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Sonic hedgehog and notch signaling can cooperate to regulate neurogenic divisions of neocortical progenitors.

Dave RK, Ellis T, Toumpas MC, Robson JP, Julian E, Adolphe C, Bartlett PF, Cooper HM, Reynolds BA, Wainwright BJ - PLoS ONE (2011)

Bottom Line: The Notch signaling pathway plays a pivotal role in the maintenance of stem/progenitor cells and the regulation of glial versus neuronal identity.To study the effect of Notch signaling on Hh-activated neural progenitors, we inactivated both Patched1 and Rbpj, a transcriptional mediator of Notch signaling, in Nestin positive cells of the neocortex.Hence, our results demonstrate that correct corticogenesis is an outcome of the interplay between the Hh and Notch signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: The Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia.

ABSTRACT

Background: Hedgehog (Hh) signaling is crucial for the generation and maintenance of both embryonic and adult stem cells, thereby regulating development and tissue homeostasis. In the developing neocortex, Sonic Hedgehog (Shh) regulates neural progenitor cell proliferation. During neurogenesis, radial glial cells of the ventricular zone (VZ) are the predominant neocortical progenitors that generate neurons through both symmetric and asymmetric divisions. Despite its importance, relatively little is known of the molecular pathways that control the switch from symmetric proliferative to differentiative/neurogenic divisions in neural progenitors.

Principal findings: Here, we report that conditional inactivation of Patched1, a negative regulator of the Shh pathway, in Nestin positive neural progenitors of the neocortex leads to lamination defects due to improper corticogenesis and an increase in the number of symmetric proliferative divisions of the radial glial cells. Hedgehog-activated VZ progenitor cells demonstrated a concomitant upregulation of Hes1 and Blbp, downstream targets of Notch signaling. The Notch signaling pathway plays a pivotal role in the maintenance of stem/progenitor cells and the regulation of glial versus neuronal identity. To study the effect of Notch signaling on Hh-activated neural progenitors, we inactivated both Patched1 and Rbpj, a transcriptional mediator of Notch signaling, in Nestin positive cells of the neocortex.

Conclusions: Our data indicate that by mid neurogenesis (embryonic day 14.5), attenuation of Notch signaling reverses the effect of Patched1 deletion on neurogenesis by restoring the balance between symmetric proliferative and neurogenic divisions. Hence, our results demonstrate that correct corticogenesis is an outcome of the interplay between the Hh and Notch signaling pathways.

