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Radial glia require PDGFD-PDGFRβ signalling in human but not mouse neocortex.

Lui JH, Nowakowski TJ, Pollen AA, Javaherian A, Kriegstein AR, Oldham MC - Nature (2014)

Bottom Line: Evolutionary expansion of the human neocortex underlies many of our unique mental abilities.However, whether or how RG gene expression varies between humans and other species is unknown.These findings highlight the requirement of PDGFD-PDGFRβ signalling for human neocortical development and suggest that local production of growth factors by RG supports the expanded germinal region and progenitor heterogeneity of species with large brains.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology and The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, California 94143, USA.

ABSTRACT
Evolutionary expansion of the human neocortex underlies many of our unique mental abilities. This expansion has been attributed to the increased proliferative potential of radial glia (RG; neural stem cells) and their subventricular dispersion from the periventricular niche during neocortical development. Such adaptations may have evolved through gene expression changes in RG. However, whether or how RG gene expression varies between humans and other species is unknown. Here we show that the transcriptional profiles of human and mouse neocortical RG are broadly conserved during neurogenesis, yet diverge for specific signalling pathways. By analysing differential gene co-expression relationships between the species, we demonstrate that the growth factor PDGFD is specifically expressed by RG in human, but not mouse, corticogenesis. We also show that the expression domain of PDGFRβ, the cognate receptor for PDGFD, is evolutionarily divergent, with high expression in the germinal region of dorsal human neocortex but not in the mouse. Pharmacological inhibition of PDGFD-PDGFRβ signalling in slice culture prevents normal cell cycle progression of neocortical RG in human, but not mouse. Conversely, injection of recombinant PDGFD or ectopic expression of constitutively active PDGFRβ in developing mouse neocortex increases the proportion of RG and their subventricular dispersion. These findings highlight the requirement of PDGFD-PDGFRβ signalling for human neocortical development and suggest that local production of growth factors by RG supports the expanded germinal region and progenitor heterogeneity of species with large brains.

