Abstract
Neural progenitor cells in the developing dorsal forebrain generate excitatory neurons followed by oligodendrocytes (OLs) and astrocytes. However, the specific mechanisms that regulate the timing of this neuron–glia switch are not fully understood. In this study, we show that the proper balance of Notch signaling in dorsal forebrain progenitors is required to generate oligodendrocytes during late stages of embryonic development. Using ex vivo and in utero approaches in mouse embryos of both sexes, we found that Notch inhibition reduced the number of oligodendrocyte lineage cells in the dorsal pallium. However, Notch overactivation also prevented oligodendrogenesis and maintained a progenitor state. These results point toward a dual role for Notch signaling in both promoting and inhibiting oligodendrogenesis, which must be fine-tuned to generate oligodendrocyte lineage cells at the right time and in the right numbers. We further identified the canonical Notch downstream factors HES1 and HES5 as negative regulators in this process. CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-mediated knockdown of Hes1 and Hes5 caused increased expression of the pro-oligodendrocyte factor ASCL1 and led to precocious oligodendrogenesis. Conversely, combining Notch overactivation with ASCL1 overexpression robustly promoted oligodendrogenesis, indicating a separate mechanism of Notch that operates synergistically with ASCL1 to specify an oligodendrocyte fate. We propose a model in which Notch signaling works together with ASCL1 to specify progenitors toward the oligodendrocyte lineage but also maintains a progenitor state through Hes-dependent repression of Ascl1 so that oligodendrocytes are not made too early, thus contributing to the precise timing of the neuron–glia switch.
SIGNIFICANCE STATEMENT Neural progenitors make oligodendrocytes after neurogenesis starts to wind down, but the mechanisms that control the timing of this switch are poorly understood. In this study, we identify Notch signaling as a critical pathway that regulates the balance between progenitor maintenance and oligodendrogenesis. Notch signaling is required for the oligodendrocyte fate, but elevated Notch signaling prevents oligodendrogenesis and maintains a progenitor state. We provide evidence that these opposing functions are controlled by different mechanisms. Before the switch, Notch signaling through Hes factors represses oligodendrogenesis. Later, Notch signaling through an unknown mechanism promotes oligodendrogenesis synergistically with the transcription factor ASCL1. Our study underscores the complexity of Notch and reveals its importance in regulating the timing and numbers of oligodendrocyte production.
Introduction
Neural progenitor cells in the brain have the tremendous task of generating many types of functionally distinct neurons and glia at specific times throughout development. As a population, progenitors in the dorsal forebrain first give rise to excitatory neurons before they start producing glial cells like astrocytes and oligodendrocytes (Sauvageot and Stiles, 2002; Guillemot, 2007; Franco and Müller, 2013). Thus, a balance between proliferation and differentiation within the progenitor pool is necessary to produce earlier-born neurons followed by later-born glia (Sauvageot and Stiles, 2002; Götz and Huttner, 2005; Huttner and Kosodo, 2005; Kriegstein et al., 2006; Oproescu et al., 2021). The timing of this neuron–glia switch is determined by both cell-intrinsic factors and position-dependent extracellular cues (Sauvageot and Stiles, 2002; Guillemot, 2007; Franco and Müller, 2013). However, the specific mechanisms that coordinate the timing and balance among progenitor maintenance, neurogenesis, and oligodendrogenesis are not fully understood.
In many contexts, the Notch signaling pathway temporally controls cell fate specification by preserving the progenitor pool and instructing differential cell fates (Artavanis-Tsakonas et al., 1999; Hatakeyama et al., 2004). Notch ligands, such as the transmembrane protein Delta-like ligand 1 (DLL1), on the surface of signal-sending cells bind to another transmembrane protein, the Notch receptor, on the surface of neighboring cells. Transactivation of the Notch signaling pathway in the cells expressing the Notch receptor causes differences in gene expression between the Notch-expressing cell and the DLL1-expressing cell, thereby instructing different fates (Artavanis-Tsakonas et al., 1999; Andersson et al., 2011). In the developing dorsal forebrain, DLL1 and DLL3 are primarily expressed in newborn neurons and intermediate progenitor cells (IPCs), whereas neural progenitors express the Notch receptors 1–3 (Kawaguchi et al., 2008; Yoon et al., 2008). These neurons and IPCs feed back to neural progenitor cells via cell–cell contact to potentiate Notch signaling, resulting in progenitor expression of Notch target genes like Hes1 and Hes5. HES1 and HES5 are transcription factors that inhibit neuronal specification genes, thus preserving a pool of progenitors that can expand as brain development proceeds (Kageyama et al., 2007; Yoon et al., 2008; Hevner, 2019; Ohtsuka and Kageyama, 2021). Accordingly, perturbing Notch signaling early in development depletes progenitors and disrupts the timing and numbers of neuron production (Hatakeyama et al., 2004; Kawaguchi et al., 2008; Bansod et al., 2017).
Studies in the developing spinal cord suggest that Notch signaling can promote oligodendrocyte specification. For example, time-dependent, reduced Notch signaling in the zebrafish spinal cord resulted in the production of excess neurons and fewer oligodendrocyte precursor cells (OPCs; Park and Appel, 2003). Although Notch signaling has been shown to have an early developmental role in progenitor maintenance and neurogenesis in the forebrain (Hatakeyama et al., 2004; Gao et al., 2009), it is not known whether Notch signaling is important for oligodendrogenesis during later stages of forebrain development.
In the current study, we tested the role of Notch signaling in oligodendrocyte fate specification in the mouse dorsal forebrain. We found that blocking Notch signaling pharmacologically or genetically, both in vitro and in vivo, disrupted the ability of dorsal forebrain progenitors to produce OPCs during the neuron–oligodendrocyte switch. Interestingly, overactivation of Notch signaling also inhibited oligodendrogenesis, suggesting that Notch signaling levels must be precisely balanced to allow dorsal progenitors to generate proper numbers of oligodendrocyte lineage cells. We then combined in utero electroporation with CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 approaches to uncover the molecular mechanisms underlying this dual role of Notch signaling. Knocking down the Notch transcriptional effectors HES1 and HES5 led to an increase in OPCs, along with an increase in expression of the pro-OPC transcription factor ASCL1. Finally, we found that increasing Notch signaling, while simultaneously increasing ASCL1 levels to overcome the inhibition by HES1/5, significantly increased oligodendrogenesis. Our study indicates a potential bimodal transcriptional mechanism by which Notch signaling regulates the balance between progenitor maintenance and OPC production for the dorsal forebrain at the right time and in the right numbers.
