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Support for the immortal strand hypothesis: neural stem cells partition DNA asymmetrically in vitro.

Karpowicz P, Morshead C, Kam A, Jervis E, Ramunas J, Ramuns J, Cheng V, van der Kooy D - J. Cell Biol. (2005)

Bottom Line: The immortal strand hypothesis proposes that asymmetrically dividing stem cells (SCs) selectively segregate chromosomes that bear the oldest DNA templates.We investigated cosegregation in neural stem cells (NSCs).It was confirmed that some BrdU-retaining cells divided actively, and that these cells exhibited some characteristics of SCs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Science, University of Toronto, Toronto, M5R 1A8, Canada. phillip.karpowicz@utoronto.ca

ABSTRACT
The immortal strand hypothesis proposes that asymmetrically dividing stem cells (SCs) selectively segregate chromosomes that bear the oldest DNA templates. We investigated cosegregation in neural stem cells (NSCs). After exposure to the thymidine analogue 5-bromo-2-deoxyuridine (BrdU), which labels newly synthesized DNA, a subset of neural precursor cells were shown to retain BrdU signal. It was confirmed that some BrdU-retaining cells divided actively, and that these cells exhibited some characteristics of SCs. This asymmetric partitioning of DNA then was demonstrated during mitosis, and these results were further supported by real time imaging of SC clones, in which older and newly synthesized DNA templates were distributed asymmetrically after DNA synthesis. We demonstrate that NSCs are unique among precursor cells in the uneven partitioning of genetic material during cell divisions.

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Live cell imaging supports nonrandom segregation of DNA. (A) Schematic showing BrdU imaging strategy. (1) Each double strand (1 chromosome) represents 10 chromosomes of a mouse cell. Cells are unlabeled for BrdU (black). (2) During DNA synthesis, BrdU (green) is taken up for exactly one division in the presence of BrdU. (3) BrdU is removed and the daughters enter a second round of DNA synthesis in the absence of BrdU. (4) Division events after the second round of DNA synthesis should show BrdU asymmetry if groups of unlabeled chromosomes are cosegregated as immortal strands into SCs. (B) A clone imaged in real time. After one division event, BrdU was removed, and colony was fixed after two further cell divisions in the absence of BrdU. Arrow indicates a cell that has cleared all BrdU signal. (i) Bright field shows clone; (ii) histone-labeled nuclei are red; (iii) BrdU is indicated by green; (iv) merge of histone and BrdU. (C) Lineage diagrams from four clones traced (ii–iv show asymmetric DNA partitioning). Clone (ii) is the same shown in B. Each lineage represents divisions of one single cell, plated in the presence of BrdU, which is taken up during the first division initially labeling daughter nuclei (green), as demonstrated in clone (i). BrdU was removed after this one division, and cells continued proliferating until analysis. Note, the presence of BrdU(+) is inferred in parental cells from their offspring. Dead cells were observed to disintegrate while imaging, before analysis. (D) Summary of clones traced. 6 out of 15 clones demonstrated asymmetric partitioning of new and old DNA.
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fig7: Live cell imaging supports nonrandom segregation of DNA. (A) Schematic showing BrdU imaging strategy. (1) Each double strand (1 chromosome) represents 10 chromosomes of a mouse cell. Cells are unlabeled for BrdU (black). (2) During DNA synthesis, BrdU (green) is taken up for exactly one division in the presence of BrdU. (3) BrdU is removed and the daughters enter a second round of DNA synthesis in the absence of BrdU. (4) Division events after the second round of DNA synthesis should show BrdU asymmetry if groups of unlabeled chromosomes are cosegregated as immortal strands into SCs. (B) A clone imaged in real time. After one division event, BrdU was removed, and colony was fixed after two further cell divisions in the absence of BrdU. Arrow indicates a cell that has cleared all BrdU signal. (i) Bright field shows clone; (ii) histone-labeled nuclei are red; (iii) BrdU is indicated by green; (iv) merge of histone and BrdU. (C) Lineage diagrams from four clones traced (ii–iv show asymmetric DNA partitioning). Clone (ii) is the same shown in B. Each lineage represents divisions of one single cell, plated in the presence of BrdU, which is taken up during the first division initially labeling daughter nuclei (green), as demonstrated in clone (i). BrdU was removed after this one division, and cells continued proliferating until analysis. Note, the presence of BrdU(+) is inferred in parental cells from their offspring. Dead cells were observed to disintegrate while imaging, before analysis. (D) Summary of clones traced. 6 out of 15 clones demonstrated asymmetric partitioning of new and old DNA.

Mentions: According to the ISH, SCs cosegregate chromosomes to retain older DNA templates in one daughter SC but not the non-SC daughter (Fig. 1). Given that DNA replication is semiconservative, cosegregated chromosomes are distinguished because they contain one older strand, albeit one that is associated with a newer strand from one preceding round of DNA synthesis. We predicted that symmetric SC divisions would randomize segregation of chromosomes between daughter cells. The ISH was investigated in neural stem cells (NSCs) using a clonal cell culture system in which brain-derived colonies, arising from a single SC, are both self renewing and multipotent (Reynolds and Weiss, 1992; Morshead et al., 1994). The halogenated thymidine analogue, 5-bromo-2-deoxyuridine (BrdU) was used to label DNA strands. We asked: (a) would SCs retain BrdU(+) DNA strands in the absence of BrdU, if they divided symmetrically many times in the presence of BrdU (see Fig. 2 A); and (b) would SCs retain their original BrdU(−) strands, in the absence of BrdU, if they divided asymmetrically once and only once in the presence of BrdU (see Fig. 7).