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PDGFD/PDGFRß signaling is necessary for normal cell cycle progression of neocortical RG in humans and sufficient to promote RG identity in micea, GW17.5 human neocortical slice cultures were treated with BrdU and DMSO (control) or an inhibitor of PDGFRß signaling (CP673451) (scale bar 50 μm). The same experiment was performed in E13.5 mouse neocortical slice cultures (slices from at least 3 individuals/litters per species). b, RG (IP) proliferation was quantified as the fraction of SOX2+ (TBR2+) cells that incorporated BrdU after 48 hours. RG slice counts: human (n = 18 [DMSO] vs. n = 17 [CP673451]); mouse (n = 13 [DMSO] vs. n = 11 [CP673451]). IP slice counts: human (n = 12 [DMSO] vs. n = 10 [CP673451]); mouse (n = 11 [DMSO] vs. n = 9 [CP673451]). Cell death was quantified in human slices as the fraction of SOX2+ or BrdU+ cells that co-stained for cleaved-caspase 3 (n = 6 [DMSO] vs. n = 7 [CP673451]). c,In utero intraventricular injection of recombinant human PDGF-DD protein (mouse E13.5-E15.5). Brain tissue was stained for SOX2 and DAPI (scale bar 50 μm). d, Quantification of data from c in dorsomedial and lateral cortex (at least n = 3 slices per embryo from 5 litters/experiments [lateral: n = 49 vehicle; n = 47 PDGF-DD; dorsomedial: n = 45 vehicle; n = 39 PDGF-DD]). The distribution of RG in the cortex (from ventricle to pia) was quantified; grey band delineates 95% confidence interval for test of equal univariate densities (n = 10,000 permutations). e,In utero electroporation of constitutively active PDGFRβ:D850V23 (mouse E13.5-E15.5). Cortex was stained for SOX2; white arrowheads indicate co-labeling with electroporated GFP cells (quantified in f: at least n = 3 slices per embryo from 2 litters; n = 15 [control], n = 18 [PDGFRβ:D850V]; scale bar 50 μm). Note disrupted epithelial structure of VZ. Error bars = mean +/- s.e.m. Statistical significance for treatment was determined by ANOVA of multiple linear regression after controlling for individual (b) or litter (d, f) (n.s. P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001). g, Schematic summarizing experimental manipulations and results. LOF: loss-offunction, GOF: gain-of-function.
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Figure 13: PDGFD/PDGFRß signaling is necessary for normal cell cycle progression of neocortical RG in humans and sufficient to promote RG identity in micea, GW17.5 human neocortical slice cultures were treated with BrdU and DMSO (control) or an inhibitor of PDGFRß signaling (CP673451) (scale bar 50 μm). The same experiment was performed in E13.5 mouse neocortical slice cultures (slices from at least 3 individuals/litters per species). b, RG (IP) proliferation was quantified as the fraction of SOX2+ (TBR2+) cells that incorporated BrdU after 48 hours. RG slice counts: human (n = 18 [DMSO] vs. n = 17 [CP673451]); mouse (n = 13 [DMSO] vs. n = 11 [CP673451]). IP slice counts: human (n = 12 [DMSO] vs. n = 10 [CP673451]); mouse (n = 11 [DMSO] vs. n = 9 [CP673451]). Cell death was quantified in human slices as the fraction of SOX2+ or BrdU+ cells that co-stained for cleaved-caspase 3 (n = 6 [DMSO] vs. n = 7 [CP673451]). c,In utero intraventricular injection of recombinant human PDGF-DD protein (mouse E13.5-E15.5). Brain tissue was stained for SOX2 and DAPI (scale bar 50 μm). d, Quantification of data from c in dorsomedial and lateral cortex (at least n = 3 slices per embryo from 5 litters/experiments [lateral: n = 49 vehicle; n = 47 PDGF-DD; dorsomedial: n = 45 vehicle; n = 39 PDGF-DD]). The distribution of RG in the cortex (from ventricle to pia) was quantified; grey band delineates 95% confidence interval for test of equal univariate densities (n = 10,000 permutations). e,In utero electroporation of constitutively active PDGFRβ:D850V23 (mouse E13.5-E15.5). Cortex was stained for SOX2; white arrowheads indicate co-labeling with electroporated GFP cells (quantified in f: at least n = 3 slices per embryo from 2 litters; n = 15 [control], n = 18 [PDGFRβ:D850V]; scale bar 50 μm). Note disrupted epithelial structure of VZ. Error bars = mean +/- s.e.m. Statistical significance for treatment was determined by ANOVA of multiple linear regression after controlling for individual (b) or litter (d, f) (n.s. P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001). g, Schematic summarizing experimental manipulations and results. LOF: loss-offunction, GOF: gain-of-function.

Mentions: We tested the requirement of PDGFD/PDGFRß signaling for hRG proliferation in GW17.5 human neocortical slice cultures, screening four chemical inhibitors of PDGFRß signaling (Sutent, Tivozanib, Imatinib, and CP673451). Three out of four PDGFRß inhibitors reduced the percentage of SOX2+ progenitors (RG) that incorporated BrdU over two days in slice culture (Extended Data Fig. 9). For replication we focused on CP673451, which exhibits the greatest selectivity for PDGFRβ over other receptors22 and caused the greatest reduction in SOX2+BrdU+ cells among tested inhibitors (Extended Data Fig. 9). PDGFRß inhibition by CP673451 in GW17.5 human neocortical slice cultures reduced the number of RG and intermediate progenitors (IP) that incorporated BrdU by >50%, affecting progenitors in the VZ and SVZ (Fig. 4a-b). The percentage of progenitor cells co-staining with cleaved caspase-3, an apoptosis marker, was slightly elevated by CP673451, but sufficiently low to attribute reduced BrdU incorporation to cell cycle dysregulation rather than cell death (Fig. 4b). Furthermore, CP673451 treatment of E13.5 mouse cortical slice cultures did not decrease BrdU incorporation or the cycling proportion (Ki67+) of RG or IP populations over multiple time points (Fig. 4b, Extended Data Fig. 9). These results indicate that PDGFRß signaling is required for hRG but not mRG to progress through the cell cycle and expand at a normal rate.