Materials and Methods
Mouse lines
The following mouse lines were obtained from The Jackson Laboratory: B6 (C57BL/6J, stock #000664), R26NZG (B6.Cg-Gt(ROSA)26Sortm1(CAG0lacZ,-EGFP)Glh/J, stock #012429), and Emx1-Cre (B6.129S2-Emx1tm1(cre)Kri/J, stock #005628). We also obtained timed pregnant Crl:CD1(ICR) mice (strain no. 022) from Charles River Laboratories, which arrived at embryonic day (E)12.5 and on which we performed electroporations at E15.5. For timed matings of B6 wild-type mice, we considered embryos to be at gestation day 0.5 on the day when the vaginal plug was detected. Emx1-Cre (cre/cre) mice were crossed with R26NZG (flox/flox) mice to generate pregnant dams carrying double heterozygous embryos for forebrain slice culture experiments at E15.5. Mouse lines were authenticated via genotyping by PCR. Animals were maintained according to the guidelines from the Institutional Animal Care and Use Committee of the University of Colorado–Anschutz Medical Campus. Sex of embryos was not determined for experiments.
Expression plasmids
A piggyBac (PB) transposase system was used for in utero electroporation experiments to permit stable integration of the reporter plasmid into electroporated progenitors (García-Moreno et al., 2014). We found this approach to be necessary to label the oligodendrocyte lineage, in line with previous reports that episomal plasmids are silenced or lost in glial lineages (García-Marqués and López-Mascaraque, 2013; Siddiqi et al., 2014).
DNA fragments for cDNAs encoding mouse ASCL1, mouse NOTCH1 Intracellular Domain (NICD; amino acids 1749–2293), and the dominant-negative (DN) version of mouse RBPJ (DN-RBPJ) were synthesized and purchased from Integrated DNA Technologies (IDT) as gBlocks. The DN-RBPJ sequence contains a mutation at R218H, which disrupts the DNA-binding activity of RBPJ but can still bind and sequester NICD (Chung et al., 1994). ASCL1, NICD, and DN-RBPJ cDNA gBlocks were cloned using the NEBuilder HiFi DNA Assembly Master Mix into a piggyBac (pPB) expression vector containing a CMV early enhancer element and chicken beta-actin (CAG) promoter, and IRES-GFP (pPB-CAG-IRES-GFP). pPB-CAG-IRES-GFP empty vector was used as the GFP control. DN-RBPJ cDNA gblock was also cloned into a pPB-CAG-IRES-mTagBFP2 backbone, in which GFP was replaced by mTagBFP2. All constructs were confirmed by restriction digest and whole-plasmid DNA sequencing (plasmidsaurus). The PBase expression plasmid CMV-mPB was described previously (Winkler et al., 2018).
CRISPR/Cas9 plasmids
We generated CRISPR/Cas9 dual guide plasmids containing two adjacent U6 promoters driving expression of two separate guide RNAs. CRISPR plasmid backbones were a gift from Joseph Brzezinski (Ran et al., 2013; Kaufman et al., 2021). We ordered paired oligonucleotides for each guide from IDT (sequences are provided in Table 1) and phosphorylated and annealed them together as described previously (Ran et al., 2013). We then cloned Guide 1 into the CRISPR plasmid backbone and Guide 2 into a shuttle plasmid backbone. The U6 and guide cassette for Guide 2 in the shuttle plasmid was then subcloned into the plasmid containing Guide 1 using the restriction enzymes XbaI and KpnI (Kaufman et al., 2021). The final plasmids also contained an EF1alpha promoter sequence and reporter sequences for cytoplasmic mCherry (Ef1alpha-pSpCas9-2A-mCherry; Kaufman et al., 2021). As controls, we used a CRISPR plasmid that expressed guide RNAs that do not target any mouse genes [CRISPR nontargeting (NT) control], cloned into the CRISPR vector expressing a nuclear localized (EF1alpha-pSpCas9-2A-H2B-mCherry) mCherry (Kaufman et al., 2021).
Oligonucleotides
CRISPR guide sequences were designed to target the protein-coding exon sequences of Hes1 and Hes5. We used the CRISPR design and Analyze Guides tools from Benchling (https://www.benchling.com) to design dual guide RNAs with on-target scores that were >70 and off-target scores that were <50. For Hes1, we selected guides that flanked Exon 1 for removal. For Hes5, we chose a guide 5′ to Exon 1 and another guide 3′ to Exon 3, for removal of Exons 1–3. We used the BLAST program to screen each guide to identify any possible off-target effects on coding sequences. Efficiency of CRISPR targeting of Hes1 and Hes5 was tested in vivo using in utero electroporation at E15.5 (see Fig. 6). We tested Hes1 targeting by IHC for HES1 protein on electroporated tissue at E17.5. As there is no commercially available antibody against HES5 that works by IHC, we further tested CRISPR targeting of Hes1 and Hes5 by fluorescence activated cell sorting (FACS) of electroporated cells at E17.5 followed by PCR of genomic DNA (see below).
In utero electroporation and injection
In utero electroporations were performed as described previously (Franco et al., 2011). Survival surgeries were performed on timed pregnant mice (E15.5), to expose their uterine horns. Approximately 1 μl of endotoxin-free plasmid DNA was injected into the lateral ventricles of each embryo. The concentration of each injection solution was the following: DN-RBPJ-IRES-GFP, DN-RBPJ-IRES-mTagBFP2, NICD-IRES-GFP, ASCL1-IRES-GFP, GFP control, Hes1 CRISPR, Hes5 CRISPR, or CRISPR NT control at 1 mg/ml and CMV-mPB at 0.3 mg/ml. For E15.5 electroporations, five pulses separated by 950 ms were applied at 45 V. Embryos were put back into the abdominal cavity, and the pregnant dams were sutured. Embryos were allowed to develop in utero for the indicated time before their brains were dissected. In utero injections were performed similarly to in utero electroporations without the electrical pulses. To account for dilution of the injection mix in the CSF, a higher concentration of DAPT and DMSO was used for in utero injections compared with slice cultures. One microliter of 10% DMSO or 50 μm DAPT in 10% DMSO was injected into the lateral ventricles of each embryo at E15.5.
