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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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Loss of Rbpj leads to disruption of neocortical patterning.Immunostaining of E14.5 neocortex using TuJ1 (red) (A–H); (green), (I–P) and Nestin (green) (A–H) or Sox2 (red) (I–P) antibodies. High magnification confocal images of the medial region of coronal sections from A–D, I–L are shown in E–H, M–P respectively. The zone of cells that are Sox2 and Nestin positive is expanded in Ptc1Lox/Lox;NestinCre and Ptc1Lox/Lox;RbpjLox/Lox;NestinCre samples. Sox2+ cells were even present in the CP (arrowheads in N). TuJ1+ cells were present throughout the RbpjLox/Lox;NestinCre neocortex, from the VZ to the pial surface. Abbreviations: CP, cortical plate; CP', choroid plexus; VZ, ventricular zone. Scale bar (A–D, I–L), 100 µm; (E–H, M–P), 20 µm.
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pone-0014680-g004: Loss of Rbpj leads to disruption of neocortical patterning.Immunostaining of E14.5 neocortex using TuJ1 (red) (A–H); (green), (I–P) and Nestin (green) (A–H) or Sox2 (red) (I–P) antibodies. High magnification confocal images of the medial region of coronal sections from A–D, I–L are shown in E–H, M–P respectively. The zone of cells that are Sox2 and Nestin positive is expanded in Ptc1Lox/Lox;NestinCre and Ptc1Lox/Lox;RbpjLox/Lox;NestinCre samples. Sox2+ cells were even present in the CP (arrowheads in N). TuJ1+ cells were present throughout the RbpjLox/Lox;NestinCre neocortex, from the VZ to the pial surface. Abbreviations: CP, cortical plate; CP', choroid plexus; VZ, ventricular zone. Scale bar (A–D, I–L), 100 µm; (E–H, M–P), 20 µm.

Mentions: Several reports have suggested the importance of Notch signaling in controlling asymmetric/symmetric divisions in neural stem and progenitor cells (reviewed in [30], [38]). Both in vivo and in vitro studies have established that Notch1 and Notch3 activation promote radial glial cell fate [14], [39] and inactivation of Notch1 signaling leads to precocious neuronal differentiation [40]. Since our data indicate that Hedgehog pathway activation may play a similar role in the same cell types we next asked whether Notch and Hedgehog signaling interact in the regulation of homeostasis in the VZ. To examine whether the increase in the number of radial glial cells in Ptc1Lox/Lox;NestinCre neocortex required Notch signaling, inactivation of Rbpj and Ptc1 was carried out concomitantly in Nestin+ neural progenitors. Forebrains from the control genotypes (Ptc1Lox/Lox, Ptc1Lox/Lox;Rbpj+/Lox, Ptc1Lox/Lox;RbpjLox/Lox) were examined and demonstrated normal neocortical development, although only Ptc1Lox/Lox neocortex samples are presented as a control in Figures 3 and 4. Whole mounts of E14.5 embryos from the RbpjLox/Lox;NestinCre and Ptc1Lox/Lox;RbpjLox/Lox;NestinCre mice showed extensive hemorrhage in the neocortex and tectum (Fig. 3C, 3D, Fig. S5), possibly due to loss of Rbpj from Nestin+ endothelial cells, however this phenotype has not been reported for the Rbpj-conditional knockout mice generated using inducible Nestin-Cre [41]. Haematoxylin and Eosin staining of E14.5 forebrains clearly demonstrated a patterning defect in the Ptc1-inactivated, Rbpj-inactivated and the double mutants (Fig. 3E–H). The diencephalon was missing in the RbpjLox/Lox;NestinCre forebrains (Fig. 3G) and the lateral ventricles of the Ptc1Lox/Lox;RbpjLox/Lox;NestinCre forebrains were enlarged (Fig. 3H). The neocortex of double mutant mice was much thinner and the neocortical structure was disrupted in contrast to the wild type neocortex, however, the VZ/SVZ was reduced compared to the Ptc1-deleted neocortex (Fig. 3L, 3P).


