Identification and culture of SOX6+/NG2+ cortical progenitors with high purity and fidelity

(A) Confocal micrograph of mouse neocortex at postnatal day 7 (P7) showing expression of SOX6 by a subset of BrdU+ proliferative cells. See also Figure S1.

(B) In situ hybridization of Neurog2 in Sox6 wild-type (wt) (left) and knockout (KO) (right) cortex at P6. (B’) Insets showing the boxed areas in B. Loss of Sox6 results in widespread ectopic expression of Neurog2 (B’).

(C) Immunofluorescence showing expression of SOX6 by DsRed+ NG2+ progenitors in cortex at P5.

(D) Immunostaining of NG2 proteoglycan in NG2-DsRed cortex at P5 shows expression of DsRed by NG2+ progenitors. Inset: a cell with strong DsRed signal in the cell body, and NG2 proteoglycan around the main cell body and in cellular processes. See also Figures S2A-S2C.

(E) Representative FACS plot of neocortical cells from NG2-DsRed transgenic cortex showing distinct DsRed-Bright, -Dim, and -negative populations.

(F) qPCR analysis of Sox6, Olig2, and NG2 (a.k.a. Cspg4) from acutely sorted DsRed-Negative, -Dim, and -Bright populations, as well as cultured DsRed-Bright cells (5DIV), demonstrates that SOX6+/NG2+ progenitors are enriched in DsRed-Bright population and maintain key gene expression in vitro. See also Figures S2D-S2G. Data are presented as mean ± SD, n=4, biological replicates, Actb normalized data relative to DsRed-negative population. ∗∗∗∗p < 0.0001, p ≥ 0.05, no statistically significant difference (n.s.); ANOVA Tukey’s post hoc test.

(G) Volcano plot comparing fold difference in average expression of progenitor genes between acutely sorted DsRed-Bright and -Dim populations (RNA-seq, n=5, biological replicates). See also Figure S3A.

(H) Representative brightfield image of cultured SOX6+/NG2+ (DsRed-Bright) progenitors at 5 DIV showing preserved progenitor multipolar morphology. See also Figures S2J-S2P.

(I and J) Cultured progenitors continue expressing the key progenitor-specific molecules NG2, SOX10 (I), OLIG2, and SOX6 (J) at 7 DIV.

(K) Quantification of TUJ1+ and GFAP+ cells at 3-, 5-, and 7 DIV shows essentially no contaminant cells in culture. Data are presented as mean ± SD, n=2, biological replicates. See also Figure S2Q.

(L) Pearson correlation analysis of progenitor genes shows high similarity between acutely sorted and cultured SOX6+/NG2+ (DsRed-Bright) progenitors (R = 0.84, p < 2.2e-16). Data points represent log2 fold differences in gene expression relative to acutely sorted DsRed-Dim population. See also Figure S3A.

(M) Heatmap of the top five marker genes for seven major cell types in brain shows that SOX6+/NG2+ progenitors are enriched in DsRed-Bright populations and that progenitor cultures are free of potential contaminants. Counts are variance-stabilizing transformed (vst) normalized data in log2 scale.

(N) Volcano plot comparing fold differences in average expression of the top 500 genes for major cell types between cultured SOX6+/NG2+ (DsRed-Bright) progenitors and acutely sorted cells. n=5/6, biological replicates. See also Figure S3. Scale bars (A, C, H) 50 µm; (D, I, J) 100 µm. cc: corpus callosum, ctx: cortex.

NVOF induces mature glutamatergic neurons from SOX6+/NG2+ cortical progenitors in vitro

(A-C) Strategy for directed differentiation of SOX6+/NG2+ progenitors into cortical output neurons (A), the NVOF multigene construct (B), and the experimental outline (C).

(D) Representative images of control- and NVOF-transfected cells at 1-, 3-, 7-, and 16-days-post-transfection (DPT). Unlike control-transfected cells, NVOF-transfected cells lose progenitor morphology at 1 DPT, and progressively exhibit complex neuronal morphology, including a primary axon-like process and multiple dendrite–like processes.