Embryonic forebrain slice culture
Whole brains from E15.5 wild-type CD-1 mice or Emx1-Cre (+/cre);NZG (+/fl) mice were dissected and placed in ice-cold Complete HBSS (1× HBSS, 2.5 mm Hepes, 30 mm D-glucose, 1 mm CaCl2, 1 mm MgSO4, 4 mm NaHCO3). Brains were embedded in 3% Low Melting Point Agarose dissolved in Complete HBSS and allowed to solidify on ice. Embedded brains were sliced using a vibratome (Leica VT1200 S) into 300-µm-thick slices and placed in Complete HBSS. Slices were transferred into uncoated Millicell cell culture membrane inserts in six-well plates and cultured in Slice Culture Media (Complete HBSS, Basal Medium Eagle, 20 mm D-glucose, 1 mm l-glutamine, penicillin-streptomycin) at 37°C, 5% CO2, and 100% humidity. For drug treatments, 100% DMSO was added to the Slice Culture Media for a final concentration of 0.1% DMSO. One hundred percent DMSO was used to make a 10 mm DAPT stock solution, which was added to the Slice Culture Media for a final concentration of 10 μm DAPT and 0.1% DMSO. Slices were plated immediately with 1.5 ml of either the DMSO- or DAPT-containing culture media. An additional 1.5 ml of DMSO- or DAPT-containing culture media was added to the slice cultures after 1 d of incubation. After 2 d in vitro (DIV), cell culture media were aspirated, and slices were washed in 1× PBS and fixed in cold 4% PFA for 30 min. Fixed slices were washed twice with 1× PBS and then used for immunohistochemical analysis as described below.
Immunohistochemistry
Embryonic brains were fixed in 4% paraformaldehyde (PFA) for 1 h at room temperature (RT). Brains were sectioned coronally at 100 µm with a vibrating microtome. Free-floating sections were placed in 24-well plates and blocked with 500 μl of 10% donkey serum and 0.2% Triton X-100 in 1× PBS for 2 h at RT. Blocking solution was then removed, and sections were incubated with primary antibodies in 10% donkey serum and 0.2% Triton X-100 in 1× PBS overnight (16 h) at 4°C. Primary antibody solution was then removed, and sections were washed at RT with 1× PBS three times for 5 min each. After washing, sections were incubated with secondary antibodies in 1× PBS for 1 h at RT. Sections were then washed again using 1× PBS three times for 5 min each. Sections were mounted on glass slides with ProLong Diamond Antifade Mountant. Images were captured using a Zeiss LSM 900 laser scanning confocal microscope in Airyscan 2 Multiplex 4Y mode at 20× or 40× magnification. Antibodies used for immunostaining are listed in Table 2. The concentration of each primary antibody used was the following: mouse anti-OLIG2 (1:500), rabbit anti-OLIG2 (1:500), goat anti-OLIG2 (1:1000), rabbit anti-ASCL1 (1:500), chicken anti-β-gal (1:1000), rat anti-PDGFRα (1:1000), rabbit anti-TagRFP to detect mTagBFP2 (1:500), rat anti-RFP to detect mCherry (1:500), rabbit anti-RFP to detect mCherry (1:500), chicken anti-GFP (1:500), rabbit anti-SATB2 (1:1000), rabbit anti-CC3 (1:500), mouse anti-KI67 (1:250), rabbit anti-TBR2 (1:500), rabbit anti-HES1 (1:500), and rabbit anti-PAX6 (1:500). Donkey secondary antibodies conjugated to Alexa Fluor 488, Rhodamine Red-X, Alexa Fluor 647, or Alexa Fluor 405 were used at 1:500.
Antibodies
Microdissection, tissue dissociation, and FACS
Mouse embryos were electroporated with CRISPR plasmids expressing mCherry at E15.5. At E17.5, electroporated brains were collected and microdissected in 1× Neurobasal-A medium under a microscope. Microdissected tissue from three to five brains were pooled for each sample and then dissociated with the Worthington Papain Dissociation System according to the Dissociation of Mouse Embryonic Neural Tissue protocol (document CG00053-Rev C) by 10x Genomics. Dissociated samples were strained with MACS SmartStrainers into 1× HBSS and 10% FBS solution, and FACS was performed to collect mCherry+ cells for each sample. Dissociated, nonelectroporated brain tissue was used as a negative control to exclude background fluorescence, and DAPI was added to each sample to exclude dead cells.
CRISPR validation by genomic DNA extraction, PCR, and sequencing of PCR product
Following FACS of mCherry+ cells, genomic DNA (gDNA) was isolated from each sample using the New England Biolabs Monarch Genomic DNA Extraction Kit. PCR primers were designed to encompass the genomic DNA within the Hes1 and Hes5 locus to be targeted by CRISPR gRNAs and to produce a product of 230 bp (Hes1) or 184 bp (Hes5). PCR was performed on isolated gDNA using Phusion DNA polymerase, and PCR products were run on 3% agarose gel by electrophoresis. A decrease in band intensity is indicative of gene deletion as one of the primers in each pair is between the two guide targets for each gene. The 230 bp PCR product for Hes1 was completely absent, indicating very efficient knockout of the targeted region in electroporated cells. Because the PCR product for Hes5 was decreased but not absent, we also tested for insertions and deletions (indels) by sequencing the PCR product. Following PCR cleanup, the Hes5 CRISPR PCR product was submitted for sequencing using the same primers used for PCR. Sequencing data were aligned with the Hes5 genomic DNA sequence from the Benchling website (https://www.benchling.com) to identify indels around the targeted area. All chemicals and reagents are listed in Table 3.
Chemicals and reagents
Experimental design and statistical analysis
All images were exported in TIFF or JPEG format with no compression. Brightness, contrast, and background were adjusted equally for the entire image between controls and mutants using the Brightness/Contrast and Levels functions from the Image/Adjustment options in Adobe Photoshop or Fiji software (Schneider et al., 2012) without any further modification.