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Hh pathway activation expands the population of cells with stem cell-like properties and promotes symmetric proliferative divisions of radial glial cells.(A) E14.5 Ptc1Lox/Lox;NestinCre neocortex (n = 10) showed an 8-fold increase (***p<0.0001) in the number of primary neurosphere colonies per cell seeded compared to littermate controls (n = 11). For RbpjLox/Lox;NestinCre and Ptc1Lox/Lox;RbpjLox/Lox;NestinCre samples, n = 3 and n = 4 respectively. * and ** p<0.005 by unpaired Student's t-test (with Welch's correction). (B) Clonal expansion of neurospheres derived from E14.5 neocortex. Cell numbers obtained at each passage were transformed into LOG and linear regression analysis was performed on resulting curves. The rate of clonal expansion of the neurospheres is represented as the slope of the line. The number of stem cells was significantly increased in Ptc1Lox/Lox;NestinCre neurospheres compared to Ptc1Lox/Lox neurospheres (slope: 0.8877±0.06774 vs 0.7060±0.03302, p<0.05). However, RbpjLox/Lox;NestinCre and Ptc1Lox/Lox;RbpjLox/Lox;NestinCre neurospheres showed a significant reduction in stem cells compared to the wild type control (slope: −1.337±3.400e+038, p<0.0001 and slope: −0.08099±0.04816, p value compared to control <0.0001, respectively). Error bars indicate standard deviation (A, B). (C–F): Co-immunofluorescence of GLAST (red) and TuJ1 (green) on neocortical progenitors isolated from E14.5 Rbpj and Ptc1 mutant and wild types neocortices. Cells were counterstained with DAPI (blue). Arrows (in C, E, F) indicate TuJ1+ cells and arrowheads (D) indicate GLAST+ radial glial cells. RG cells from Ptc1Lox/Lox;NestinCre neocortex rarely differentiate into TuJ1+ neurons after 24 hours (D, G), whereas concomitant loss of the Rbpj and Ptc1 alleles results in an increased rate of neurogenesis in Ptc1Lox/Lox;RbpjLox/Lox;NestinCre neocortical cells (F, G). On the other hand, cells from RbpjLox/Lox;NestinCre neocortex mostly differentiate into TuJ1+ neurons after 24 hours. Also, (G): Quantitative analysis for GLAST and TuJ1 revealed that the percentage of RG cells undergoing symmetric neurogenic divisions was significantly increased (p<0.05) in the Ptc1Lox/Lox;RbpjLox/Lox;NestinCre compared to the Ptc1Lox/Lox;NestinCre neocortices. On the other hand, there was a reduction in the number of symmetric proliferative cell divisions in Ptc1Lox/Lox;RbpjLox/Lox;NestinCre compared to Ptc1Lox/Lox;NestinCre (p<0.05) samples. Bars represent standard errors. ***p<0.0001. Scale bar (C–F), 10 µm.
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pone-0014680-g002: Hh pathway activation expands the population of cells with stem cell-like properties and promotes symmetric proliferative divisions of radial glial cells.(A) E14.5 Ptc1Lox/Lox;NestinCre neocortex (n = 10) showed an 8-fold increase (***p<0.0001) in the number of primary neurosphere colonies per cell seeded compared to littermate controls (n = 11). For RbpjLox/Lox;NestinCre and Ptc1Lox/Lox;RbpjLox/Lox;NestinCre samples, n = 3 and n = 4 respectively. * and ** p<0.005 by unpaired Student's t-test (with Welch's correction). (B) Clonal expansion of neurospheres derived from E14.5 neocortex. Cell numbers obtained at each passage were transformed into LOG and linear regression analysis was performed on resulting curves. The rate of clonal expansion of the neurospheres is represented as the slope of the line. The number of stem cells was significantly increased in Ptc1Lox/Lox;NestinCre neurospheres compared to Ptc1Lox/Lox neurospheres (slope: 0.8877±0.06774 vs 0.7060±0.03302, p<0.05). However, RbpjLox/Lox;NestinCre and Ptc1Lox/Lox;RbpjLox/Lox;NestinCre neurospheres showed a significant reduction in stem cells compared to the wild type control (slope: −1.337±3.400e+038, p<0.0001 and slope: −0.08099±0.04816, p value compared to control <0.0001, respectively). Error bars indicate standard deviation (A, B). (C–F): Co-immunofluorescence of GLAST (red) and TuJ1 (green) on neocortical progenitors isolated from E14.5 Rbpj and Ptc1 mutant and wild types neocortices. Cells were counterstained with DAPI (blue). Arrows (in C, E, F) indicate TuJ1+ cells and arrowheads (D) indicate GLAST+ radial glial cells. RG cells from Ptc1Lox/Lox;NestinCre neocortex rarely differentiate into TuJ1+ neurons after 24 hours (D, G), whereas concomitant loss of the Rbpj and Ptc1 alleles results in an increased rate of neurogenesis in Ptc1Lox/Lox;RbpjLox/Lox;NestinCre neocortical cells (F, G). On the other hand, cells from RbpjLox/Lox;NestinCre neocortex mostly differentiate into TuJ1+ neurons after 24 hours. Also, (G): Quantitative analysis for GLAST and TuJ1 revealed that the percentage of RG cells undergoing symmetric neurogenic divisions was significantly increased (p<0.05) in the Ptc1Lox/Lox;RbpjLox/Lox;NestinCre compared to the Ptc1Lox/Lox;NestinCre neocortices. On the other hand, there was a reduction in the number of symmetric proliferative cell divisions in Ptc1Lox/Lox;RbpjLox/Lox;NestinCre compared to Ptc1Lox/Lox;NestinCre (p<0.05) samples. Bars represent standard errors. ***p<0.0001. Scale bar (C–F), 10 µm.

