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The strength of SMAD1/5 activity determines the mode of stem cell division in the developing spinal cord.

Le Dréau G, Saade M, Gutiérrez-Vallejo I, Martí E - J. Cell Biol. (2014)

Bottom Line: However, the mechanisms controlling such events are not fully understood.Characterizing these three modes of division during interneuron generation in the developing chick spinal cord, we demonstrated that they correlate to different levels of activity of the canonical bone morphogenetic protein effectors SMAD1/5.Together, these results lead us to propose that the strength of SMAD1/5 activity dictates the mode of stem cell division during spinal interneuron generation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Instituto de Biología Molecular de Barcelona, Consejo Superior de Investigaciones Científicas, Parc Científic de Barcelona, Barcelona 08028, Spain.

ABSTRACT
The different modes of stem cell division are tightly regulated to balance growth and differentiation during organ development and homeostasis. However, the mechanisms controlling such events are not fully understood. We have developed markers that provide the single cell resolution necessary to identify the three modes of division occurring in a developing nervous system: self-expanding, self-renewing, and self-consuming. Characterizing these three modes of division during interneuron generation in the developing chick spinal cord, we demonstrated that they correlate to different levels of activity of the canonical bone morphogenetic protein effectors SMAD1/5. Functional in vivo experiments showed that the premature neuronal differentiation and changes in cell cycle parameters caused by SMAD1/5 inhibition were preceded by a reduction of self-expanding divisions in favor of self-consuming divisions. Conversely, SMAD1/5 gain of function promoted self-expanding divisions. Together, these results lead us to propose that the strength of SMAD1/5 activity dictates the mode of stem cell division during spinal interneuron generation.

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Analysis of the cell cycle distribution of PP, PN, and NN progenitors. (A) Illustration of the methodology used to analyze the cell cycle distribution of PP, PN, and NN divisions by flow cytometry in dissociated cells processed for pH3 staining or Hoechst incorporation 20 hpe with pTis21:RFP, pSox2:EGFP, and control or Smad1/5 shRNA vectors. (B) Percentages of mitotic (pH3+) GFP+;RFP− (PP), GFP+;RFP+ (PN), and GFP−;RFP+ (NN) cells electroporated with control (Ctrl) or Smad1/5 shRNA (sh-S1/5). (C) Mitotic indices of GFP+;RFP− (PP), GFP+;RFP+ (PN), and GFP−;RFP+ (NN) cells electroporated with control or Smad1/5 shRNA (sh-S1/5). (D–F) Overlays of representative DNA content profiles obtained after Hoechst incorporation for GFP+;RFP− (D, PP), GFP+;RFP+ (E, PN), and GFP−;RFP+ (F, NN) cells electroporated with control or sh-S1/5 constructs. Mean values of the percentages of cells in G1, S, and G2–M phases are presented. EP, electroporation. Error bars show means ± SEM. **, P < 0.01.
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fig7: Analysis of the cell cycle distribution of PP, PN, and NN progenitors. (A) Illustration of the methodology used to analyze the cell cycle distribution of PP, PN, and NN divisions by flow cytometry in dissociated cells processed for pH3 staining or Hoechst incorporation 20 hpe with pTis21:RFP, pSox2:EGFP, and control or Smad1/5 shRNA vectors. (B) Percentages of mitotic (pH3+) GFP+;RFP− (PP), GFP+;RFP+ (PN), and GFP−;RFP+ (NN) cells electroporated with control (Ctrl) or Smad1/5 shRNA (sh-S1/5). (C) Mitotic indices of GFP+;RFP− (PP), GFP+;RFP+ (PN), and GFP−;RFP+ (NN) cells electroporated with control or Smad1/5 shRNA (sh-S1/5). (D–F) Overlays of representative DNA content profiles obtained after Hoechst incorporation for GFP+;RFP− (D, PP), GFP+;RFP+ (E, PN), and GFP−;RFP+ (F, NN) cells electroporated with control or sh-S1/5 constructs. Mean values of the percentages of cells in G1, S, and G2–M phases are presented. EP, electroporation. Error bars show means ± SEM. **, P < 0.01.

Mentions: It appears that modulating SMAD1/5 activity within spinal progenitors alters both their mode of division and their cell cycle kinetics. We next established an experimental design to discriminate which of these two events is directly controlled by SMAD1/5. Accordingly, we coelectroporated the two reporters (pTis21:RFP and pSox2:GFP) with the sh-S1/5 or control vectors, and we then dissociated the neural tube cells at 20 hpe, stained them for pH3, and analyzed them by flow cytometry (Fig. 7 A). The assessment of the proportions of PP, PN, and NN divisions among the mitotic (pH3+) electroporated cells confirmed that Smad1/5 knockdown increased the percentage of NN divisions at the expense of PP ones, without significantly altering PN divisions (Fig. 7 B). In the same set of experiments, we calculated the mitotic index for each of the three progenitor subpopulations. After control electroporation, although the percentages of mitotic PP and PN cells were comparable, that of mitotic NN progenitors was significantly higher (percentages of pH3+ cells were 6.5 ± 0.5 for PP, 9.1 ± 0.4 for PN, and 17.6 ± 1.9 for NN in control conditions; P < 0.01; Fig. 7 C), suggesting that NN divisions were faster. Importantly, interfering with SMAD1/5 activity did not affect the mitotic indices of any of the three progenitor populations compared with their respective controls (percentages of pH3+ cells were 4.3 ± 0.9 for PP, 8.9 ± 1.0 for PN, and 19.3 ± 3.7 for NN after sh-S1/5 electroporation; Fig. 7 C).