For all immunostainings, three or more histologic sections at three distinct rostral–caudal z-planes from each of three to five different animals (at least nine sections total for each condition) were analyzed in the entire dorsal pallium. Biological replicates are individual animals. For slice cultures, biological replicates were individual slices from three to four different animals that were histologically matched at the rostral–caudal level between untreated and treated groups. Confocal, single-plane optical sections were used for quantification. Cells were analyzed in columns spanning the entire dorsal pallium across the lateral–medial axis. Total number of marker-positive cells were quantified from these columns in Fiji and then divided by the area of a column to get cell density (cells/mm2). The cell density was averaged for each animal. To determine the number of dorsal progenitors undergoing cell death in slice cultures, the number of cells that were CC3+ per mm2 was counted in the ventricular and subventricular zones across the dorsal pallium of each brain slice and averaged. Cell death in the oligodendrocyte lineage was analyzed by quantifying the percentage of OLIG2+ and OLIG2+ PDGFRα+ cells present in the dorsal pallium that were positive for CC3. For electroporations, the percentage of electroporated cells that were positive for a marker was calculated and averaged for each animal. For independent two-group experiments, an unpaired two-tailed Student’s t test was used to determine statistical significance between two groups with equal variance. For comparisons between two groups with unequal variance, Welch’s t test was used. For analysis involving three or more independent groups, a one-way ANOVA was used followed by Tukey’s post hoc test. Values were considered statistically significant at p < 0.05.
Results
The Notch signaling pathway regulates oligodendrogenesis in the dorsal forebrain
We previously showed that neural progenitors in the dorsal forebrain begin producing OLIG2+ cells during late embryonic stages and are the primary source of OLIG2+ cells from E15.5 and on (Kessaris et al., 2006; Winkler et al., 2018). These OLIG2+ cells then express PDGFR α, at which point they are defined as OPCs (Pringle and Richardson, 1993; Zhou et al., 2000). To test whether Notch signaling is important for generating OPCs in the mouse dorsal forebrain, we inhibited Notch signaling activity using an ex vivo slice culture system (Polleux and Ghosh, 2002). We cultured forebrain slices from E15.5 embryos, a time just before dorsal forebrain progenitors start making OPCs (Winkler et al., 2018). Notch-mediated transcriptional activation requires cleavage and release of the NICD from the plasma membrane by γ-secretases (Andersson et al., 2011). We treated forebrain slice cultures with the γ-secretase inhibitor DAPT at 10 μm for 2DIV before fixing and staining forebrain slices for OLIG2 and PDGFR α to identify cells of the oligodendrocyte lineage (Fig. 1A). After 2DIV, control forebrain slices treated with DMSO vehicle control produced many OLIG2+ cells (Fig. 1B). However, treatment with DAPT resulted in fewer OLIG2+ cells present in the dorsal pallium (Fig. 1B,C). Although CC3 staining indicated some cell death occurred in neurons in the cortical plate in both DMSO- and DAPT-treated slices, we did not observe any differences in the number of CC3+ progenitors in the ventricular and subventricular zones (Fig. 2A,B). These findings suggested a role for the Notch signaling pathway in generating the oligodendrocyte lineage from dorsal progenitors.
Notch pathway inhibition blocks dorsal OPC production in forebrain slice cultures. A, Schematic of ex vivo forebrain slice culture approach. Brains of E15.5 mouse embryos were sectioned and cultured with or without DAPT. At 2DIV, forebrain slices were fixed and stained for OLIG2 and PDGFR α to identify oligodendrocyte lineage cells. B, Representative images of OLIG2+ PDGFR α+ oligodendrocyte lineage cells in the pallium of forebrain slices cultured in DMSO control or 10 μm DAPT. Dotted lines outline the dorsal (top) and ventral (bottom) limits of the pallium. Scale bar, 100 µm. C–E, Quantification of oligodendrocyte lineage cells from cultured slices. Total numbers of OLIG2+ cells (C), OLIG2+ PDGFRα− cells (D), and OLIG2+ PDGFR α+ cells (E) were counted (per mm2) in the pallium of DMSO control and 10 μm DAPT-treated forebrain slice cultures. Graphs show the average ± SEM among biological replicates; N = 4 forebrain slices for each condition. F, Schematic of genetic lineage tracing approach to identify dorsally derived and ventrally derived cells. Emx1-Cre mice were crossed to R26-NZG reporter mice to permanently label all EMX1+ progenitors and their progeny with β-gal. Forebrain slice cultures from Emx1-Cre;NZG mouse embryos were prepared at E15.5 and treated with DMSO or 10 μm DAPT. G, Representative images of DMSO-treated and 10 μm DAPT-treated forebrain slice cultures. Left, in each condition, Overview; right, zoomed-in images. Dorsally derived (β-gal+) oligodendrocyte lineage cells are outlined in white in the DMSO control images. Ventrally derived (β-gal−) oligodendrocyte lineage cells are outlined in yellow in DAPT. Scale bars: left, overview, 100 µm; right, zoomed in, 50 µm. H, Graph of the average percentage (±SEM among biological replicates) of total OLIG2+ PDGFR α+ cells in the pallium that were dorsally derived (β-gal+) versus ventrally derived (β-gal−) at 2DIV. I, Quantification of the average ± SEM among biological replicates for β-gal+ OLIG2+ PDGFR α+ cells per mm2 (Dorsal OPCs). DMSO control, N = 4; 10 μm DAPT, N = 3. SEM, standard error of the mean; n.s., not significant.
