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

Abstract

Evolutionary expansion of the human neocortex underlies many of our unique mental abilities. This expansion has been attributed to the increased proliferative potential1,2 of radial glia (RG; neural stem cells) and their subventricular dispersion from the periventricular niche3-5 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 receptor6,7 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.

Headnote

Evolutionary expansion of the human neocortex underlies many of our unique mental abilities. This expansion has been attributed to the increased proliferative potential1,2 of radial glia (RG; neural stem cells) and their subventricular dispersion from the periventricular niche3-5 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 receptor6,7 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.

Radial glia are the physical substrate8 and progenitor population that underlie production of most cells in human neocortex2. We sought to determine a general transcriptional 'signature' of human neocortical RG (hRG) as a starting point for identifying genes that may regulate uniquely human aspects of cortical development. We and others have previously shown that gene co-expression analysis of heterogeneous tissue samples can deconvolve transcriptional signatures of distinct cell types without cell isolation or purification9,10. Because prenatal samples of human neo- cortex are scarce, we developed a novel strategy called Gene Co-expression Analysis of Serial Sections (GCASS) that exploits variation in cellular abundance across serial sections of a single tissue sample to reveal cell- type-specific patterns of gene expression (Fig. la-c and Extended Data Fig. 1; see Supplementary Information for methods, rationale and fur- ther discussion). We applied GCASS to 87 150-pm sections of a single human cortical sample from gestational week 14.5 (GW14.5, corres- ponding to peak layer V neurogenesis11; Supplementary Table 1) and identified 55 modules of co-expressed genes. Six modules overlapped significantly with a set of genes that we determined were expressed sig- nificantly higher in fluorescence-activated cell sorting (FACS)-sorted mouse RG (mRG) versus intermediate progenitor cells (FACS mRG; Extended Data Fig. 1 and Supplementary Table 2), suggesting that they might represent transcriptional signatures of hRG (Fig. Id). Analysis of laser-microdissected samples from three independent transcriptomic data sets12,13 confirmed that genes in these modules are most highly expressed in the ventricular zone (VZ) and subventricular zone (SVZ) of developing human neocortex, where both ventricular (vRG) and outer subventricular (oRG) subtypes of RG reside4 (Extended Data Fig. 2).

To produce a consensus transcriptional signature for GW 14.5 hRG, we first summarized each of these six modules by its first principal com- ponent/module eigengene14,15 (ME) and calculated the Weighted Gene Co-expression Network Analysis16 measure of intramodular gene con- nectivity, /cme10,14 (see Fig. lc). kME quantifies the extent to which a gene conforms to the characteristic expression pattern of a module and can predict gene expression specificity for individual cell types10. /cME values for the six modules were combined into a single measure (ZhRG), with higher values predicting greater expression specificity for hRG (Fig. le). Genes with high ZhRG values included markers of neocortical RG such as SLC1A3 (GLAST1), VIM, SOX2y NOTCH1 and PAX6 (Fig. le, blue lines). Genes with low ZhRG values included markers of committed neu- ronal lineages such as TBRly FEZF2y RELN and SATB2 (Fig. le, black lines). We performed in situ hybridization (ISH) and immunostaining on independent prenatal human neocortical samples for genes with high ZhRG values that have not, to our knowledge, previously been implicated in RG biology (Fig. le, red lines and Extended Data Fig. 3). In all cases, expression of these genes was restricted to the VZ/SVZ (Fig. If and Extended Data Fig. 3). These results indicate that GCASS can discern a general transcriptional signature of hRG from a single, heterogeneous tissue sample without cell labelling, isolation, or purification. Moreover, because the sample derives from a single individual, this strategy impli- citly controls for genotype and developmental stage and has broad impli- cations for the molecular analysis of rare tissue samples.

To establish the robustness of the hRG transcriptional signature, we analysed four additional prenatal human cortex gene expression data sets12,13 (see also http://www.brainspan.org/rnaseq/search/index.html) that were generated with diverse sampling strategies and technology plat- forms (Extended Data Table 1). In parallel, we also analysed three embry- onic mouse cortex gene expression data sets12,17,18 (largely embryonic day (E) 14-14.5, corresponding to peak layer V neurogenesis1 Extended Data Table 1) to establish a robust mRG transcriptional signature. For each data set, we constructed an unsupervised co-expression network, identified the module with the most significant overlap with the FACS mRG gene set, and calculated kME values (RG kME) for every gene with respect to this RG module (Supplementary Table 3). To facilitate com- parisons of RG transcriptional signatures, we mapped all probe sets/ transcripts to a common identifier (HomoloGene ID) and converted RG kME values for each data set into percentile ranks (RG PR; workflow schematic, Extended Data Fig. 4). Genome-wide correlations of RG PR among all data sets ranged from 0.36 to 0.95 (human) and 0.47 to 0.70 (mouse) (P< 2.2 X 10-16; Extended Data Fig. 5), demonstrating the robustness of RG transcriptional signatures in both species.

