 2011
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    ABSTRACT: We have examined expression of the Myc target gene Misu (NSUN2) in breast cancer. There was extensive copy number gain, and increased mRNA and protein levels, of Misu in approximately one third of breast cancer cell lines and primary tumours examined, irrespective of tumour subtype. Genes on 5p15.31-33, where Misu is located, showed evolutionary synteny. siRNA-mediated knockdown of Misu reduced cell number in over half of the cell lines tested, irrespective of estrogen receptor status. We conclude that Misu is up-regulated in a substantial proportion of breast cancers and has therapeutic potential as a drug target.
    Full-text · Article · Oct 2009 · Cancer letters
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    ABSTRACT: Myc-induced SUN domain-containing protein (Misu or NSun2) is a nucleolar RNA methyltransferase important for c-Myc-induced proliferation in skin, but the mechanisms by which Misu contributes to cell cycle progression are unknown. In this study, we demonstrate that Misu translocates from the nucleoli in interphase to the spindle in mitosis as an RNA-protein complex that includes 18S ribosomal RNA. Functionally, depletion of Misu caused multiple mitotic defects, including formation of unstructured spindles, multipolar spindles, and chromosome missegregation, leading to aneuploidy and cell death. The presence of both RNA and Misu is required for correct spindle assembly, and this process is independent of active translation. Misu might mediate its function at the spindle by recruiting nucleolar and spindle-associated protein (NuSAP), an essential microtubule-stabilizing and bundling protein. We further identify NuSAP as a novel direct target gene of c-Myc. Collectively, our results suggest a novel mechanism by which c-Myc promotes proliferation by stabilizing the mitotic spindle in fast-dividing cells via Misu and NuSAP.
    Full-text · Article · Aug 2009 · The Journal of Cell Biology

Publication Stats

146 Citations
29.45 Total Impact Points

Institutions

  • 2011-2014
    • University of Cambridge
      • Stem Cell Institute
      Cambridge, England, United Kingdom
  • 2009
    • Wellcome Trust
      Londinium, England, United Kingdom
    • Goethe-Universität Frankfurt am Main
      Frankfurt, Hesse, Germany