Radial glia require PDGFD-PDGFRβ signalling in human but not mouse neocortex.

Lui JH, Nowakowski TJ, Pollen AA, Javaherian A, Kriegstein AR, Oldham MC - Nature (2014)

PDGFD/PDGFRß signaling is necessary for normal cell cycle progression of neocortical RG in humans and sufficient to promote RG identity in micea, GW17.5 human neocortical slice cultures were treated with BrdU and DMSO (control) or an inhibitor of PDGFRß signaling (CP673451) (scale bar 50 μm). The same experiment was performed in E13.5 mouse neocortical slice cultures (slices from at least 3 individuals/litters per species). b, RG (IP) proliferation was quantified as the fraction of SOX2+ (TBR2+) cells that incorporated BrdU after 48 hours. RG slice counts: human (n = 18 [DMSO] vs. n = 17 [CP673451]); mouse (n = 13 [DMSO] vs. n = 11 [CP673451]). IP slice counts: human (n = 12 [DMSO] vs. n = 10 [CP673451]); mouse (n = 11 [DMSO] vs. n = 9 [CP673451]). Cell death was quantified in human slices as the fraction of SOX2+ or BrdU+ cells that co-stained for cleaved-caspase 3 (n = 6 [DMSO] vs. n = 7 [CP673451]). c,In utero intraventricular injection of recombinant human PDGF-DD protein (mouse E13.5-E15.5). Brain tissue was stained for SOX2 and DAPI (scale bar 50 μm). d, Quantification of data from c in dorsomedial and lateral cortex (at least n = 3 slices per embryo from 5 litters/experiments [lateral: n = 49 vehicle; n = 47 PDGF-DD; dorsomedial: n = 45 vehicle; n = 39 PDGF-DD]). The distribution of RG in the cortex (from ventricle to pia) was quantified; grey band delineates 95% confidence interval for test of equal univariate densities (n = 10,000 permutations). e,In utero electroporation of constitutively active PDGFRβ:D850V23 (mouse E13.5-E15.5). Cortex was stained for SOX2; white arrowheads indicate co-labeling with electroporated GFP cells (quantified in f: at least n = 3 slices per embryo from 2 litters; n = 15 [control], n = 18 [PDGFRβ:D850V]; scale bar 50 μm). Note disrupted epithelial structure of VZ. Error bars = mean +/- s.e.m. Statistical significance for treatment was determined by ANOVA of multiple linear regression after controlling for individual (b) or litter (d, f) (n.s. P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001). g, Schematic summarizing experimental manipulations and results. LOF: loss-offunction, GOF: gain-of-function.
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Figure 13: PDGFD/PDGFRß signaling is necessary for normal cell cycle progression of neocortical RG in humans and sufficient to promote RG identity in micea, GW17.5 human neocortical slice cultures were treated with BrdU and DMSO (control) or an inhibitor of PDGFRß signaling (CP673451) (scale bar 50 μm). The same experiment was performed in E13.5 mouse neocortical slice cultures (slices from at least 3 individuals/litters per species). b, RG (IP) proliferation was quantified as the fraction of SOX2+ (TBR2+) cells that incorporated BrdU after 48 hours. RG slice counts: human (n = 18 [DMSO] vs. n = 17 [CP673451]); mouse (n = 13 [DMSO] vs. n = 11 [CP673451]). IP slice counts: human (n = 12 [DMSO] vs. n = 10 [CP673451]); mouse (n = 11 [DMSO] vs. n = 9 [CP673451]). Cell death was quantified in human slices as the fraction of SOX2+ or BrdU+ cells that co-stained for cleaved-caspase 3 (n = 6 [DMSO] vs. n = 7 [CP673451]). c,In utero intraventricular injection of recombinant human PDGF-DD protein (mouse E13.5-E15.5). Brain tissue was stained for SOX2 and DAPI (scale bar 50 μm). d, Quantification of data from c in dorsomedial and lateral cortex (at least n = 3 slices per embryo from 5 litters/experiments [lateral: n = 49 vehicle; n = 47 PDGF-DD; dorsomedial: n = 45 vehicle; n = 39 PDGF-DD]). The distribution of RG in the cortex (from ventricle to pia) was quantified; grey band delineates 95% confidence interval for test of equal univariate densities (n = 10,000 permutations). e,In utero electroporation of constitutively active PDGFRβ:D850V23 (mouse E13.5-E15.5). Cortex was stained for SOX2; white arrowheads indicate co-labeling with electroporated GFP cells (quantified in f: at least n = 3 slices per embryo from 2 litters; n = 15 [control], n = 18 [PDGFRβ:D850V]; scale bar 50 μm). Note disrupted epithelial structure of VZ. Error bars = mean +/- s.e.m. Statistical significance for treatment was determined by ANOVA of multiple linear regression after controlling for individual (b) or litter (d, f) (n.s. P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001). g, Schematic summarizing experimental manipulations and results. LOF: loss-offunction, GOF: gain-of-function.
Mentions: We tested the requirement of PDGFD/PDGFRß signaling for hRG proliferation in GW17.5 human neocortical slice cultures, screening four chemical inhibitors of PDGFRß signaling (Sutent, Tivozanib, Imatinib, and CP673451). Three out of four PDGFRß inhibitors reduced the percentage of SOX2+ progenitors (RG) that incorporated BrdU over two days in slice culture (Extended Data Fig. 9). For replication we focused on CP673451, which exhibits the greatest selectivity for PDGFRβ over other receptors22 and caused the greatest reduction in SOX2+BrdU+ cells among tested inhibitors (Extended Data Fig. 9). PDGFRß inhibition by CP673451 in GW17.5 human neocortical slice cultures reduced the number of RG and intermediate progenitors (IP) that incorporated BrdU by >50%, affecting progenitors in the VZ and SVZ (Fig. 4a-b). The percentage of progenitor cells co-staining with cleaved caspase-3, an apoptosis marker, was slightly elevated by CP673451, but sufficiently low to attribute reduced BrdU incorporation to cell cycle dysregulation rather than cell death (Fig. 4b). Furthermore, CP673451 treatment of E13.5 mouse cortical slice cultures did not decrease BrdU incorporation or the cycling proportion (Ki67+) of RG or IP populations over multiple time points (Fig. 4b, Extended Data Fig. 9). These results indicate that PDGFRß signaling is required for hRG but not mRG to progress through the cell cycle and expand at a normal rate.

