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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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Activated Hh pathway leads to neocortical defects in Ptc1Lox/Lox;NestinCre dorsal brain.In situ hybridisation on E14.5 sagittal sections demonstrating that both Ptc1 (A, B, Ai, Bi) and Gli1 (C, D, Ci, Di) transcripts are upregulated in VZ of Ptc1Lox/Lox;NestinCre neocortex compared to wild type Ptc1Lox/Lox littermate. (Ai–Di): shows a higher magnification of the wild type and mutant neocortex in A–D (boxed area). E: Taqman RT-PCR for detection of total Ptc1 transcript in microdissected VZ of wild type and mutant neocortex. The expression of Ptc1 was upregulated in E14.5 Ptc1Lox/Lox;NestinCre VZ compared to Ptc1Lox/Lox VZ, due to negative feedback mechanism. The data was normalised based on the expression of Gapdh and is presented as three independent pooled samples of each genotype. (F, G): Haematoxylin and Eosin stained coronal sections of E14.5 neocortex. Ptc1Lox/Lox;NestinCre neocortex is extensively folded and GE are distorted compared to Ptc1Lox/Lox (F). The regions of thickened neuroepithelium in the mutant are not consistent even in the same rostral/caudal plane (arrows) with regions of normal thickness also present (arrowheads). Scale bars (A–D, F–G), 100 µm; (Ai–Di), 50 µm. Abbreviations: CP, choroid plexus; GE, ganglionic eminence; LV, lateral ventricle; NC, neocortex; VZ, ventricular zone.
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pone-0014680-g001: Activated Hh pathway leads to neocortical defects in Ptc1Lox/Lox;NestinCre dorsal brain.In situ hybridisation on E14.5 sagittal sections demonstrating that both Ptc1 (A, B, Ai, Bi) and Gli1 (C, D, Ci, Di) transcripts are upregulated in VZ of Ptc1Lox/Lox;NestinCre neocortex compared to wild type Ptc1Lox/Lox littermate. (Ai–Di): shows a higher magnification of the wild type and mutant neocortex in A–D (boxed area). E: Taqman RT-PCR for detection of total Ptc1 transcript in microdissected VZ of wild type and mutant neocortex. The expression of Ptc1 was upregulated in E14.5 Ptc1Lox/Lox;NestinCre VZ compared to Ptc1Lox/Lox VZ, due to negative feedback mechanism. The data was normalised based on the expression of Gapdh and is presented as three independent pooled samples of each genotype. (F, G): Haematoxylin and Eosin stained coronal sections of E14.5 neocortex. Ptc1Lox/Lox;NestinCre neocortex is extensively folded and GE are distorted compared to Ptc1Lox/Lox (F). The regions of thickened neuroepithelium in the mutant are not consistent even in the same rostral/caudal plane (arrows) with regions of normal thickness also present (arrowheads). Scale bars (A–D, F–G), 100 µm; (Ai–Di), 50 µm. Abbreviations: CP, choroid plexus; GE, ganglionic eminence; LV, lateral ventricle; NC, neocortex; VZ, ventricular zone.

Mentions: To demonstrate activation of Hh signaling in mice homozygous for Nestin+ cell specific inactivation of the Ptc1 gene (Ptc1Lox/Lox;NestinCre ), the expression of Ptc1 and Gli1 transcripts [33] was analysed. Both Ptc1 and Gli1 are universal Hh targets and upon Ptc1 inactivation in Ptc1Lox/Lox;NestinCre mice an upregulation of both Ptc1 and Gli1 transcripts in the VZ of the developing E14.5 neocortex was observed (Fig. 1B, 1D, 1Ai–Di). Although we were unable to detect Ptc1 and Gli1 expression in the neocortex of Ptc1Lox/Lox mice by section in situ hybridisation (Fig. 1A, 1C), total Ptc1 transcript was detected by Taqman RT-PCR in microdissected VZ (Fig. 1E) and the deleted or mutant transcript was detected by SYBR Green RT-PCR (Fig. S1B). These results demonstrate that Ptc1 is normally expressed in the cortical VZ neural progenitors at low levels that are sufficient to reduce the Hh pathway activity and a consequence of Ptc1 inactivation is the generation of a robust physiological cell-autonomous Hh response.


