NEUROG2 function in RGC of the gyrencephalic ferret cortex

The developing ferret and human cerebral cortices share several key features including stereotyped sulci and gyri, a dramatically expanded subventricular zone (SVZ), and an abundance of ORG, making the ferret an attractive model for the study of cortical neurogenesis^9,28,29. We first confirmed the expression of NEUROG2 in ferret RGC (Fig. 2a,b), identifying a marked “salt-and-pepper” pattern of NEUROG2 immunoreactivity in both the VZ and SVZ. We then used in vivo electroporation of the newborn ferret dorsal cortical VZ to express a NEUROG2-VP16 fusion protein, which links the DNA-binding domain of NEUROG2 to the VP16 constitutive transcriptional activator domain²⁵, thus activating all direct downstream targets of NEUROG2 in ferret apical RGC. Following delivery of the Neurog2-VP16 expression construct, we allowed ferret kits to develop for up to ten days, during which time many neurons of the upper cortical layers are generated^30–32 (Fig. 2c). In both control (pCAG-GFP) and Neurog2-VP16 brains, we observed numerous GFP⁺SOX2⁺ and GFP⁺TBR2⁺ cells in the VZ, including cells in the basal VZ and inner SVZ with a characteristic ORG morphology (Fig. 2d,e); as well many GFP⁺ cells in the SVZ and intermediate zone (IZ), with a small number reaching the cortical plate (CP) after longer survival times (up to ten days post-electroporation [DPE]) (Fig. 2f). After 7–9 DPE, NEUROG2-VP16 induced a significant shift in the proportion of GFP⁺ cells from the VZ to the SVZ/IZ, and a concomitant reduction in the proportion of GFP⁺ cells co-expressing SOX2 (Fig. 2g), with the majority of NEUROG2-VP16+ cells displaying the morphology of radially migrating postmitotic neurons in the outer SVZ and IZ. In addition, we FACS-purified electroporated cells from the ferret cortex and performed qRT-PCR analysis of ORG-enriched candidate genes identified in humans, and found that nearly all human ORG-enriched NEUROG2 downstream targets were highly upregulated by the NEUROG2-VP16 construct in ferret, relative to control GFP-expressing cells, while Sox2 was repressed (Supplementary Fig. 4). Notably, NEUROG2-VP16 expression in ferret RGC in vivo also resulted in increased expression of the ferret orthologs of several other human ORG-enriched genes, including Gadd45g and Ttyh2 (Supplementary Fig. 4), further suggesting that NEUROG2 is a critical regulator of a conserved radial progenitor development program in species with abundant ORG. Altogether, our ferret functional experiments demonstrate a conserved role for NEUROG2 transcriptional targets in driving delamination from the ventricular neuroepithelium, which is a key step in the production of ORG, while additional downstream effectors initiate repression of Sox2 and activation of a neuronal differentiation program, including radial migration, as previously described in mice. Future studies will be required to identify the specific factors downstream of NEUROG2 that regulate neuroepithelial integration, and elucidate the molecular mechanisms that permit a subset of NEUROG2-expressing ferret and human RGC to remain integrated in the VZ, while others detach and migrate into the SVZ.

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Figure 2

NEUROG2 regulates progenitor morphology and molecular identity in thedeveloping cortex of the gyrencephalic ferret