(E) Percentage of control-GFP and NVOF-transfected cells with neuronal morphology and TUJ1 expression (∼42% at 3 DPT and ∼74% at 7 DPT for NVOF, n=4, >200 cells/n).

(F) Quantification of primary process length for NVOF-induced neurons at 3- and 7 DPT (n=3, >100 cells/n).

(G) Representative morphology of NVOF-induced, TUJ1+ neurons at 7 DPT. Note the single axon, dendrite-like structures, and multiple axonal collaterals.

(H) Representative images of NVOF-induced neurons at 16 DPT showing acquisition of elaborate dendritic morphology and highly intercalated axonal processes.

(I) High-power representative images of individual NVOF-induced neurons at 16 DPT showing dendritic complexity and a single primary axon-like process for each neuron (red arrows).

(J) Representative images of Neurog2-induced neurons with multiple atypical axon-like processes. (GFP is pseudocolored for enhanced clarity of cell morphology.)

(K) Representative images of Neurog2-induced neurons expressing the axonal marker ANKYRIN-G (ANK3) by multiple neurites.

(L) Quantification of neurons with single versus multiple axons in Neurog2- and NVOF-induced neurons. At 7 DPT, 49% ±16% of Neurog2-induced neurons have multiple, long axon-like processes, whereas a small number of such neurons exist after NVOF induction (9% ±5%) (n=5, >100 cell). See methods for details.

(M-N) Representative images of NVOF-induced neurons at 7 DPT showing compartmentalized expression of the somato-dendritic marker MAP2, the somato-axonal marker Neurofilament-M, and the mature neuronal marker, NeuN.

(O) Quantification of NVOF-induced, TUJ1+ neurons expressing MAP2 at 3 DPT (∼48%, n=3, >200 cells) and 7 DPT (∼93%, n=4, >200 cells), as well as NeuN at 7 DPT (66% ±16%, n=4, >100 cells).

(Q) Volcano plot showing upregulation of neuronal genes in NVOF-induced neurons compared to control-transfected cells at 7 DPT (RNA-seq, n=3, biological replicates).

(Q) Bar graph of RNA-seq data displaying upregulation of neuronal genes and downregulation of progenitor genes in NVOF-induced neurons at 7 DPT. Neurons exclusively upregulate glutamatergic genes, but not genes specific to alternate neuronal identities.

Scale bars (D, G, H, J, M, N) 100 µm; (I) 50 µm. Error bars show standard deviations. ∗∗∗∗p < 0.0001, ***p < 0.001, **p < 0.01, t-test in (E, F, L). n.f. (no TUJ1+ cell found).

NVOF-induced neurons are electrically active, and have spontaneous synaptic currents

(A-B) Representative high-magnification images of NVOF-induced neurons at 14 DPT (pseudo-colored GFP) with and without co-culture of forebrain primary neurons and astrocyte-conditioned media. (A’-B’) Insets showing the boxed areas. Note differences in morphology of presumptive synaptic structures between the two conditions. See methods for details.

(C) Representative high-magnification image of a primary cortical neuron at 14 DPT (pseudo-colored tdTomato) from in utero electroporated wild-type mice as a positive control. (C’) Note similarity in morphology of presumptive synaptic structures between primary neurons and NVOF-induced neurons in B’. See methods for details.

(D) Representative high-magnification image of a SYNAPSIN-positive NVOF-induced neuron co-cultured with forebrain neurons, indicating abundant connections from surrounding neurons. Arrows show the presumptive single primary axon with no synapsin staining.

(E) A representative NVOF-induced neuron at 10 DPT showing depolarizing steps evoking a train of action potentials (red highlighted trace: step 6, 50 pA). 10 minutes after break-in, or following a resting Vm stabilization greater than 1 minute, cells were injected with 10 current steps ranging from -40 pA to 95 pA in 15 pA increments, for a duration of 500 ms each.

(F) The first evoked action potential in response to positive current injections for 10 individual cells, overlaid (10 DPT). Waveforms are aligned at threshold for comparison.