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)

Activated Hh pathway leads to neocortical defects in Ptc1Lox/Lox;NestinCre dorsal brain.In situ hybridisation on E14.5 sagittal sections demonstrating that both Ptc1 (A, B, Ai, Bi) and Gli1 (C, D, Ci, Di) transcripts are upregulated in VZ of Ptc1Lox/Lox;NestinCre neocortex compared to wild type Ptc1Lox/Lox littermate. (Ai–Di): shows a higher magnification of the wild type and mutant neocortex in A–D (boxed area). E: Taqman RT-PCR for detection of total Ptc1 transcript in microdissected VZ of wild type and mutant neocortex. The expression of Ptc1 was upregulated in E14.5 Ptc1Lox/Lox;NestinCre VZ compared to Ptc1Lox/Lox VZ, due to negative feedback mechanism. The data was normalised based on the expression of Gapdh and is presented as three independent pooled samples of each genotype. (F, G): Haematoxylin and Eosin stained coronal sections of E14.5 neocortex. Ptc1Lox/Lox;NestinCre neocortex is extensively folded and GE are distorted compared to Ptc1Lox/Lox (F). The regions of thickened neuroepithelium in the mutant are not consistent even in the same rostral/caudal plane (arrows) with regions of normal thickness also present (arrowheads). Scale bars (A–D, F–G), 100 µm; (Ai–Di), 50 µm. Abbreviations: CP, choroid plexus; GE, ganglionic eminence; LV, lateral ventricle; NC, neocortex; VZ, ventricular zone.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040755&req=5

pone-0014680-g001: Activated Hh pathway leads to neocortical defects in Ptc1Lox/Lox;NestinCre dorsal brain.In situ hybridisation on E14.5 sagittal sections demonstrating that both Ptc1 (A, B, Ai, Bi) and Gli1 (C, D, Ci, Di) transcripts are upregulated in VZ of Ptc1Lox/Lox;NestinCre neocortex compared to wild type Ptc1Lox/Lox littermate. (Ai–Di): shows a higher magnification of the wild type and mutant neocortex in A–D (boxed area). E: Taqman RT-PCR for detection of total Ptc1 transcript in microdissected VZ of wild type and mutant neocortex. The expression of Ptc1 was upregulated in E14.5 Ptc1Lox/Lox;NestinCre VZ compared to Ptc1Lox/Lox VZ, due to negative feedback mechanism. The data was normalised based on the expression of Gapdh and is presented as three independent pooled samples of each genotype. (F, G): Haematoxylin and Eosin stained coronal sections of E14.5 neocortex. Ptc1Lox/Lox;NestinCre neocortex is extensively folded and GE are distorted compared to Ptc1Lox/Lox (F). The regions of thickened neuroepithelium in the mutant are not consistent even in the same rostral/caudal plane (arrows) with regions of normal thickness also present (arrowheads). Scale bars (A–D, F–G), 100 µm; (Ai–Di), 50 µm. Abbreviations: CP, choroid plexus; GE, ganglionic eminence; LV, lateral ventricle; NC, neocortex; VZ, ventricular zone.
Mentions: To demonstrate activation of Hh signaling in mice homozygous for Nestin+ cell specific inactivation of the Ptc1 gene (Ptc1Lox/Lox;NestinCre ), the expression of Ptc1 and Gli1 transcripts [33] was analysed. Both Ptc1 and Gli1 are universal Hh targets and upon Ptc1 inactivation in Ptc1Lox/Lox;NestinCre mice an upregulation of both Ptc1 and Gli1 transcripts in the VZ of the developing E14.5 neocortex was observed (Fig. 1B, 1D, 1Ai–Di). Although we were unable to detect Ptc1 and Gli1 expression in the neocortex of Ptc1Lox/Lox mice by section in situ hybridisation (Fig. 1A, 1C), total Ptc1 transcript was detected by Taqman RT-PCR in microdissected VZ (Fig. 1E) and the deleted or mutant transcript was detected by SYBR Green RT-PCR (Fig. S1B). These results demonstrate that Ptc1 is normally expressed in the cortical VZ neural progenitors at low levels that are sufficient to reduce the Hh pathway activity and a consequence of Ptc1 inactivation is the generation of a robust physiological cell-autonomous Hh response.

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