a, Co-expression of RGC marker SOX2 and proneural marker NEUROG2 in developing ferret cortex. Numerous SOX2⁺NEUROG2⁺ cells are found in both the VZ and SVZ at early postnatal ages, when these germinal zones are populated respectively by large numbers of apical and non-apical RGC producing neurons destined for the upper cortical layers. b, Higher magnification of SOX2⁺NEUROG2⁺ progenitors in the SVZ co-expressing the RGC neurofilament protein Vimentin (VIM), which labels the basal radial process (yellow arrowhead). c, Genetic manipulation of apical RGC is achieved by in vivo intraventricular injection and electroporation in neonatal ferret kits. After several days post-electroporation (DPE), GFP⁺ cells are observed throughout the developing cortical wall, with GFP⁺ radial fibers extending from the germinal zones to the pial surface and newborn neurons migrating through the SVZ and intermediate zone (IZ) into the cortical plate (CP). d, Littermates electroporated at postnatal day 1 and harvested 7 days later (P1:P8) with NEUROG2 gain-of-function (pCAG-Neurog2-VP16) or GFP control (pCAG-GFP) expression constructs, analyzed for the distribution of GFP⁺ cells and their co-expression of SOX2. ISVZ, inner SVZ; OSVZ, outer SVZ; MZ, marginal zone. e, At all survival time-points, we identified numerous GFP⁺SOX2⁺ apical RGC in the VZ as well as occasional GFP⁺SOX2⁺ ORG with soma at the VZ/SVZ border (insets). f, Higher magnification of the boxed area from (d) shows GFP⁺ cells in the SVZ/IZ that are SOX2-negative with the morphology of radially migrating newborn neurons. g, At 7 to 9 DPE, NEUROG2 gain-of-function induced a significant shift of GFP⁺ cells from the VZ into the SVZ/IZ compared to the control (asterisk denotes p < 0.05, paired t-test; exact p-values: VZ=0.026, SVZ/IZ=0.026, CP=0.034; n=3 animals per condition; 3–4 brain sections counted per animal; data represented as mean ± SEM), with a concomitant loss of RGC morphology and SOX2 expression, demonstrating that the NEUROG2 proneural network promotes delamination of daughter cells from the ventricular surface, migration into the SVZ, and neuronal differentiation. Scale bars: 50 µm (a), 1 mm (c), 100 µm (d), 20 µm (e,f).

Single-cell analysis of species-specific RGC heterogeneity

Both our human RNA-seq and ferret immunofluorescence data demonstrate NEUROG2⁺ RGC subpopulations in both the VZ and SVZ, intermingled with NEUROG2⁻ progenitors, exemplifying the heterogeneity that confounds population-level transcriptome comparisons, so we turned to single cell analysis to compare the subpopulations of radial progenitors in human, ferret, and mouse. We first sorted RGC from human fetal cortex (n=6, 16–21 WG; Supplementary Table 1) into 96-well plates and performed microfluidics-based, highly multiplexed single-cell qRT-PCR to simultaneously assay several dozen genes that included markers for all RGC (PAX6, SOX2, GLAST, BLBP, VIM, NES) and apical RGC (PROM1, PARD3, MPP5, TJP1); proneural Neurogenin pathway TFs; and additional validated LG⁺Pr^lo, ORG-enriched genes (Supplementary Fig. 3). Of 546 sorted single human progenitors, PAX6, SOX2, GLAST, BLBP, and VIM were detected in virtually all cells (93±3% detection rate; Supplementary Fig. 5a), confirming their radial glial identity. Hierarchical clustering revealed several distinct transcriptional states characterized by the combinatorial expression of apical markers and proneural factors, such that cells fell into one of four main subpopulations which we refer to as apical/multipotent; apical/proneural; non-apical/multipotent; and non-apical/proneural (Fig. 3a and Supplementary Fig. 5b). Apical RGC subpopulations (clusters I, II, V in Fig. 3a; 71±8% detection rate for apical genes) could be divided into apical/multipotent (I) and apical/proneural (II and V) subpopulations based on their lesser (21±5%) or greater (81±7%) expression of proneural Neurogenin pathway TFs, respectively. ORG-like non-apical subpopulations (clusters III and IV) with lower detection rates of apical complex transcripts (31±6%) were similarly subdivided according to lower or higher rates of proneural gene expression (22±7% vs. 65±15%). Notably, most non-apical/proneural cells were NEUROG2⁻ (cluster III), consistent with observations in the mouse that NEUROG2 represses apical identity and is then downregulated upon delamination²³. Finally, both apical and non-apical proneural RGC were further subdivided by expression of other LG⁺Pr^lo-enriched genes (TTYH2, PLCB4, SSTR2, RASGRP1) that define additional transcriptional heterogeneity among human cortical progenitors. These results demonstrate significant multigenic transcriptional diversity within cortical radial glial progenitors, and are characteristic of the previously unappreciated heterogeneity recently revealed by single-cell analyses in other non-neural stem cell niches^33,34.