Mentions: In vivo and in vitro loss of function studies have shown that Hh signaling is required for the maintenance of stem cells in the developing neocortex [9], [11], [12], [21], [34]. To directly address the consequences of sustained Hh signaling in neocortical precursors, the neurosphere culture assay was utilised to test for stem and progenitor cell properties [29]. We observed a substantial (approximately 8-fold) increase in sphere forming potential of cells derived from the Ptc1Lox/Lox;NestinCre neocortex (2.154±0.267%) compared to Ptc1Lox/Lox neocortex (0.27±0.047%, p<0.0001,) (Fig. 2A) indicating that Hh pathway activation in vivo can regulate the total number of stem and progenitor cells. Next, the neurospheres were passaged multiple times to select against lineage-committed progenitor cells not capable of self-renewal. The rate of clonal expansion of the neurospheres is represented as the slope of the line. There was a clear difference in the rate of fold increase between Ptc1Lox/Lox and Ptc1Lox/Lox;NestinCre neocortical samples (clonal expansion of 0.7060±0.03302 vs. 0.8877±0.06774, p<0.05) (Fig. 2B) indicating that Hh pathway activation is mitogenic for cells that have a capacity to self-renew over an extended period of time. We also investigated whether the enlargement of the neocortical VZ progenitor population was a result of changes in cell cycle kinetics due to Ptc1 inactivation. If enlargement of the progenitor pool is due to shortening of the cell cycle, the proportion of progenitor cells labelled by a single 2-hour pulse of BrdU in the total population of cycling progenitors (Ki67+) would increase. Indeed, a significant shortening of the cell cycle in the mutant E14.5 neocortex was detected confirming that Ptc1 inactivated progenitor cells divide significantly faster than wild type progenitors (Fig. S3B). In the Ptc1Lox/Lox;NestinCre neocortex, the proportion of VZ progenitors co-labelled with BrdU/Ki67 was approximately two-fold greater when compared to wild type littermates (Fig. S3D). Altogether, our results suggest that Hh pathway activation can regulate neocortical stem and/or progenitor cell divisions and the rate of cell division in vivo.


Sonic hedgehog and notch signaling can cooperate to regulate neurogenic divisions of neocortical progenitors.

Dave RK, Ellis T, Toumpas MC, Robson JP, Julian E, Adolphe C, Bartlett PF, Cooper HM, Reynolds BA, Wainwright BJ - PLoS ONE (2011)

