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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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SMAD1/5 inhibition triggers premature differentiation in a cell-autonomous manner. (A) Proportions of electroporated cells differentiated into neurons (H2B-RFP+;HuC/D+) obtained after electroporation of HH14 embryos with control (Ctrl) and Smad1/5 shRNA (sh-S1 and sh-S5) constructs, in sections of neural tubes recovered at the times indicated (hpe). (B) Representative sections stained for HuC/D expression 24 h after coelectroporation with the Tubb3enh:EGFP reporter, the control H2B-RFP vector, and control or Smad1/5 shRNA (sh-S1/5). (C) Quantification of luciferase activity (expressed in relative luciferase units [rlu]) driven by the pNeuroD reporter at 24 hpe with the Smad1/5 shRNA (sh-S1 or sh-S5) or control vectors. (D) Representative transverse sections of a chick neural tube at 48 hpe with control or Smad1/5 shRNA (sh-S1/5). DAPI, HuC/D, and H2B-RFP stain nuclei, differentiating neurons and electroporated cells. (E) Analysis of the ratios of the areas occupied by the VZ (Sox2+) and MZ (HuC/D+) measured for the electroporated side and standardized to their contralateral controls. EP, electroporation. Error bars show means ± SEM. *, P < 0.05; **, P < 0.01. Bars: (B) 25 µM; (D) 50 µM.
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fig5: SMAD1/5 inhibition triggers premature differentiation in a cell-autonomous manner. (A) Proportions of electroporated cells differentiated into neurons (H2B-RFP+;HuC/D+) obtained after electroporation of HH14 embryos with control (Ctrl) and Smad1/5 shRNA (sh-S1 and sh-S5) constructs, in sections of neural tubes recovered at the times indicated (hpe). (B) Representative sections stained for HuC/D expression 24 h after coelectroporation with the Tubb3enh:EGFP reporter, the control H2B-RFP vector, and control or Smad1/5 shRNA (sh-S1/5). (C) Quantification of luciferase activity (expressed in relative luciferase units [rlu]) driven by the pNeuroD reporter at 24 hpe with the Smad1/5 shRNA (sh-S1 or sh-S5) or control vectors. (D) Representative transverse sections of a chick neural tube at 48 hpe with control or Smad1/5 shRNA (sh-S1/5). DAPI, HuC/D, and H2B-RFP stain nuclei, differentiating neurons and electroporated cells. (E) Analysis of the ratios of the areas occupied by the VZ (Sox2+) and MZ (HuC/D+) measured for the electroporated side and standardized to their contralateral controls. EP, electroporation. Error bars show means ± SEM. *, P < 0.05; **, P < 0.01. Bars: (B) 25 µM; (D) 50 µM.

Mentions: We reasoned that the concomitant decrease in PP and increase in NN divisions, which are caused by SMAD1/5 inhibition should alter the pace of neuronal differentiation. We thus analyzed the time course of neuronal differentiation after interfering with SMAD1/5 activity. The proportion of electroporated cells (H2B-RFP+) differentiated into neurons (HuC/D+) was significantly higher from 24 h after Smad1/5 knockdown onwards (percentages of H2B-RFP+;HuC/D+ cells at 38 hpe were 31 ± 3 for control, 55 ± 4 for sh-S1, and 54 ± 3 for sh-S5; P < 0.01; Figs. 5 A and S3 A), indicating that the loss of SMAD1/5 activity caused the premature differentiation of neural progenitors in a cell-autonomous manner. To confirm this result, we took advantage of the Tubb3enh:EGFP reporter (Bergsland et al., 2011), which contains an EGFP cassette driven by an enhancer of the β3-tubulin gene, the expression of which is activated during neuronal differentiation. More H2B-RFP+;GFP+ cells were observed in embryos electroporated with sh-S1 and sh-S5 than in the controls at 24 hpe (Fig. 5 B). At 24 hpe, Smad1/5 knockdown had moreover triggered an approximately threefold increase in the activity of a luciferase (Luc) reporter driven by a fragment of the NeuroD promoter, a basic helix–loop–helix factor known to activate the panneurogenic differentiation program (pNeuroD:Luc; 14.0 ± 0.8 for control, 41.3 ± 4.2 for sh-S1, and 40.4 ± 4.1 for sh-S5; P < 0.01; Fig. 5 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)