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Figure 3

Single-cell gene expression of human and mouse progenitors reveals species-specific RGC subpopulations

a, Multiplexed gene expression profiling of 546 single human RGC reveals distinct transcriptional states defined by the presence or absence of transcripts encoding apical membrane-specific proteins, proneural transcription factors downstream of NEUROG2 such as NEUROD1 and NEUROD4, and additional LG⁺Pr^lo-enriched genes such as TTYH2 and PLCB4. Hierarchical clustering and heatmap representation of single-cell qRT-PCR data (left) indicates the co-expression patterns of these genes, and a schematic representation (right) of the four main subpopulations of RGC identified: “multipotent” RGC (blue) are found as subsets of both the apical (cluster I) and non-apical RGC (cluster IV), as are the “proneural” NEUROG2/TBR2⁺ RGC (clusters II, III, V). In addition, proneural RGC can be further subdivided according to their expression of downstream factors and additional LG⁺Pr^lo-enriched genes (e.g., compare clusters II and V). b, The same genes assayed in 226 RGC from E16-E17 mouse cortex yield only three subpopulations: apical multipotent (i); apical proneural (ii); and non-apical proneural (iii). Schematic representation of these subpopulations (right) highlights the major species differences, namely, the mouse has fewer non-apical cells overall; few if any multipotent (NEUROG2⁻TBR2⁻) non-apical cells, suggesting the absence of a significant subpopulation of proliferative ORG; and very few cells expressing other human subset-enriched genes (e.g., TTYH1, PLCB4). c, Violin plots of gene expression distributions for apical complex, NEUROG2 network, and ORG-enriched genes in human and mouse single RGC. Several ORG-enriched genes appear to be abundantly expressed in subsets of human but not mouse RGC including NEUROD4, GADD45G, PLCB4, TTYH2, SSTR2, RASGRP1.

In contrast to the human analysis, single RGC from the embryonic day (E)16–17 mouse cortex showed fewer distinct transcriptional states (Fig. 3b and Supplementary Fig. 5a), and rarely expressed many of the genes that defined subsets of human cells (Fig. 3b,c). Virtually all mouse single cells (n=226) expressed the RGC markers Sox2, Vim, Blbp, and Glast (93±8%), and the vast majority (88%) of cells also expressed some apical complex genes, confirming that ORG are rare in the mouse. Hierarchical clustering yielded only three subpopulations (Fig. 3b), corresponding to the apical/multipotent (cluster i), apical/proneural (ii), and non-apical/proneural (iii) subsets observed in the human cortex. Although a significant subset of mouse RGC co-expressed proneural TFs, the proportion of cells was significantly smaller than in human (27% in mouse vs. 47% in human; p=9.57E-8, Fisher’s exact test). Importantly, the absence of a significant non-apical/multipotent subpopulation (human cluster IV) suggests a critical species difference in the proliferative potential of ORG, which could underlie the paucity of ORG in the mouse. Most notably, orthologs of human ORG-enriched genes that contributed markedly to human RGC heterogeneity, including Plcb4, Gadd45g, Ttyh2, Rasgrp1, and Sstr2, were detected in a rare and uncorrelated minority of mouse RGC (<10%, compared to 45–55% in human) (Fig. 3b,c), further highlighting the species-specificity of RGC transcriptional heterogeneity.