Notch inhibition does not increase cell death of dorsal forebrain progenitors or oligodendrocyte lineage cells in slice cultures. A, Representative images of CC3+ cells in the dorsal pallium VZ/SVZ of forebrain slices cultured in DMSO control or 10 μm DAPT. Bottom, Dotted lines outline the ventral limits of the pallium. Scale bar, 50 µm. B, Total numbers of CC3+ cells were counted (per mm2) in the VZ/SVZ of the dorsal pallium, where dorsal progenitors reside. Graphs show the average number (±SEM among biological replicates) of CC3+ cells (per mm2); N = 3 forebrain slices for each condition. VZ/SVZ, Ventricular/subventricular zone. C, Representative images of OPCs in DMSO control and 10 μm DAPT-treated forebrain slice cultures at 2DIV. Yellow arrowheads show OLIG2+ PDGFR α+ cells that were CC3−. Scale bar, 50 µm. D, E, Quantification of CC3+ oligodendrocyte lineage cells from cultured slices. Graphs show the average percentage (±SEM among biological replicates) of total OLIG2+ cells (D) and OLIG2+ PDGFR α+ cells (E) in the pallium that expressed CC3 at 2DIV. N = 3 forebrain slices for each condition. SEM, standard error of the mean; n.s., not significant.
Many of the OLIG2+ cells at this stage are pre-OPCs that have not yet started expressing PDGFR
To test whether Notch signaling is important for OPC genesis in vivo, we performed in utero injections of DAPT or DMSO vehicle control into the lateral ventricles of E15.5 mouse embryos (Fig. 3A). At E17.5, we fixed, sectioned, and stained injected brains for the OPC markers OLIG2 and PDGFR α. Two days after DMSO control treatment, the dorsal pallium was populated by OLIG2+ PDGFR α+ OPCs, as we have previously shown (Winkler et al., 2018). However, DAPT injection reduced the number of OPCs in the dorsal pallium by about threefold (Fig. 3B,C). Together with our ex vivo slice culture experiments, these results indicate that the Notch signaling pathway is important for specifying dorsal forebrain progenitors toward the oligodendrocyte lineage.
Notch inhibition reduces the number of OPCs in the pallium in vivo. A, Schematic of in utero drug injection approach. DAPT or DMSO was injected into the lateral ventricles of mouse embryos at E15.5, then analyzed at E17.5 by staining for OLIG2 and PDGFR α to identify oligodendrocyte lineage cells. B, Representative images of the pallium of DMSO- and DAPT-treated brains. C, Graph of the average number (±SEM among biological replicates) of OLIG2+ PDGFR α+ cells (per mm2). N = 3 brains for each condition. Scale bar, 100 µm. SEM, standard error of the mean.
The Notch cofactor RBPJ is cell autonomously required for dorsal progenitors to generate OPCs
As γ-secretase processes the cleavage of multiple proteins, the reduced OPC phenotype resulting from DAPT treatment may not be specific to the Notch signaling pathway (Barthet et al., 2012). Therefore, we used in utero electroporation to specifically target the Notch pathway in a spatially and temporally controlled manner. We used a piggyBAC transposase system to stably integrate plasmids into the genome of electroporated progenitors, thus allowing us to additionally track progenitors and their progeny using GFP fluorescence (García-Moreno et al., 2014).
Following activation and γ-secretase-dependent cleavage of the Notch receptor, NICD forms a transcriptional complex with the cofactor RBPJ, which binds to DNA and activates transcription of target genes such as Hes1 and Hes5 (Andersson et al., 2011). Notch signaling can be inhibited by a DN mutation in RBPJ (DN-RBPJ) that disrupts the DNA-binding domain but still binds NICD, thereby blocking downstream transcriptional activation of Notch target genes (Chung et al., 1994). We used in utero electroporation to express DN-RBPJ-IRES-GFP or GFP alone in dorsal progenitors at E15.5, allowed the embryos to mature to E18.5, and analyzed the electroporated cells for expression of OLIG2 and PDGFR α (Fig. 4A,B). We found many electroporated GFP+ cells in control brains that were colabeled with the OPC markers OLIG2 and PDGFR α (Fig. 4C). However, very few cells expressing DN-RBPJ-IRES-GFP were OPCs (Fig. 4D). We quantified the percentage of GFP+ cells that were OLIG2+ and found approximately a twofold reduction (Fig. 4E), with about a threefold reduction in GFP+ cells that were OLIG2+ PDGFR α+ (Fig. 4F). These results demonstrate that the Notch cofactor RBPJ is cell autonomously required for dorsal progenitors to generate OPCs. We also found an increase in the number of cells expressing the upper-layer neuron marker SATB2 (Fig. 4G,H). This increase in neuronal production (71.0 to 76.6% = +5.57%) roughly corresponded to the decrease in OLIG2+ cells (5.24 to 1.55% = −3.69%), although the increase in neurons was not statistically significant, likely because of the high percentage of electroporated cells that normally make SATB2+ neurons. Together, these data suggest that Notch signaling is important for the gradual transition from neurogenesis to oligodendrogenesis from E15.5 to 18.5.
Notch signaling activity is cell autonomously required for dorsal OPC genesis through the cofactor RBPJ. A, Schematic of in utero electroporation approach. Mouse embryos were electroporated with GFP control or DN-RBPJ-IRES-GFP at E15.5 then dissected at E18.5 and analyzed by IHC for OLIG2 and PDGFR α to identify oligodendrocyte lineage cells. B, Representative overview images of the pallium of E18.5 brains electroporated with GFP control or DN-RBPJ-IRES-GFP. Boxed area is enlarged in C and D. Scale bar, 200 µm. C, D, Representative higher-magnification images of brains electroporated with GFP control (C) and DN-RBPJ-IRES-GFP (D). Left, Boxed areas in the overview are shown enlarged at right. White arrowheads denote GFP+ OLIG2+ PDGFR α+ cells, yellow arrowheads denote GFP+ OLIG2− PDGFR α− cells. EP, electroporated. Scale bars: left, overview, 100 µm; right, zoomed in, 50 µm. E, F, Quantification of oligodendrocyte lineage cells among electroporated cells. Graphs show the average percentage (±SEM among biological replicates) of electroporated (GFP+) cells that were OLIG2+ (E) or OLIG2+ PDGFR α+ (F). N = 5 brains for each condition. G, Representative images of SATB2 expression in GFP control and DN-RBPJ-IRES-GFP brains. Scale bar, 100 µm. H, Graphs show the average (±SEM among biological replicates) percentage of total GFP+ cells that were SATB2+ or OLIG2+; *p = 0.01. SEM, standard error of the mean; n.s., not significant.