(G) Corresponding dV/dt traces for action potentials shown in F.

(H) Representative sag current, indicating presence of Ih, induced with a 500 ms current injection of -40 pA (average of 10 sweeps).

(I) Cell membrane resistance decreases over time (10 DPT, n=10; 16 DPT, n=10), and is substantially lower without NVOF (GFP, n=2).

(J) Resting membrane voltage for each condition (10 DPT, n=10; 16 DPT, n=10; GFP, n=2).

(K-M) Action potential threshold, amplitude, and width at 10 DPT (n=10) and 16 DPT (n=10).

(N) Sag current at 10 DPT (n=9) and 16 DPT (n=8).

(O) Representative spontaneous outward synaptic currents recorded at -70mV in NVOF+ cells at 16 DPT.

(P) Representative spontaneous inward synaptic currents recorded at -70mV in NVOF-induced neurons at 16 DPT.

Scale bars (A-D) 50 µm; (F) 50 mV; (G) 50 mV/ms. For all graphs I-N, open circles are individual cells, filled boxes are mean ± s.e.m.

NVOF-induced neurons exhibit molecular hallmarks of corticospinal neurons in vitro

(A-F) Representative immunocytochemistry images of NVOF-induced neurons expressing the subcerebral projection neuron (SCPN) transcriptional controls CTIP2 (A) and PCP4 (B), the corticothalamic projection neuron (CThPN) transcriptional controls FOG2 (C) and FOXP2 (D), but not the callosal projection neuron (CPN) molecular controls SATB2 (E) and CUX1 (F). Scale bars (A-F) 100 µm.

(G) Percentage of NVOF-induced, TUJ1+ neurons expressing CTIP2 (56% ±20%, n=5), PCP4 (77% ±14%, n=3), FOG2 (81% ±13%, n=3), and SATB2 (0%, n=3). Error bars show standard deviations.

(H) Violin plot shows mean intensities of CTIP2, PCP4, and FOG2 fluorescence signals in nuclei of NVOF-induced neurons. Plotted values are mean nuclear intensity of individual neurons normalized to the average intensity of the three lowest-expressing neurons. Red line shows median expression, and dark gray lines show quartile expressions.

(I) Bar plot showing percentages of CTIP2-negative, -dim, and -bright neurons at 1-, 3-, 7-, and 12-DPT.

(J-K) Volcano plots and heatmaps of neurons transfected with control GFP and NVOF 7 DPT, displaying the 862 genes enriched in SCPN primary neurons compared to control-transfected cells. See methods for details.

(L) Bar plot of RNA-seq data showing upregulation of SCPN (purple) and CThPN (blue) marker genes, and no activation or downregulation of CPN (green) genes by NVOF-induced neurons relative to neurons transfected with control GFP at 7 DPT.

Unlike NVOF-induced neurons, Neurog2-induced neurons exhibit unresolved subtype-specific molecular features

(A) Pearson correlation analysis shows high similarity between NVOF-induced neurons at 7 DPT and primary SCPN at P2 (R: 0.87). Compared to NVOF, Neurog2-induction (7 DPT) leads to decreased similarity with primary SCPN at P2 (R: 0.77). Data points are log2 fold differences of gene expression at 7 DPT by NVOF- or Neurog2-induced neurons (on X-axis) and by SCPN (on Y-axis) compared to progenitors transfected with control GFP.

(B) Volcano plot showing fold differences of SCPN-enriched genes between NVOF- and Neurog2-induced neurons.

(C) Volcano plot showing fold differences of CPN-enriched genes between NVOF- and Neurog2-induced neurons.

(D-E) Direct comparison of NVOF-versus Neurog2-induced neurons at 7 DPT for select developmental genes with key roles in specification and differentiation of SCPN, CPN, and CThPN. Scatter plot (D) and bar graph (E) shows fold differences in gene expression.

Identification of SOX6+ cortical progenitors in postnatal and adult neocortex

(A) Schematic of BrdU cumulative labeling experiments.