Finally, we performed RNA-seq and single-cell profiling of ferret radial glial progenitors and found that they share some of the key transcriptional states of human RGC. In the absence of working antibodies against ferret Prominin, we first validated LeX and Glast antibodies by immunohistochemistry in ferret brain sections as well as by FACS (data not shown), then collected LG⁺ and LG⁻ cells from neonatal ferret cortex, at which time middle and upper layers of cortex are being generated^30,31, roughly corresponding to mouse E16–17 or human 16–20 WG, and performed population-level RNA-seq. Ferret LG⁺ cells were enriched for most previously described RGC marker genes, and showed transcriptome-wide expression patterns similar to LG⁺ cells from human and mouse cortex (Fig. 4a). Having validated that LG⁺ cells comprise a substantial proportion of ferret RGC, we performed single-cell profiling on 185 single LG⁺ cells from the neonatal ferret cortex, and interestingly found an intermediate degree of heterogeneity compared to the human and mouse progenitors (Fig. 4b). The vast majority of sorted ferret cells expressed classic RGC markers, confirming the specificity of the sorting, while a subset co-expressed Tbr2 (ferret clusters iv and v, and a number of cells in cluster i), and a smaller subset of those were positive for Neurod4 and Neurod1 (clusters iv and v). Interestingly, the ferret homologs of some human ORG-enriched genes (e.g., Rasgrp1) were preferentially expressed in these proneural cells, as in human, whereas most human ORG genes were more homogeneously expressed in most ferret cells (Ttyh2, Sstr2), and conversely, some genes were heterogeneously expressed in novel subsets of cells that did not correspond to those seen in human (Foxn2). Finally, we noted that a greater proportion of ferret cells expressed the gliogenic marker Gfap compared to human cells, which is consistent with evidence that ferret ORG have astrogliogenic potential⁹. Taken together, our single-cell analyses from human, ferret, and mouse implicate a large number of genes acting in a coordinated network that may be responsible for the evolution of novel progenitor transcriptional states critical for human cortical development.

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Figure 4

Population-level whole-transcriptome RNA-seq and single-cell expression analysis of ferret RGC

a, Expression heatmaps of known progenitor and neuronal marker genes, as well as selected human RGC-enriched gene sets, from LG⁺ and LG⁻ cells isolated by FACS from the P2 developing ferret cortex (n=2). Enrichment of classic RGC markers and a high degree of similarity between gene sets enriched in human and ferret LG⁺ cells validate the use of LeX and Glast to select RGC from the developing ferret cortex. Notably, however, several genes (black bullets) show distinct expression patterns between the two species (e.g., CXCL12, UNC5B, NTNG2, SEMA5A), suggesting that certain growth factor and other pathways may be expressed in a species-specific manner in RGC. b, Single-cell gene expression profiling of 185 single ferret LG⁺ progenitors was performed using the same gene panel as shown in Figure 3 for human and mouse RGC. As in humans, a substantial fraction of ferret cells in clusters i, iv, and v co-express both RGC markers and Tbr2/Neurog2, consistent with our immunohistochemcial analysis of NEUROG2 expression in the ferret (Fig. 2a) and suggesting this “proneural” RGC transcriptional state is conserved. Similar to human RGC, a subset of these proneural cells also express the downstream factors NEUROD1 and NEUROD4. However, some human ORG-enriched genes (e.g. Rasgrp1) are expressed in fewer ferret RGC, while others (e.g. Plcb4, Sstr2, Gadd45g, Ttyh2) appear more homogenous across all cells. Interestingly, Foxn2, which was detected in nearly all human RGC, appears to mark a distinct subpopulation of ferret apical RGC (clusters i and iv). Overall, while ferret RGC exhibit more diversity of transcriptional states than mouse and generally more similarity to human, they are nonetheless distinct in their relative proportions and composition.