Because our Notch pathway loss-of-function experiments indicated a role for Notch signaling in promoting an OPC fate, we next wanted to test whether Notch gain of function could increase oligodendrocyte fate specification from dorsal progenitors. We used in utero electroporation to express NICD-IRES-GFP in progenitors at E15.5 and analyzed brains at E18.5 (Fig. 5A). Nearly all NICD-expressing cells were found in the ventricular zone and expressed the progenitor marker PAX6, consistent with previous studies showing that NICD overexpression promotes radial glia identity and prevents neurogenesis (Gaiano et al., 2000; Mizutani et al., 2007; Fig. 5B,D). Compared with controls expressing GFP alone, progenitors overexpressing NICD produced about twofold fewer OLIG2+ cells (Fig. 5B,C). Thus, rather than promoting oligodendrocyte fate, overactivation of Notch signaling inhibited oligodendrogenesis. Together with our loss-of-function studies, these results indicate that Notch signaling levels must be precisely balanced to allow dorsal progenitors to generate proper numbers of oligodendrocyte lineage cells.
NICD overexpression prevents generation of OLIG2+ cells from dorsal progenitors. A, Schematic of constructs for GFP control and NICD-IRES-GFP. Mouse embryos were electroporated at E15.5 and stained for OLIG2 to identify oligodendrocyte lineage cells at E18.5. B, Representative images of brains electroporated with GFP control or NICD-IRES-GFP. Bottom, Dotted lines outline the ventral limits of the pallium. White arrowheads denote GFP+ OLIG2+ cells, yellow arrowheads denote GFP+ OLIG2− cells. Scale bar, 50 µm. C, Graph of the average percentage (±SEM among biological replicates) of electroporated (GFP+) cells that were OLIG2+. N = 3 brains for each condition. D, Representative images of PAX6 expression in GFP control and NICD-IRES-GFP brains. Scale bar, 100 µm. SEM, standard error of the mean.
The Notch downstream transcription factor HES1 inhibits OPC production from dorsal progenitors
Our results suggested that the Notch pathway is required for an oligodendrocyte fate but too much Notch signaling prevents oligodendrogenesis. We therefore wanted to investigate the molecular mechanisms underlying this dual role of Notch signaling. Following Notch pathway activation, the NICD-RBPJ complex induces transcription of Hes1 and Hes5, which encode for the canonical Notch pathway basic helix-loop-helix (bHLH) transcription factors (Ohtsuka et al., 1999). Previous studies showed that HES1 and HES5 are important for the production of different cortical neuron subtypes at the proper times, as well as the differentiation of astrocytes (Wu et al., 2003; Chenn, 2009; Bansod et al., 2017). However, the roles of HES1 and HES5 in oligodendrocyte fate specification are unknown. We reasoned that knocking down HES1 and HES5 would distinguish their roles in either the positive or negative regulation of oligodendrogenesis.
We used a plasmid-based CRISPR/Cas9 approach (Kaufman et al., 2021) to knock down expression of Hes1 or Hes5 by in utero electroporation of dorsal forebrain progenitors. We designed plasmids that targeted either Exon 1 of Hes1 (Hes1 CRISPR) or Exons 1–3 of Hes5 (Hes5 CRISPR). The plasmids also included an mCherry reporter to identify the electroporated cells (Fig. 6A). As controls, we electroporated a CRISPR plasmid that expressed guide RNAs that do not target any mouse genes (CRISPR NT control). Efficiency of targeting was confirmed by IHC showing loss of HES1 protein in the electroporated region (Fig. 6A,B), and by PCR on genomic DNA from electroporated cells demonstrating that the targeted regions were either absent (Hes1) or decreased and mutated (Hes5) compared with CRISPR NT control (Fig. 6C–F).
Validation of Hes1 and Hes5 CRISPR. A, Schematic of in utero electroporation with dual guide CRISPR/Cas9 approach. Mouse embryos were electroporated with a CRISPR plasmid that targeted Hes1 and expressed cytoplasmic mCherry or with a nontargeting CRISPR plasmid that expressed nuclear mCherry. B, Representative images of HES1 protein expression in CRISPR NT control or Hes1 CRISPR. In CRISPR NT control, HES1 was expressed in the ventricular zone within the electroporated region (magenta bar). In Hes1 CRISPR, HES1 expression in the ventricular zone was normal within the nonelectroporated region (yellow bar), but absent in the ventricular zone of the electroporated region (magenta bar). Dotted lines outline the ventricular wall of the dorsal pallium. Scale bar, 100 µm. C, Workflow for validating CRISPR targeting by PCR on gDNA of FACSorted electroporated cells. D, E, Representative images of PCR products run on agarose gels, showing that the expected band (boxed in pink) for the targeted region was not present in Hes1 CRISPR (D) or was reduced in Hes5 CRISPR (E). A decrease in PCR product was indicative of excision of the genomic region between the two guide RNAs targeting each gene. F, DNA sequencing traces of PCR products from E for Control and Hes5 CRISPR. The DNA sequence in Control mapped perfectly to the reference genomic sequence for Hes5 exon 3. The DNA sequence in Hes5 CRISPR showed mismatches (highlighted in red) at the targeted sequence, indicating indels were present within the genomic region targeted by Hes5 CRISPR Guide 2. EP’d, electroporated; non-EP’d, nonelectroporated.
We first electroporated Hes5 CRISPR into E15.5 mouse embryos. At E18.5, we dissected and fixed electroporated brains and then sectioned and stained for OLIG2. Knocking down Hes5 did not affect the percentage of mCherry+ cells that were OLIG2+ (Fig. 7B,C). However, when we knocked down Hes1 in dorsal progenitors at E15.5, we found about a twofold increase in the proportion of electroporated cells that were OLIG2+ at E18.5 (Fig. 7B,D). These results indicate that the Notch downstream transcription factor HES1 normally inhibits oligodendrogenesis from dorsal forebrain progenitors during late embryonic stages.