To explore the global extent of gene expression conservation between hRG and mRG, we calculated the mean RG specificity for each gene across the five human (hRG PR) three mouse (mRG PR) and all eight (RG PR) data sets (Supplementary Table 3). For genes present in at least one data set in each species (n = 15,576), the correlation between hRG PR and mRG PR was 0.48 (P < 2.2X10-16; Fig. 2a), indicating broad conservation of transcriptional programs that are active during cortical development in humans and mice. Genes with hRG PR and mRG PR >80 (Fig. 2a, +RG box) or <20 (Fig. 2a, - RG box) were significantly enriched with sets of genes expressed by RG or neurons, respectively (Supplementary Table 4). Furthermore, the most conserved set of core RG genes (hRG PR and mRG PR >95; Fig. 2a, green box) included the canonical RG markers VIM and PAX6y and SOX family members implicated in nervous system development such as SOX2y SOX3y SOX9 and SOX21 (Fig. 2b). Also present among this core set were elements of the Notch (NOTCH2 and HES1) and Wnt-ß-catenin (SFRPly FZD8 and LRP4) signalling pathways, which critically regulate neurogenesis2. In addition, we observed a cohesive subgroup of genes involved in cell cycle regulation, including CKS2y RACGAPR MELKy CCNBly CCNA2y ASPM and MKI67. Finally, several genes in this group have not, to our knowledge, previously been implicated in RG biology, including PSATly DDAHly AIFlLy PSRC1 and ACSSL Together, these results provide a framework for identifying conserved and distinct aspects of gene expression in hRG and mRG.

To identify evolutionary changes in RG gene expression that might underlie neurodevelopmental differences between human and mouse, we assessed homologous genes based on predicted differences in RG expression specificity between the species (differential RG specificity (DS) = hRG PR - mRG PR). Because such differences could emerge from gene expression changes in RG or neighbouring cell types (Fig. 2c), we also compared the relative expression levels of homologous genes between the species (differential expression (DE) = hEx PR - mEx PR ). Genome-wide analysis of DS/DE revealed four distinct quadrants of genes that differed substantially between human and mouse (Fig. 2d). Consistent with the proposed model (Fig. 2c), these quadrants were enriched with sets of genes expressed in distinct patterns in developing human and mouse cortex (Supplementary Table 4).

We reasoned that quadrant 1 might contain genes with 'Boolean5 expression differences (that is, ON in hRG and OFF in mRG) required for human but not mouse neocortical development. We applied stringent criteria to identify and validate 18 candidate genes in this quadrant with strong and consistent evidence of expression in hRG and no evidence of expression in mRG: ABHD3y ASAP3y BMP7y C5y C8orf4y FAM107Ay FOXN4y ITGA2y LRIG3y LRRC17y PAMy PDGFDy PDLIM3y RFTN2y SLC2A10, SP110y STOX1 an<3ZC3HAVl (Fig. 2d, cyan circles; see also Extended Data Fig. 6). Because secreted growth factors could alter the size of the developing neocortex by influencing proliferation, we focused on PDGFD (Fig. 2d (right panel), green square), which has not, to our knowledge, previously been implicated in cortical development of any species. Compared to genes with the highest RG PR (conserved in both species), PDGFD expression was highly correlated in human, but not mouse (Fig. 3a, b). ISH confirmed that PDGFD was expressed by RG throughout the VZ of GW 14.5 human neocortex (Fig. 3c). In contrast, Pdgfd was not detected in RG (or any cell type) in El5.5 mouse neocor- tex (Fig. 3d). These expression differences were consistent for multiple ages in human (GW14.5-GW18.2) and mouse (E14.5-E17.5) prenatal cortex (Extended Data Fig. 7).

The effects of PDGFD are specifically mediated by the PDGFRß receptor, which upon phosphorylation can trigger signalling pathways that promote cell proliferation6,7,19. Although PDGFRB did not meet the same stringent criteria as PDGFD, its location on the DS/DE plot was proximal to PDGFD (Fig. 2d (right panel), red square), also suggesting species differences in PDGFRB expression. Compared to genes with the highest RG PR, PDGFRB expression was moderately correlated in human, but not mouse (Fig. 3e, f). Immunostaining for PDGFRß in GW 14.5 human brain revealed strong expression throughout the telen- cephalic germinal zones (VZ/SVZ) and in vascular pericytes, with highest levels in dorsolateral cortical progenitors and the lateral ganglionic emi- nence (Fig. 3g). In contrast, immunostaining for PDGFRß in El5.5 mouse brain revealed expression in lateral ganglionic eminence pro- genitors but no evidence of expression in cortical progenitors (Fig. 3h). However, we did observe very low levels of Pdgfrb transcript in the VZ of lateral mouse cortex20 (Extended Data Fig. 8), leaving open the pos- sibility of modest, region-specific function. Collectively, these results indicate that expression patterns of PDGFD and PDGFRß in develop- ing neocortex have diverged considerably during human and mouse evolution, despite retaining amino acid sequences that are -85% iden- tical between the species.