Support for the immortal strand hypothesis: neural stem cells partition DNA asymmetrically in vitro.

Karpowicz P, Morshead C, Kam A, Jervis E, Ramunas J, Ramuns J, Cheng V, van der Kooy D - J. Cell Biol. (2005)

Live cell imaging supports nonrandom segregation of DNA. (A) Schematic showing BrdU imaging strategy. (1) Each double strand (1 chromosome) represents 10 chromosomes of a mouse cell. Cells are unlabeled for BrdU (black). (2) During DNA synthesis, BrdU (green) is taken up for exactly one division in the presence of BrdU. (3) BrdU is removed and the daughters enter a second round of DNA synthesis in the absence of BrdU. (4) Division events after the second round of DNA synthesis should show BrdU asymmetry if groups of unlabeled chromosomes are cosegregated as immortal strands into SCs. (B) A clone imaged in real time. After one division event, BrdU was removed, and colony was fixed after two further cell divisions in the absence of BrdU. Arrow indicates a cell that has cleared all BrdU signal. (i) Bright field shows clone; (ii) histone-labeled nuclei are red; (iii) BrdU is indicated by green; (iv) merge of histone and BrdU. (C) Lineage diagrams from four clones traced (ii–iv show asymmetric DNA partitioning). Clone (ii) is the same shown in B. Each lineage represents divisions of one single cell, plated in the presence of BrdU, which is taken up during the first division initially labeling daughter nuclei (green), as demonstrated in clone (i). BrdU was removed after this one division, and cells continued proliferating until analysis. Note, the presence of BrdU(+) is inferred in parental cells from their offspring. Dead cells were observed to disintegrate while imaging, before analysis. (D) Summary of clones traced. 6 out of 15 clones demonstrated asymmetric partitioning of new and old DNA.
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Related In: Results  -  Collection

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fig7: Live cell imaging supports nonrandom segregation of DNA. (A) Schematic showing BrdU imaging strategy. (1) Each double strand (1 chromosome) represents 10 chromosomes of a mouse cell. Cells are unlabeled for BrdU (black). (2) During DNA synthesis, BrdU (green) is taken up for exactly one division in the presence of BrdU. (3) BrdU is removed and the daughters enter a second round of DNA synthesis in the absence of BrdU. (4) Division events after the second round of DNA synthesis should show BrdU asymmetry if groups of unlabeled chromosomes are cosegregated as immortal strands into SCs. (B) A clone imaged in real time. After one division event, BrdU was removed, and colony was fixed after two further cell divisions in the absence of BrdU. Arrow indicates a cell that has cleared all BrdU signal. (i) Bright field shows clone; (ii) histone-labeled nuclei are red; (iii) BrdU is indicated by green; (iv) merge of histone and BrdU. (C) Lineage diagrams from four clones traced (ii–iv show asymmetric DNA partitioning). Clone (ii) is the same shown in B. Each lineage represents divisions of one single cell, plated in the presence of BrdU, which is taken up during the first division initially labeling daughter nuclei (green), as demonstrated in clone (i). BrdU was removed after this one division, and cells continued proliferating until analysis. Note, the presence of BrdU(+) is inferred in parental cells from their offspring. Dead cells were observed to disintegrate while imaging, before analysis. (D) Summary of clones traced. 6 out of 15 clones demonstrated asymmetric partitioning of new and old DNA.
Mentions: According to the ISH, SCs cosegregate chromosomes to retain older DNA templates in one daughter SC but not the non-SC daughter (Fig. 1). Given that DNA replication is semiconservative, cosegregated chromosomes are distinguished because they contain one older strand, albeit one that is associated with a newer strand from one preceding round of DNA synthesis. We predicted that symmetric SC divisions would randomize segregation of chromosomes between daughter cells. The ISH was investigated in neural stem cells (NSCs) using a clonal cell culture system in which brain-derived colonies, arising from a single SC, are both self renewing and multipotent (Reynolds and Weiss, 1992; Morshead et al., 1994). The halogenated thymidine analogue, 5-bromo-2-deoxyuridine (BrdU) was used to label DNA strands. We asked: (a) would SCs retain BrdU(+) DNA strands in the absence of BrdU, if they divided symmetrically many times in the presence of BrdU (see Fig. 2 A); and (b) would SCs retain their original BrdU(−) strands, in the absence of BrdU, if they divided asymmetrically once and only once in the presence of BrdU (see Fig. 7).

Bottom Line: The immortal strand hypothesis proposes that asymmetrically dividing stem cells (SCs) selectively segregate chromosomes that bear the oldest DNA templates.We investigated cosegregation in neural stem cells (NSCs).It was confirmed that some BrdU-retaining cells divided actively, and that these cells exhibited some characteristics of SCs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Science, University of Toronto, Toronto, M5R 1A8, Canada. phillip.karpowicz@utoronto.ca

ABSTRACT
The immortal strand hypothesis proposes that asymmetrically dividing stem cells (SCs) selectively segregate chromosomes that bear the oldest DNA templates. We investigated cosegregation in neural stem cells (NSCs). After exposure to the thymidine analogue 5-bromo-2-deoxyuridine (BrdU), which labels newly synthesized DNA, a subset of neural precursor cells were shown to retain BrdU signal. It was confirmed that some BrdU-retaining cells divided actively, and that these cells exhibited some characteristics of SCs. This asymmetric partitioning of DNA then was demonstrated during mitosis, and these results were further supported by real time imaging of SC clones, in which older and newly synthesized DNA templates were distributed asymmetrically after DNA synthesis. We demonstrate that NSCs are unique among precursor cells in the uneven partitioning of genetic material during cell divisions.

Show MeSH
Related in: MedlinePlus