Purification of Cortical Progenitors

Cortical tissue was separated from remaining brain tissue in ice-cold HBSS medium and manually disrupted using a sterile razor blade down to ~1-mm³ pieces. The tissue was then dissociated into a single cell suspension using the trypsin Neural Dissociation Kit (Miltenyi Biotec) according to manufacturer’s instructions. Cells were placed into FACS “pre-sort” media (Neurobasal media, 0.25% HEPES, 0.5% FBS, rhEGF, rhFGF) for labeling with cell surface antibodies. Cells were labeled in aliquots of 500ul containing up to 40 million cells with anti-CD15-FITC (BD Biosciences 560997) at 1:10,000; anti-GLAST-PE (Miltenyi Biotec 130-098-804) at 1:10,000; and anti-CD133-APC (Miltenyi Biotec 130-098-829) at 1:1,000 for 30 minutes at +4°C and washed twice with pre-sort media before FACS. Alternatively, minced tissue was cryopreserved prior to enzymatic dissociation by storing in HBSS + 10% DMSO, cooled gradually in a cryochamber to −80°C overnight, and transferred to −150°C for long-term storage.

RNA Isolation, Processing, and RNA sequencing

Cells were sorted directly into RNA stabilizing lysis buffer followed by total RNA extraction (Qiagen). Next-generation sequencing libraries were prepared using Illumina TruSeq v2 according to manufacturer’s instructions and sequencing was performed on an Illumina HighSeq 2000. Data were analyzed primarily with the Tuxedo software suite (bowtie/tophat/cufflinks/cummerbund)⁴⁷ using the hg19 genome and UCSC KnownGene transcriptome references. Additional R/Bioconductor packages were used for principle component analysis, clustering, and the generation of heatmaps. Gene set enrichment analyses were performed using DAVID (http://david.abcc.ncifcrf.gov/) and comparison of non-reference cufflinks transcripts to published lncRNA catalogs was done in Galaxy (http://main.g2.bx.psu.edu/).

Ferret Electroporation

Timed-pregnant ferrets (Mustela putorius furo) were obtained from Marshall Bioresources. Neonatal ferret kits were anesthetized with 5% and maintained at 3% isoflurane utilizing a nose cone during the entire procedure. A small incision was made on the skin at the dorsomedial part of the head using a surgical blade and a hole was opened anterior to the bregma on the left side of the skull, above the lateral ventricle, using an insulin syringe needle. 3–5µl of DNA construct (1µg/µl) was injected into the lateral ventricle using a pulled glass micropipette inserted through the craniotomy and the overlying cortical wall. 150V electric pulses were passed 5 times at 1s intervals using paddle electrodes positioned outside the animal’s head. The skin incision was closed using VetBond (3M) tissue adhesive and kits were returned to the nest after recovering from anesthesia. Kits were deeply anesthetized prior to transcardial perfusion with cold PBS and 4% PFA, and brains were extracted and placed in 4% PFA at +4° overnight prior to processing for immunohistochemistry.

Immunohistochemistry

Ferret brains were embedded in 4% low-melting-point agarose and sectioned at 70µm on a vibrating microtome. Sections were washed in cold 0.1M PB followed by antigen retrieval in 10mM citric acid (pH 6.0) + 0.05% Tween-20 at 80°C for 30 minutes. Sections were then cooled to room temperature and washed in cold 0.1M PB. Sections were blocked for at least 1 hour at room temperature (10% Normal Donkey Serum, 0.1% Triton X-100, 0.2% gelatin in PBS). Primary antibodies were incubated at 4°C in 0.2X blocking buffer for at least 48 hours. Primary antibodies included chicken anti-Vimentin 1:250 (Abcam ab24525), chicken anti-Tbr2 1:250 (Millipore AB15894), rabbit anti-GFP 1:1,000 (Abcam ab290), goat anti-Sox2 1:250 (Santa Cruz sc-17320). Sections were washed in PBS and then incubated for two hours in 0.2X blocking buffer containing AlexaFluor secondary antibodies (Life Technologies). Slices were then rinsed and coverslipped with Fluoromount-G (Southern Biotech) containing Hoechst 1:1,000 (Roche). Images were obtained with Zeiss LSM700 confocal microscope and Leica MZ16 F fluorescence stereomicroscope. For quantification, tiled confocal images spanning the entire cortical wall were captured at 20x, stitched, and exported to Photoshop. Three to four pairs of coronal sections from three control (GFP) and three NEUROG2-VP16 electroporated hemispheres, matched for the level of section and spatial extent of the electroporation, were imaged and the images were then coded and quantified blind to experimental condition. The Hoechst nuclear counterstain was used to demarcate the borders between VZ, SVZ, IZ, and CP, and the numbers of GFP+ cell bodies in each zone counted. The percentages of GFP+ cells in each zone were calculated separately for each image and averaged across the images for each brain. The averages for each replicate were then compared across experimental conditions using a paired student’s t-test.