CRISPR-mediated knockdown of Hes1 and Hes5 promotes production of oligodendrocyte lineage cells from dorsal progenitors. A, Schematic of in utero electroporation and dual guide CRISPR/Cas9 approach. E15.5 mouse embryos were electroporated with CRISPR plasmids that targeted Hes1 or Hes5 and expressed cytoplasmic mCherry or with a nontargeting CRISPR plasmid that expressed nuclear mCherry. At E18.5, brains were analyzed by IHC for OLIG2 to identify oligodendrocyte lineage cells that were electroporated and labeled by mCherry. B, Representative images showing mCherry+ OLIG2+ cells (white arrowheads) in CRISPR NT control, Hes5 CRISPR, Hes1 CRISPR, and Hes1 + Hes5 dKD. Left, Overview; right, zoomed-in image. Scale bars: left, overview, 200 µm; right, zoomed in, 50 µm. C–E, Quantification of oligodendrocyte lineage cells among electroporated cells. The average percentage (±SEM among biological replicates) of mCherry+ cells that were OLIG2+ was quantified for Hes5 CRISPR (C), Hes1 CRISPR (D), and Hes1 + Hes5 dKD. Control data in D was regraphed in E. N = 4 brains for Control; N = 3 brains for Hes1 CRISPR, Hes5 CRISPR, and Hes1 + Hes5 dKD. SEM, standard error of the mean; n.s., not significant.
We also performed double knockdown electroporations with CRISPR plasmids targeting both Hes1 and Hes5 [Hes1 + Hes5 double knockdown (dKD)]. Targeting both genes increased the percentage of OLIG2+ cells about five times more than controls (Fig. 7B,E). This increase was over two times more than Hes1 single knockdowns, indicating that HES1 and HES5 may partially compensate for each other in the single knockdowns. We did not observe any differences in the percentage of mCherry+ OLIG2+ cells that coexpressed KI67 between dKDs and controls. Therefore, the increase in OLIG2+ cells was not the result of increased proliferation (Fig. 8A,D). Importantly, Hes1 + Hes5 dKD increased the proportion of mCherry+ cells that were PDGFR α+ OPCs (Fig. 8C,F). Together, these results suggest that the canonical Notch downstream effectors, HES1 and HES5, are responsible for the negative regulation of OPC production by Notch signaling.
Hes1 + Hes5 CRISPR double knockdown promotes OPC specification and not proliferation. A, Representative images showing mCherry+ OLIG2+ cells that were KI67+ (white arrowheads) or KI67− (yellow arrowheads) in CRISPR NT control and Hes1 + Hes5 dKD. B, Representative images showing mCherry+ ASCL1+ cells (white arrowheads) in CRISPR NT control and Hes1 + Hes5 dKD. C, Representative images showing mCherry+ cells that were PDGFR α+ (white arrowheads) in CRISPR NT control and Hes1 + Hes5 dKD. D–F, Quantification of marker-positive cells among electroporated cells. Graphs of the average percentage (±SEM among biological replicates) of electroporated (mCherry+) cells that were KI67+ (D), ASCL1+ (E), and PDGFR α+ (F). N = 3 brains for each condition for graphs in D and E. N = 4 brains for each condition for graph in F. Scale bars: 50 µm. SEM, standard error of the mean; n.s., not significant.
Hes transcription factors inhibit ASCL1 expression
How might HES1/5 be preventing an OPC fate? We hypothesized that the Hes proteins may be repressing expression of Ascl1, a bHLH transcription factor that is important for the fate specification of the oligodendrocyte lineage in the spinal cord, ventral forebrain, and postnatal telencephalon (Parras et al., 2007; Sugimori et al., 2008; Nakatani et al., 2013; Vue et al., 2014; Kelenis et al., 2018). HES1 and HES5 both inhibit transcription of Ascl1 by binding to the promoter (Kageyama et al., 2007). To determine whether loss of Hes genes increased ASCL1 expression, we coelectroporated the Hes1 and Hes5 CRISPR plasmids into brains at E15.5 and stained for ASCL1 protein at E18.5. Indeed, Hes1 + Hes5 dKD brains exhibited a significant increase in the percentage of mCherry+ cells that were ASCL1+, compared with CRISPR NT controls (Fig. 8B,E). These data suggest that one mechanism by which increased Notch signaling can inhibit oligodendrogenesis is by repression of ASCL1 expression by Notch downstream effectors HES1 and HES5.
Notch activation and ASCL1 together drive production of oligodendrocyte lineage cells
If Notch signaling can promote an oligodendrocyte fate while simultaneously preventing oligodendrocyte production by repressing Ascl1, we reasoned that activating Notch while also increasing Ascl1 expression would robustly increase production of oligodendrocyte lineage cells. To test this hypothesis, we coelectroporated NICD together with an ASCL1 expression plasmid (Fig. 9A). ASCL1 overexpression alone increased the percentage of electroporated cells that were OLIG2+ compared with GFP control (Fig. 9B,E), consistent with the role of ASCL1 in promoting an oligodendrocyte fate (Parras et al., 2007; Sugimori et al., 2008; Nakatani et al., 2013). Moreover, coelectroporation of ASCL1 with NICD led to an even greater production of OLIG2+ cells from dorsal progenitors (Fig. 9B,E). Neither ASCL1 nor ASCL1 plus NICD changed the percentage of GFP+ OLIG2+ cells that were KI67+ (Fig. 10A,B), indicating that the increase was likely because of specification, not proliferation. None of the OLIG2+ cells in any of the conditions coexpressed markers of the neuronal lineage, including TBR2 (Fig. 11), suggesting that the increase was not because of the generation of confused cells but rather was a true fate shift toward the oligodendrocyte lineage.