Hh pathway activation expands the population of cells with stem cell-like properties and promotes symmetric proliferative divisions of radial glial cells.(A) E14.5 Ptc1Lox/Lox;NestinCre neocortex (n = 10) showed an 8-fold increase (***p<0.0001) in the number of primary neurosphere colonies per cell seeded compared to littermate controls (n = 11). For RbpjLox/Lox;NestinCre and Ptc1Lox/Lox;RbpjLox/Lox;NestinCre samples, n = 3 and n = 4 respectively. * and ** p<0.005 by unpaired Student's t-test (with Welch's correction). (B) Clonal expansion of neurospheres derived from E14.5 neocortex. Cell numbers obtained at each passage were transformed into LOG and linear regression analysis was performed on resulting curves. The rate of clonal expansion of the neurospheres is represented as the slope of the line. The number of stem cells was significantly increased in Ptc1Lox/Lox;NestinCre neurospheres compared to Ptc1Lox/Lox neurospheres (slope: 0.8877±0.06774 vs 0.7060±0.03302, p<0.05). However, RbpjLox/Lox;NestinCre and Ptc1Lox/Lox;RbpjLox/Lox;NestinCre neurospheres showed a significant reduction in stem cells compared to the wild type control (slope: −1.337±3.400e+038, p<0.0001 and slope: −0.08099±0.04816, p value compared to control <0.0001, respectively). Error bars indicate standard deviation (A, B). (C–F): Co-immunofluorescence of GLAST (red) and TuJ1 (green) on neocortical progenitors isolated from E14.5 Rbpj and Ptc1 mutant and wild types neocortices. Cells were counterstained with DAPI (blue). Arrows (in C, E, F) indicate TuJ1+ cells and arrowheads (D) indicate GLAST+ radial glial cells. RG cells from Ptc1Lox/Lox;NestinCre neocortex rarely differentiate into TuJ1+ neurons after 24 hours (D, G), whereas concomitant loss of the Rbpj and Ptc1 alleles results in an increased rate of neurogenesis in Ptc1Lox/Lox;RbpjLox/Lox;NestinCre neocortical cells (F, G). On the other hand, cells from RbpjLox/Lox;NestinCre neocortex mostly differentiate into TuJ1+ neurons after 24 hours. Also, (G): Quantitative analysis for GLAST and TuJ1 revealed that the percentage of RG cells undergoing symmetric neurogenic divisions was significantly increased (p<0.05) in the Ptc1Lox/Lox;RbpjLox/Lox;NestinCre compared to the Ptc1Lox/Lox;NestinCre neocortices. On the other hand, there was a reduction in the number of symmetric proliferative cell divisions in Ptc1Lox/Lox;RbpjLox/Lox;NestinCre compared to Ptc1Lox/Lox;NestinCre (p<0.05) samples. Bars represent standard errors. ***p<0.0001. Scale bar (C–F), 10 µm.
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pone-0014680-g002: Hh pathway activation expands the population of cells with stem cell-like properties and promotes symmetric proliferative divisions of radial glial cells.(A) E14.5 Ptc1Lox/Lox;NestinCre neocortex (n = 10) showed an 8-fold increase (***p<0.0001) in the number of primary neurosphere colonies per cell seeded compared to littermate controls (n = 11). For RbpjLox/Lox;NestinCre and Ptc1Lox/Lox;RbpjLox/Lox;NestinCre samples, n = 3 and n = 4 respectively. * and ** p<0.005 by unpaired Student's t-test (with Welch's correction). (B) Clonal expansion of neurospheres derived from E14.5 neocortex. Cell numbers obtained at each passage were transformed into LOG and linear regression analysis was performed on resulting curves. The rate of clonal expansion of the neurospheres is represented as the slope of the line. The number of stem cells was significantly increased in Ptc1Lox/Lox;NestinCre neurospheres compared to Ptc1Lox/Lox neurospheres (slope: 0.8877±0.06774 vs 0.7060±0.03302, p<0.05). However, RbpjLox/Lox;NestinCre and Ptc1Lox/Lox;RbpjLox/Lox;NestinCre neurospheres showed a significant reduction in stem cells compared to the wild type control (slope: −1.337±3.400e+038, p<0.0001 and slope: −0.08099±0.04816, p value compared to control <0.0001, respectively). Error bars indicate standard deviation (A, B). (C–F): Co-immunofluorescence of GLAST (red) and TuJ1 (green) on neocortical progenitors isolated from E14.5 Rbpj and Ptc1 mutant and wild types neocortices. Cells were counterstained with DAPI (blue). Arrows (in C, E, F) indicate TuJ1+ cells and arrowheads (D) indicate GLAST+ radial glial cells. RG cells from Ptc1Lox/Lox;NestinCre neocortex rarely differentiate into TuJ1+ neurons after 24 hours (D, G), whereas concomitant loss of the Rbpj and Ptc1 alleles results in an increased rate of neurogenesis in Ptc1Lox/Lox;RbpjLox/Lox;NestinCre neocortical cells (F, G). On the other hand, cells from RbpjLox/Lox;NestinCre neocortex mostly differentiate into TuJ1+ neurons after 24 hours. Also, (G): Quantitative analysis for GLAST and TuJ1 revealed that the percentage of RG cells undergoing symmetric neurogenic divisions was significantly increased (p<0.05) in the Ptc1Lox/Lox;RbpjLox/Lox;NestinCre compared to the Ptc1Lox/Lox;NestinCre neocortices. On the other hand, there was a reduction in the number of symmetric proliferative cell divisions in Ptc1Lox/Lox;RbpjLox/Lox;NestinCre compared to Ptc1Lox/Lox;NestinCre (p<0.05) samples. Bars represent standard errors. ***p<0.0001. Scale bar (C–F), 10 µm.
Mentions: In vivo and in vitro loss of function studies have shown that Hh signaling is required for the maintenance of stem cells in the developing neocortex [9], [11], [12], [21], [34]. To directly address the consequences of sustained Hh signaling in neocortical precursors, the neurosphere culture assay was utilised to test for stem and progenitor cell properties [29]. We observed a substantial (approximately 8-fold) increase in sphere forming potential of cells derived from the Ptc1Lox/Lox;NestinCre neocortex (2.154±0.267%) compared to Ptc1Lox/Lox neocortex (0.27±0.047%, p<0.0001,) (Fig. 2A) indicating that Hh pathway activation in vivo can regulate the total number of stem and progenitor cells. Next, the neurospheres were passaged multiple times to select against lineage-committed progenitor cells not capable of self-renewal. The rate of clonal expansion of the neurospheres is represented as the slope of the line. There was a clear difference in the rate of fold increase between Ptc1Lox/Lox and Ptc1Lox/Lox;NestinCre neocortical samples (clonal expansion of 0.7060±0.03302 vs. 0.8877±0.06774, p<0.05) (Fig. 2B) indicating that Hh pathway activation is mitogenic for cells that have a capacity to self-renew over an extended period of time. We also investigated whether the enlargement of the neocortical VZ progenitor population was a result of changes in cell cycle kinetics due to Ptc1 inactivation. If enlargement of the progenitor pool is due to shortening of the cell cycle, the proportion of progenitor cells labelled by a single 2-hour pulse of BrdU in the total population of cycling progenitors (Ki67+) would increase. Indeed, a significant shortening of the cell cycle in the mutant E14.5 neocortex was detected confirming that Ptc1 inactivated progenitor cells divide significantly faster than wild type progenitors (Fig. S3B). In the Ptc1Lox/Lox;NestinCre neocortex, the proportion of VZ progenitors co-labelled with BrdU/Ki67 was approximately two-fold greater when compared to wild type littermates (Fig. S3D). Altogether, our results suggest that Hh pathway activation can regulate neocortical stem and/or progenitor cell divisions and the rate of cell division in vivo.