(B-D) Confocal photomicrographs of cortex sagittal sections showing BrdU+ proliferative cells at P7 (B), a subset of which express Sox6 in the caudal cortical SVZ and cortical parenchyma (C and D). BrdU was injected from P3 to P7 in wild-type c57bl/6 mouse (50 µg/mg of body weight).

(E-G) Same experiment as (B-D), but BrdU administration is from P23-P28. Fewer BrdU-labeled cells are labeled in P28 neocortex (E), subset of which express SOX6 (F and G).

(H-J) After five-week administration of BrdU in drinking water, SOX6+/BrdU+ cells are identified in adult brain.

Scale bars (A, D, G) 150 µm; (B, C, E, F, H, I) 25 µm. Ctx: cortex, LV: lateral ventricle, SVZ: subventricular zone, P: postnatal-day, BrdU: Bromodeoxyuridine.

Characterization, FACS isolation, and culture of cortical SOX6+/NG2+ progenitors

(A-B) Photomicrograph of NG2-DsRed coronal section at P5 showing expression of DsRed by uniformly distributed progenitor cells and pericytes (see also Figures S3H-S3L). Schematic in A depicts a coronal section; boxed region in A identifies the approximate region shown in B. Ctx: cortex.

(C) Immunostaining of NG2 and SOX10 shows uniform distribution of NG2+ progenitors in cortex at P5.

(D) Immunostaining confirms expression of NG2 and DsRed by cultured DsRed-Bright cells after FACS purification (4 hours). n=2, biological replicates.

(E) qPCR of Gfap, Tuj1, and Mbp demonstrates that acutely sorted and cultured DsRed-Bright cells do not contain neurons or astrocytes, and that culture conditions do not promote oligodendrocyte differentiation. n=4, biological replicates, Actb normalized data relative to negative population.

(F-G) Immunofluorescence shows that DsRed-Dim populations contain a mixture of NG2+ cells (with relatively low NG2 expression compared to DsRed-Bright; see S2L) (F) and GFAP+/NESTIN+/NG2+ glial progenitors (G).

(H) Quantification of progenitor proliferation in response to PDGF-A, FGF2, and SHH at 5 DIV. (Due to potential ventralization by SHH, only PDGF-A and FGF2 were used in subsequent experiments.)

(I) Cultured SOX6+/NG2+ cortical progenitors proliferate robustly in response to PDGF-A and FGF2. Cell number increases 7-fold through 7 DIV. n=2, biological replicates.

(J-L) Representative images of cultured progenitors shows proliferation and continued expression of NG2 and Olig2 over time.

(M and N) Low-magnification immunofluorescence and bright-field images of progenitors in culture show expression of NG2 proteoglycan, and maintenance of multipolar morphology through 5 DIV.

(O-P) Representative high-magnification (40x) images of progenitors in culture shows preservation of characteristic non-overlapping processes by SOX6+/NG2+ progenitors.

(Q) qPCR shows that serum causes cultured progenitors to decrease expression of the key progenitor genes Sox6 and NG2, and increase expression of the astroglial marker Gfap. n=4, biological replicates, Actb normalized data relative to negative population.

Scale bars (C, D, F, G, J, K, L, N) 100 µm, (M) 500 µm, (O, P) 10 µm. Data are presented as mean ± SD. ∗∗∗∗p < 0.0001, ***p < 0.001, **p < 0.01, *p ≥ 0.05, no statistically significant difference (n.s.); ANOVA Tukey’s post hoc test in (E), t-test in (Q).

SOX6+/NG2+ progenitors maintain molecular characteristics in vitro, and cultures are free of DsRed+ pericytes

(A-E) Heatmap of the top-500 genes enriched in major cell lineages63 for acutely sorted DsRed-Negative, -Dim, and -Bright cells, as well as cultured DsRed-Bright cells (5-DIV). Counts are variance-stabilizing transformed (vst) normalized data in log2 scale.

(F) qPCR of the pericyte markers Pdgfrb and Mcam (CD146) shows that pericytes are FACS-purified with SOX6+/NG2+ progenitors in DsRed-Bright populations, but they are not present in culture at 5 DIV. Data are presented as mean ± SD, n=4, biological replicates, Actb normalized data relative to negative population.