Single Cell mRNA Expression Profiling

Following cell labeling, single cells were sorted by FACS into skirted 96 well PCR plates containing Pre-Amplification solution (Cells Direct Kit, Life Technologies) and appropriate mixtures of Taqman assays (for human and mouse) or validated primer pairs (for ferret). Plates were transported on ice and spun down before pre-amplification (94°C 10 minutes, 50°C 60 minutes, 94°C 30 seconds, 50°C 3 minutes × 28 cycles). Target-specific cDNA from single cells was harvested, screened for expression of housekeeping genes ACTB and GAPDH, and then loaded onto a Biomark chip (Fluidigm) for expression profiling with the panel of qRT-PCR assays. Expression data was processed and analyzed using the Singular Analysis Toolset (Fluidigm) and gplots packages in R. Hierarchical clustering was performed using complete linkage based on Euclidean distance and clusters of cells were defined by cutting the single-cell dendrogram at the same height for all three species.

Comparative Genomics Analysis of Novel lncRNA Loci

Comparative evolutionary analysis of lncRNAs was performed using a modified version of the recently published “Forward Genomics” approach⁴⁸. Briefly, multi-sequence fasta files were generated for all conserved regions located within novel lncRNAs using existing 100-way vertebrate multiple alignment files available from the UCSC genome browser. Next we generated ancestral sequences for the common ancestor of human, mice, and ferrets using the prequel algorithm (--keep-gaps --no-probs --msa-format PHYLIP), part of the PHAST tools⁴⁹. The percent identities of sequences from all species were determined by alignment to the corresponding ancestral sequence using Needleall, part of the EMBOSS tools⁵⁰. Species with low quality or missing sequence information were excluded from the analysis. Finally, the number of identical bases from all regions within each lncRNA were calculated to yield the %ID to the common ancestor.

We thank J. Partlow for coordinating human tissue protocols; D. Gonzalez for animal protocol and technical experimental assistance; Suzan Lazo-Kallanian for single-cell FACS assistance; and all members of the Walsh lab for comments and discussion. The Neurog2-VP16 construct was a generous gift from Carol Schuurmans (University of Calgary). This work was supported by grants to C.A.W. from the National Institutes of Neurological Disease and Stroke (R01 NS032457) and the Paul G. Allen Family Foundation. M.B.J. was supported by a fellowship from the Nancy Lurie Marks Family Foundation. P.P.W. is a Stuart H.Q. & Victoria Quan Fellow at Harvard Medical School. Single-cell expression profiling experiments were performed at the Molecular Genetics Core at Boston Children’s Hospital (BCH IDDRC, P30 HD18655). Transcriptome analysis was performed using Harvard Medical School’s Orchestra high-performance computing cluster, which is partially supported by NIH grant NCRR 1S10RR028832-01. C.A.W. is a Distinguished Investigator of the Paul G. Allen Family Foundation, and an Investigator of the Howard Hughes Medical Institute.