The strength of SMAD1/5 activity determines the mode of stem cell division in the developing spinal cord.

Le Dréau G, Saade M, Gutiérrez-Vallejo I, Martí E - J. Cell Biol. (2014)

Analysis of the cell cycle distribution of PP, PN, and NN progenitors. (A) Illustration of the methodology used to analyze the cell cycle distribution of PP, PN, and NN divisions by flow cytometry in dissociated cells processed for pH3 staining or Hoechst incorporation 20 hpe with pTis21:RFP, pSox2:EGFP, and control or Smad1/5 shRNA vectors. (B) Percentages of mitotic (pH3+) GFP+;RFP− (PP), GFP+;RFP+ (PN), and GFP−;RFP+ (NN) cells electroporated with control (Ctrl) or Smad1/5 shRNA (sh-S1/5). (C) Mitotic indices of GFP+;RFP− (PP), GFP+;RFP+ (PN), and GFP−;RFP+ (NN) cells electroporated with control or Smad1/5 shRNA (sh-S1/5). (D–F) Overlays of representative DNA content profiles obtained after Hoechst incorporation for GFP+;RFP− (D, PP), GFP+;RFP+ (E, PN), and GFP−;RFP+ (F, NN) cells electroporated with control or sh-S1/5 constructs. Mean values of the percentages of cells in G1, S, and G2–M phases are presented. EP, electroporation. Error bars show means ± SEM. **, P < 0.01.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3926951&req=5

fig7: Analysis of the cell cycle distribution of PP, PN, and NN progenitors. (A) Illustration of the methodology used to analyze the cell cycle distribution of PP, PN, and NN divisions by flow cytometry in dissociated cells processed for pH3 staining or Hoechst incorporation 20 hpe with pTis21:RFP, pSox2:EGFP, and control or Smad1/5 shRNA vectors. (B) Percentages of mitotic (pH3+) GFP+;RFP− (PP), GFP+;RFP+ (PN), and GFP−;RFP+ (NN) cells electroporated with control (Ctrl) or Smad1/5 shRNA (sh-S1/5). (C) Mitotic indices of GFP+;RFP− (PP), GFP+;RFP+ (PN), and GFP−;RFP+ (NN) cells electroporated with control or Smad1/5 shRNA (sh-S1/5). (D–F) Overlays of representative DNA content profiles obtained after Hoechst incorporation for GFP+;RFP− (D, PP), GFP+;RFP+ (E, PN), and GFP−;RFP+ (F, NN) cells electroporated with control or sh-S1/5 constructs. Mean values of the percentages of cells in G1, S, and G2–M phases are presented. EP, electroporation. Error bars show means ± SEM. **, P < 0.01.
Mentions: It appears that modulating SMAD1/5 activity within spinal progenitors alters both their mode of division and their cell cycle kinetics. We next established an experimental design to discriminate which of these two events is directly controlled by SMAD1/5. Accordingly, we coelectroporated the two reporters (pTis21:RFP and pSox2:GFP) with the sh-S1/5 or control vectors, and we then dissociated the neural tube cells at 20 hpe, stained them for pH3, and analyzed them by flow cytometry (Fig. 7 A). The assessment of the proportions of PP, PN, and NN divisions among the mitotic (pH3+) electroporated cells confirmed that Smad1/5 knockdown increased the percentage of NN divisions at the expense of PP ones, without significantly altering PN divisions (Fig. 7 B). In the same set of experiments, we calculated the mitotic index for each of the three progenitor subpopulations. After control electroporation, although the percentages of mitotic PP and PN cells were comparable, that of mitotic NN progenitors was significantly higher (percentages of pH3+ cells were 6.5 ± 0.5 for PP, 9.1 ± 0.4 for PN, and 17.6 ± 1.9 for NN in control conditions; P < 0.01; Fig. 7 C), suggesting that NN divisions were faster. Importantly, interfering with SMAD1/5 activity did not affect the mitotic indices of any of the three progenitor populations compared with their respective controls (percentages of pH3+ cells were 4.3 ± 0.9 for PP, 8.9 ± 1.0 for PN, and 19.3 ± 3.7 for NN after sh-S1/5 electroporation; Fig. 7 C).

Bottom Line: However, the mechanisms controlling such events are not fully understood.Characterizing these three modes of division during interneuron generation in the developing chick spinal cord, we demonstrated that they correlate to different levels of activity of the canonical bone morphogenetic protein effectors SMAD1/5.Together, these results lead us to propose that the strength of SMAD1/5 activity dictates the mode of stem cell division during spinal interneuron generation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Instituto de Biología Molecular de Barcelona, Consejo Superior de Investigaciones Científicas, Parc Científic de Barcelona, Barcelona 08028, Spain.

ABSTRACT
The different modes of stem cell division are tightly regulated to balance growth and differentiation during organ development and homeostasis. However, the mechanisms controlling such events are not fully understood. We have developed markers that provide the single cell resolution necessary to identify the three modes of division occurring in a developing nervous system: self-expanding, self-renewing, and self-consuming. Characterizing these three modes of division during interneuron generation in the developing chick spinal cord, we demonstrated that they correlate to different levels of activity of the canonical bone morphogenetic protein effectors SMAD1/5. Functional in vivo experiments showed that the premature neuronal differentiation and changes in cell cycle parameters caused by SMAD1/5 inhibition were preceded by a reduction of self-expanding divisions in favor of self-consuming divisions. Conversely, SMAD1/5 gain of function promoted self-expanding divisions. Together, these results lead us to propose that the strength of SMAD1/5 activity dictates the mode of stem cell division during spinal interneuron generation.

Show MeSH
Related in: MedlinePlus