(G-I) Heatmaps of commonly known pericyte markers (G), a conservative list of 241 pericyte-enriched genes (H), and an extended list of 785 genes that are enriched in brain mural cells (I)161, confirming absence of pericytes in culture. See Supplementary Table S1 for a list of genes used in these plots.

(J-L) qPCR (J), microscopy (K), and immunocytochemistry (L) of DsRed-Bright cells cultured in standard proliferation media with or without serum (see methods) at 5 DIV showing that pericytes only survive in the presence of serum. qPCR data are presented as mean ± SD, n=4, biological replicates, Actb normalized data relative to no serum condition.

Scale bars (K) 50 µm, (L) 100 µm. ∗∗∗∗p < 0.0001, ***p < 0.001, **p < 0.01; ANOVA Tukey’s post hoc test in (F), t-test in (J).

Expression and function of individual NVOF components

(A) Representative image of Neurog2-GFP-induced neurons showing TUJ1 expression (A’).

(B, C) Representative images of control-GFP versus VP16:Olig2-GFP-transfected SOX6+/NG2+ progenitors. Overexpression of VP16:Olig2 represses T3-mediated differentiation of progenitors into oligodendrocytes, causing transfected cells to acquire morphology of immature neuroblasts (n=3).

(D, E) Representative images of Fezf2-GFP transfected cells. Fezf2 overexpression in progenitors does not fully support neuronal differentiation, and many cells adopt a ‘chimeric’ morphology with glial-cell-like soma structures and long processes. (n=2)

(F, G) Representative images of Ctip2-GFP transfected cells. Ctip2 overexpression in SOX6+/NG2+progenitors induces a striking, TUJ1+/OLIG2-axon-like neurite at 3 DPT, demonstrating inherent plasticity and neuronal competency in SOX6+/NG2+ progenitors. (H-J) Immunofluorescence of HEK293 cells transfected with NVOF confirms that GFP, NEUROG2 (H), and FEZF2-HA (I) are expressed appropriately. Immunocytochemistry for 2A peptides reports expression of GFP, NEUROG2, and VP16:OLIG2 (J).

(K-N) Testing NVOF in embryonic dorsal progenitors at E15.5 via in utero electroporation. E15.5 embryos were electroporated with control vector (GFP) (K, L) and NVOF (M, N), and analyzed at P7. Neurons electroporated with control vector project to contralateral cortex (K”), but not to subcortical targets (K’, K’’’, L’). After NVOF electroporation, many GFP+ axons descend through internal capsule (M’) towards thalamus (M’’’), and some of these axons even reach cerebral peduncle by P7 (N). Some NVOF-electroporated neurons still send axons to contralateral hemisphere through the corpus callosum (M”). Together, these data show that NVOF redirects axons of later-born upper-layer neurons from contralateral targets to subcortical targets, akin to deep-layer cortical output neurons. (n=4) Scale bars (A-G) 100 µm; (H-J) 50 µm.

NVOF-transduced progenitors rapidly lose progenitor identity and acquire cardinal features of mature functional neurons

(A-D) Representative images of NVOF-transfected progenitors at 3- and 6 DPT showing acquisition of neuronal morphology coupled with TUJ1 expression (filled arrows) and downregulation of the progenitor markers NG2 (a.k.a. Cspg4) and SOX10 (empty arrows).

(E) Representative low-magnification image of NVOF-induced neurons at 7 DPT. (E’) Pseudocolored GFP for better visualization of cell morphology.

(F-J) NVOF-induced neurons at 7 DPT express the cell adhesion molecule PSA-NCAM (F), which has a punctate distribution at 14 DPT (G), and the presynaptic molecules SYNAPSIN (Syn1/2) (H), SYNAPTOPHYSIN (Syp) (I) and VGLUT1 (J), which are key molecular features of glutamatergic neurons. Note the localization of SYNAPSIN and VGLUT1 to filopodial, presumptive presynaptic structures along axonal compartments.