Bottom Line: Evolutionary expansion of the human neocortex underlies many of our unique mental abilities.However, whether or how RG gene expression varies between humans and other species is unknown.These findings highlight the requirement of PDGFD-PDGFRβ signalling for human neocortical development and suggest that local production of growth factors by RG supports the expanded germinal region and progenitor heterogeneity of species with large brains.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology and The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, California 94143, USA.

ABSTRACT
Evolutionary expansion of the human neocortex underlies many of our unique mental abilities. This expansion has been attributed to the increased proliferative potential of radial glia (RG; neural stem cells) and their subventricular dispersion from the periventricular niche during neocortical development. Such adaptations may have evolved through gene expression changes in RG. However, whether or how RG gene expression varies between humans and other species is unknown. Here we show that the transcriptional profiles of human and mouse neocortical RG are broadly conserved during neurogenesis, yet diverge for specific signalling pathways. By analysing differential gene co-expression relationships between the species, we demonstrate that the growth factor PDGFD is specifically expressed by RG in human, but not mouse, corticogenesis. We also show that the expression domain of PDGFRβ, the cognate receptor for PDGFD, is evolutionarily divergent, with high expression in the germinal region of dorsal human neocortex but not in the mouse. Pharmacological inhibition of PDGFD-PDGFRβ signalling in slice culture prevents normal cell cycle progression of neocortical RG in human, but not mouse. Conversely, injection of recombinant PDGFD or ectopic expression of constitutively active PDGFRβ in developing mouse neocortex increases the proportion of RG and their subventricular dispersion. These findings highlight the requirement of PDGFD-PDGFRβ signalling for human neocortical development and suggest that local production of growth factors by RG supports the expanded germinal region and progenitor heterogeneity of species with large brains.

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Related in: MedlinePlus