SMAD1/5 inhibition triggers premature differentiation in a cell-autonomous manner. (A) Proportions of electroporated cells differentiated into neurons (H2B-RFP+;HuC/D+) obtained after electroporation of HH14 embryos with control (Ctrl) and Smad1/5 shRNA (sh-S1 and sh-S5) constructs, in sections of neural tubes recovered at the times indicated (hpe). (B) Representative sections stained for HuC/D expression 24 h after coelectroporation with the Tubb3enh:EGFP reporter, the control H2B-RFP vector, and control or Smad1/5 shRNA (sh-S1/5). (C) Quantification of luciferase activity (expressed in relative luciferase units [rlu]) driven by the pNeuroD reporter at 24 hpe with the Smad1/5 shRNA (sh-S1 or sh-S5) or control vectors. (D) Representative transverse sections of a chick neural tube at 48 hpe with control or Smad1/5 shRNA (sh-S1/5). DAPI, HuC/D, and H2B-RFP stain nuclei, differentiating neurons and electroporated cells. (E) Analysis of the ratios of the areas occupied by the VZ (Sox2+) and MZ (HuC/D+) measured for the electroporated side and standardized to their contralateral controls. EP, electroporation. Error bars show means ± SEM. *, P < 0.05; **, P < 0.01. Bars: (B) 25 µM; (D) 50 µM.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig5: SMAD1/5 inhibition triggers premature differentiation in a cell-autonomous manner. (A) Proportions of electroporated cells differentiated into neurons (H2B-RFP+;HuC/D+) obtained after electroporation of HH14 embryos with control (Ctrl) and Smad1/5 shRNA (sh-S1 and sh-S5) constructs, in sections of neural tubes recovered at the times indicated (hpe). (B) Representative sections stained for HuC/D expression 24 h after coelectroporation with the Tubb3enh:EGFP reporter, the control H2B-RFP vector, and control or Smad1/5 shRNA (sh-S1/5). (C) Quantification of luciferase activity (expressed in relative luciferase units [rlu]) driven by the pNeuroD reporter at 24 hpe with the Smad1/5 shRNA (sh-S1 or sh-S5) or control vectors. (D) Representative transverse sections of a chick neural tube at 48 hpe with control or Smad1/5 shRNA (sh-S1/5). DAPI, HuC/D, and H2B-RFP stain nuclei, differentiating neurons and electroporated cells. (E) Analysis of the ratios of the areas occupied by the VZ (Sox2+) and MZ (HuC/D+) measured for the electroporated side and standardized to their contralateral controls. EP, electroporation. Error bars show means ± SEM. *, P < 0.05; **, P < 0.01. Bars: (B) 25 µM; (D) 50 µM.
Mentions: We reasoned that the concomitant decrease in PP and increase in NN divisions, which are caused by SMAD1/5 inhibition should alter the pace of neuronal differentiation. We thus analyzed the time course of neuronal differentiation after interfering with SMAD1/5 activity. The proportion of electroporated cells (H2B-RFP+) differentiated into neurons (HuC/D+) was significantly higher from 24 h after Smad1/5 knockdown onwards (percentages of H2B-RFP+;HuC/D+ cells at 38 hpe were 31 ± 3 for control, 55 ± 4 for sh-S1, and 54 ± 3 for sh-S5; P < 0.01; Figs. 5 A and S3 A), indicating that the loss of SMAD1/5 activity caused the premature differentiation of neural progenitors in a cell-autonomous manner. To confirm this result, we took advantage of the Tubb3enh:EGFP reporter (Bergsland et al., 2011), which contains an EGFP cassette driven by an enhancer of the β3-tubulin gene, the expression of which is activated during neuronal differentiation. More H2B-RFP+;GFP+ cells were observed in embryos electroporated with sh-S1 and sh-S5 than in the controls at 24 hpe (Fig. 5 B). At 24 hpe, Smad1/5 knockdown had moreover triggered an approximately threefold increase in the activity of a luciferase (Luc) reporter driven by a fragment of the NeuroD promoter, a basic helix–loop–helix factor known to activate the panneurogenic differentiation program (pNeuroD:Luc; 14.0 ± 0.8 for control, 41.3 ± 4.2 for sh-S1, and 40.4 ± 4.1 for sh-S5; P < 0.01; Fig. 5 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