(K) Representative low-magnification image of NVOF-induced neurons (green, GFP) co-cultured with primary neurons isolated from wild-type forebrain at P0 (red, Tuj1) in astrocyte-conditioned media, showing that NVOF-induced neurons intercalate with primary neurons at 14 DPT.

(L, M) Representative images of co-culture showing considerable morphological maturation and axon elongation of NVOF-induced neurons. Note dendrite size (filled arrows) and single axons protruding from cell bodies (empty arrows).

(N) Representative image of an NVOF-induced neuron with highly elongated dendritic structures, and abundant SYNAPSIN-positive presynaptic contacts from nearby primary neurons (N’).

Scale bars (A, B, C, D, I, J, L, M) 100 µm; (E) 200 µm; (F, G, H, N) 50 µm.

NVOF induces glutamatergic neurons from SOX6+/NG2+ progenitors with high fidelity and reproducibility.

(A, B) Volcano and heatmap of differentially expressed genes between NVOF-induced neurons and cells transfected with control-GFP.

(C) Volcano plot showing downregulation of progenitor genes in NVOF-induced neurons compared to control-GFP transfected cells at 7 DPT (n=3, biological replicates).

(D) Gene ontology (GO) term enrichment analysis of genes upregulated in NVOF-induced neurons for biological processes and cellular components.

(E-M) Representative images of NVOF-induced neurons with negative immunoreactivity for the non-glutamatergic neuronal markers GABA for GABAergic interneurons (E, E’), DARPP32 for striatal projection neurons (G, G’), 5-HT for serotonergic neurons (I, I’), TH for dopaminergic neurons (K, K’), and ISL1 for spinal motor neurons (M, M’), demonstrating that NVOF-induced neurons appropriately do not co-exhibit multiple neuronal identities. Cultured P2 primary neurons were used as a positive control for immunocytochemistry staining (F, H, J, L, N). Scale bars (E-N) 100 µm.

Synthetic mRNA mediates neuronal induction from SOX6+/NG2+ cortical progenitors

(A-B) Cultured progenitors are transfected with GFP RNA at higher efficiency (A) than GFP DNA (B).

(C) Co-transfection of GFP RNA and tdTomato DNA is highly efficient.

(D, G) Time course of gene expression in cultured progenitors transfected with GFP RNA shows that GFP expression begins by 6 hours-post-transfection (D), peaks between 12 and 24 hours (E, F), then declines (G).

(H-I) One pulse of Neurog2 RNA induces morphologically complex, TUJ1+ neurons from SOX6+/NG2+ cortical progenitors (H), albeit at a lower efficiency than Neurog2 or NVOF DNA constructs (I). Percentage of TUJ1+, neuron-like cells in cells transfected with GFP control RNA, Neurog2 RNA, and Neurog2 DNA relative to the number of TUJ1+ cells after NVOF transfection (I). Error bars show standard deviations. ∗∗∗∗p < 0.0001, **p < 0.01, *p < 0.05, t-test. (n=3)

(J) Co-transfection of Neurog2 RNA and Fezf2-GFP DNA induces TUJ1+ neurons from SOX6+/NG2+ progenitors. Scale bars (A-J) 100 µm.

Neurog2 is not sufficient to induce molecular hallmarks of cortical output neurons

(A) Percentage of Neurog2- and NVOF-induced neurons expressing CTIP2, PCP4, FOG2, SATB2, and CTIP1 at 7 DPT.

(B) Quantification of mean fluorescence intensity for CTIP2 (n=∼700), PCP4 (n=∼450), FOG2 (n=∼450), and CTIP1 (n=∼150) in Neurog2- and NVOF-induced neurons at 7 DPT. Plotted values are mean nuclear intensity of individual neurons normalized to the average intensity of the three lowest-expressing neurons.

(C-D) Representative images of CTIP1 expression by Neurog2- (C) and NVOF-induced neurons (D). Scale bar (C, D) 100 µm. Error bars show standard deviations. ∗∗∗∗p < 0.0001, t-test in (B).