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)

Loss of Rbpj leads to disruption of neocortical patterning.Immunostaining of E14.5 neocortex using TuJ1 (red) (A–H); (green), (I–P) and Nestin (green) (A–H) or Sox2 (red) (I–P) antibodies. High magnification confocal images of the medial region of coronal sections from A–D, I–L are shown in E–H, M–P respectively. The zone of cells that are Sox2 and Nestin positive is expanded in Ptc1Lox/Lox;NestinCre and Ptc1Lox/Lox;RbpjLox/Lox;NestinCre samples. Sox2+ cells were even present in the CP (arrowheads in N). TuJ1+ cells were present throughout the RbpjLox/Lox;NestinCre neocortex, from the VZ to the pial surface. Abbreviations: CP, cortical plate; CP', choroid plexus; VZ, ventricular zone. Scale bar (A–D, I–L), 100 µm; (E–H, M–P), 20 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040755&req=5

pone-0014680-g004: Loss of Rbpj leads to disruption of neocortical patterning.Immunostaining of E14.5 neocortex using TuJ1 (red) (A–H); (green), (I–P) and Nestin (green) (A–H) or Sox2 (red) (I–P) antibodies. High magnification confocal images of the medial region of coronal sections from A–D, I–L are shown in E–H, M–P respectively. The zone of cells that are Sox2 and Nestin positive is expanded in Ptc1Lox/Lox;NestinCre and Ptc1Lox/Lox;RbpjLox/Lox;NestinCre samples. Sox2+ cells were even present in the CP (arrowheads in N). TuJ1+ cells were present throughout the RbpjLox/Lox;NestinCre neocortex, from the VZ to the pial surface. Abbreviations: CP, cortical plate; CP', choroid plexus; VZ, ventricular zone. Scale bar (A–D, I–L), 100 µm; (E–H, M–P), 20 µm.
Mentions: Several reports have suggested the importance of Notch signaling in controlling asymmetric/symmetric divisions in neural stem and progenitor cells (reviewed in [30], [38]). Both in vivo and in vitro studies have established that Notch1 and Notch3 activation promote radial glial cell fate [14], [39] and inactivation of Notch1 signaling leads to precocious neuronal differentiation [40]. Since our data indicate that Hedgehog pathway activation may play a similar role in the same cell types we next asked whether Notch and Hedgehog signaling interact in the regulation of homeostasis in the VZ. To examine whether the increase in the number of radial glial cells in Ptc1Lox/Lox;NestinCre neocortex required Notch signaling, inactivation of Rbpj and Ptc1 was carried out concomitantly in Nestin+ neural progenitors. Forebrains from the control genotypes (Ptc1Lox/Lox, Ptc1Lox/Lox;Rbpj+/Lox, Ptc1Lox/Lox;RbpjLox/Lox) were examined and demonstrated normal neocortical development, although only Ptc1Lox/Lox neocortex samples are presented as a control in Figures 3 and 4. Whole mounts of E14.5 embryos from the RbpjLox/Lox;NestinCre and Ptc1Lox/Lox;RbpjLox/Lox;NestinCre mice showed extensive hemorrhage in the neocortex and tectum (Fig. 3C, 3D, Fig. S5), possibly due to loss of Rbpj from Nestin+ endothelial cells, however this phenotype has not been reported for the Rbpj-conditional knockout mice generated using inducible Nestin-Cre [41]. Haematoxylin and Eosin staining of E14.5 forebrains clearly demonstrated a patterning defect in the Ptc1-inactivated, Rbpj-inactivated and the double mutants (Fig. 3E–H). The diencephalon was missing in the RbpjLox/Lox;NestinCre forebrains (Fig. 3G) and the lateral ventricles of the Ptc1Lox/Lox;RbpjLox/Lox;NestinCre forebrains were enlarged (Fig. 3H). The neocortex of double mutant mice was much thinner and the neocortical structure was disrupted in contrast to the wild type neocortex, however, the VZ/SVZ was reduced compared to the Ptc1-deleted neocortex (Fig. 3L, 3P).

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