Bottom Line: The Notch signaling pathway plays a pivotal role in the maintenance of stem/progenitor cells and the regulation of glial versus neuronal identity.To study the effect of Notch signaling on Hh-activated neural progenitors, we inactivated both Patched1 and Rbpj, a transcriptional mediator of Notch signaling, in Nestin positive cells of the neocortex.Hence, our results demonstrate that correct corticogenesis is an outcome of the interplay between the Hh and Notch signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: The Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia.

ABSTRACT

Background: Hedgehog (Hh) signaling is crucial for the generation and maintenance of both embryonic and adult stem cells, thereby regulating development and tissue homeostasis. In the developing neocortex, Sonic Hedgehog (Shh) regulates neural progenitor cell proliferation. During neurogenesis, radial glial cells of the ventricular zone (VZ) are the predominant neocortical progenitors that generate neurons through both symmetric and asymmetric divisions. Despite its importance, relatively little is known of the molecular pathways that control the switch from symmetric proliferative to differentiative/neurogenic divisions in neural progenitors.

Principal findings: Here, we report that conditional inactivation of Patched1, a negative regulator of the Shh pathway, in Nestin positive neural progenitors of the neocortex leads to lamination defects due to improper corticogenesis and an increase in the number of symmetric proliferative divisions of the radial glial cells. Hedgehog-activated VZ progenitor cells demonstrated a concomitant upregulation of Hes1 and Blbp, downstream targets of Notch signaling. The Notch signaling pathway plays a pivotal role in the maintenance of stem/progenitor cells and the regulation of glial versus neuronal identity. To study the effect of Notch signaling on Hh-activated neural progenitors, we inactivated both Patched1 and Rbpj, a transcriptional mediator of Notch signaling, in Nestin positive cells of the neocortex.

Conclusions: Our data indicate that by mid neurogenesis (embryonic day 14.5), attenuation of Notch signaling reverses the effect of Patched1 deletion on neurogenesis by restoring the balance between symmetric proliferative and neurogenic divisions. Hence, our results demonstrate that correct corticogenesis is an outcome of the interplay between the Hh and Notch signaling pathways.

Show MeSH
Related in: MedlinePlus