We tested the requirement of PDGFD-PDGFRß signalling for hRG proliferation in GW 17.5 human neocortical slice cultures, screening four chemical inhibitors of PDGFRß signalling (Sutent, tivozanib, imatinib and CP673451 ). Three out of four PDGFRß inhibitors reduced the per- centage of SOX2+ progenitors (RG) that incorporated 5-bromodeox- yuridine (BrdU) over 2 days in slice culture (Extended Data Fig. 9). For replication we focused on CP673451, which exhibits the greatest select- ivity for PDGFRß over other receptors21 and caused the greatest reduction in SOX2+BrdU+ cells among tested inhibitors (Extended Data Fig. 9). PDGFRß inhibition by CP673451 in GW 17.5 human neocortical slice cultures reduced the number of RG and intermediate progenitors 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 dysrégulation rather than cell death (Fig. 4b). Furthermore, CP673451 treatment of El 3.5 mouse cortical slice cultures did not decrease BrdU incorporation or the cycling proportion (Ki67+) ofRG or intermediate progenitor populations over multiple time points (Fig. 4b and Extended Data Fig. 9). These results indicate that PDGFRß signalling is required for hRG but not mRG to progress through the cell cycle and expand at a normal rate.

We next investigated whether introducing PDGFD into embryonic mouse cortex, where it is normally absent, could promote mRG prolif- eration. We injected recombinant PDGF-DD protein into the lateral ventricles of E13.5 mouse embryos, bypassing the need for PDGFD to be generated and dimerized in vivo, and analysed the number and spa- tial distribution of SOX2+ progenitors at El5.5 in lateral cortex. Relative to vehicle, PDGF-DD increased the proportion of RG (SOX2+DAPI+) in lateral cortex by ~ 10% (Fig. 4c, d). Furthermore, PDGF-DD induced a modest subventricular shift in the distribution of mRG in the develop- ing cortical wall (Fig. 4d). These effects were not observed in dorsomedial cortex, where PDGFRß was not detected in RG (Fig. 4c, d and Extended Data Fig. 8).

To test whether mRG can respond to PDGFRß activation, we ectopicaUy expressed two forms of constitutively active PDGFRß (PDGFRß(D850V) (homologous to human D849V (ref. 22)) or the TEL-PDGFRß (ref. 23) fusion protein) in mRG by in utero electroporation at E13.5. By E15.5, expression of PDGFRß(D850V) approximately doubled the propor- tion of SOX2+ or Ki67+ progenitors among electroporated (GFP+) cells and markedly dispersed SOX2 + progenitors in the basal direction (Fig. 4e, f and Extended Data Fig. 9). Similar but less marked effects were observed after TEL-PDGFRß electroporation (Extended Data Fig. 9). These results indicate that PDGFRß signalling in mRG can function analogously to its known role as an oncogenic pathway that promotes proliferation and epithelial-mesenchymal transition19. We therefore propose that physiological levels of PDGFD-PDGFRß signalling in hRG may contribute to the proliferation and subventricular dispersion of neural progenitors that characterize OSVZ formation4-5.

Mouse studies have demonstrated that the size and shape of cerebral cortex depend on precise regulation of molecular pathways controlling RG proliferation and differentiation1-2. While many of these pathways are probably conserved in humans, mouse studies alone cannot reveal uniquely human aspects of cortical development24-29. By analysing human tissue as a starting point, we found that PDGFD-PDGFRß signalling is required for normal RG proliferation in developing human but not mouse neocortex, and is sufficient to promote some 'humanizing' char- acteristics in mouse. Our analysis has also identified other genes that probably contribute to differences between human and mouse cortical development. BMP7, another secreted growth factor, was predicted and validated to be expressed by hRG but not mRG (Fig. 2d and Extended Data Fig. 6), raising the possibility that local production of growth fac- tors by hRG may be necessary to support the expanded germinal region and progenitor heterogeneity of developing human neocortex. We anti- cipate that the analytical and experimental strategies described here will help determine the extent to which these and other pathways are shared among primates or uniquely required for human cortical development.

Online Content Methods, along with any additional Extended Data display Items and Source Data, are available In the online version of the paper; references unique to these sections appear only In the online paper.

Received 15 April; accepted 16 October 2014.

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