NICD and ASCL1 operate synergistically to promote oligodendrogenesis. A, Schematic of constructs for ASCL1-IRES-GFP and NICD-IRES-GFP. Mouse embryos were electroporated at E15.5 and analyzed at E18.5 by IHC for OLIG2 and PDGFR α to identify oligodendrocyte lineage cells that were electroporated. B, Representative images showing electroporated (GFP+) cells that were OLIG2+ PDGFR α+ (white arrowheads). C, Schematic of constructs for ASCL1-IRES-GFP and DN-RBPJ-IRES-mTagBFP2. Brains were coelectroporated with ASCL1 + DN-RBPJ at E15.5 and analyzed at E18.5. Cells that were coelectroporated (GFP+ BFP+) were counted for OLIG2 and PDGFR α expression. D, Representative images of electroporated (GFP+ BFP+) cells that were double negative for OLIG2 and PDGFR α (yellow arrowheads). Scale bars: 50 µm. E, Quantification of the average percentage (±SEM among biological replicates) of electroporated cells that were OLIG2+ in GFP control, ASCL1 OE, ASCL1 OE + NICD, and ASCL1 OE + DN-RBPJ; one-way ANOVA, p = 4.10E-06, Tukey’s post hoc test, *p = 0.01, **p = 0.002, ****p = 0.000007. Statistical comparisons not shown in graph are GFP control with ASCL1 OE + NICD, p = 0.000009; GFP control with ASCL1 OE + DN-RBPJ, n.s.; ASCL1 OE with ASCL1 OE + DN-RBPJ, p = 0.004. GFP control, N = 5; ASCL1 OE, N = 4; ASCL1 OE + NICD, N = 4; ASCL1 OE + DN-RBPJ, N = 3. F, Quantification of the average percentage (±SEM among biological replicates) of electroporated cells that were OLIG2+ PDGFR α+ OPCs in GFP control, ASCL1 OE, ASCL1 OE + NICD, and ASCL1 OE + DN-RBPJ; one-way ANOVA, p = 3.00E-06, Tukey’s post hoc test, ***p = 0.0005, ****p = 0.00003. Statistical comparisons not shown in graph are GFP control with ASCL1 OE + NICD, p = 0.0001; GFP control with ASCL1 OE + DN-RBPJ, n.s.; ASCL1 OE with ASCL1 OE + DN-RRBPJ, p = 0.03. N = 3 brains for each condition. Some brains in E were also used for counting OLIG2+ PDGFR α+ cells as shown in F. OE, overexpression; EP’d, electroporated. SEM, standard error of the mean; n.s., not significant.
ASCL1 overexpression with and without NICD does not increase proliferation. A, Representative images of electroporated GFP+ OLIG2+ cells that were KI67+ (white arrowheads) or KI67− (yellow arrowheads) in GFP control, ASCL1-IRES-GFP, and ASCL1 + NICD. Scale bar, 50 µm. B, Graph shows average percentage (±SEM among biological replicates) of GFP+ OLIG2+ cells that were KI67+. N = 3 brains for each condition. SEM, standard error of the mean; n.s., not significant.
ASCL1 overexpression with and without NICD does not produce confused OLIG2+ cells. Representative images of electroporated (GFP+) cells that were OLIG2+ and TBR2− (yellow arrowheads) in GFP control, ASCL1-IRES-GFP, and ASCL1 + NICD-IRES-GFP. Bottom, Dotted lines outline the ventral limits of the pallium. Scale bar, 50 µm.
Interestingly, ASCL1 overexpression alone did not increase the proportion of GFP+ cells that were OLIG2+ PDGFR α+, but ASCL1 combined with NICD produced about two to three times more OLIG2+ PDGFR α+ OPCs compared with controls or ASCL1 alone (Fig. 9F). Thus, not only did ASCL1 coexpression rescue the ability of NICD-overexpressing progenitors to produce oligodendrocyte lineage cells, but NICD plus ASCL1 appeared to act synergistically to promote oligodendrogenesis. Importantly, combining DN-RBPJ with ASCL1 (Fig. 9C) did not have the same synergistic effect as NICD + ASCL1 (Fig. 9D–F), although expression of DN-RBPJ or NICD alone each inhibited OPC production (Figs. 4, 5). This result suggests the inhibitory effects of DN-RBPJ on OPC genesis are independent of ASCL1.
Altogether, our results point to a model in which Notch signaling through RBPJ is required for normal oligodendrocyte production from dorsal forebrain progenitors but that signaling through HES1 and HES5 also keeps oligodendrogenesis in check by repressing Ascl1 expression (Fig. 12).
Proposed model for Notch signaling in regulating the neuron-to-glia switch. A, During neurogenesis, dorsal forebrain progenitor cells that have lower Notch signaling activity differentiate into IPCs, which give rise to upper-layer excitatory neurons. Meanwhile, higher Notch signaling in other progenitors inhibits neurogenesis and promotes self-renewal to maintain a progenitor state. As oligodendrogenesis begins, Notch (+) progenitors give rise to pre-OPCs and OPCs. OPCs proliferate and populate the pallium. B, The canonical Notch pathway controls progenitor maintenance during neurogenesis. Binding of the Notch ligand DLL1 from Notch (−) cells to the NOTCH1 receptor in Notch (+) cells leads to cleavage of NICD by γ-secretase. NICD translocates to the nucleus and forms a transcriptional complex with RBPJ to activate transcription of unknown factors that promote oligodendrogenesis. NICD-RBPJ complex also activates transcription of Hes1 and Hes5. HES1/5 then represses Ascl1 transcription to prevent OPC genesis and maintain the progenitor state. C, During oligodendrogenesis, HES1/5 action on ASCL1 is repressed by an unknown factor. Additionally, factors induced by the NICD-RBPJ complex cooperate with ASCL1 expression to initiate fate specification of OPCs from dorsal forebrain progenitors or perhaps through direct induction of ASCL1 expression.
Discussion
Oligodendrogenesis temporally follows neurogenesis during forebrain development, and the molecular controls over the timing of this neuron–glia switch have remained elusive (Sauvageot and Stiles, 2002; Guillemot, 2007; Franco and Müller, 2013). Notch signaling is important in the developing mouse forebrain for maintaining the progenitor pool during neurogenesis and for instructing differential fates in neighboring cells (Artavanis-Tsakonas et al., 1999; Hatakeyama et al., 2004; Gao et al., 2009). However, the role of Notch during later stages, specifically during oligodendrogenesis, has not been explored. In this study, we focused on whether and how Notch signaling regulates oligodendrogenesis from dorsal forebrain progenitors. We show that (1) Notch signaling through NICD and RBPJ is required for normal production of oligodendrocyte lineage cells during the neuron–glia switch, (2) too much Notch signaling is inhibitory to oligodendrocyte production, and (3) the balance between these dual roles of Notch signaling depends on the ability of Notch effectors HES1 and HES5 to inhibit expression of the pro-oligodendrocyte factor ASCL1.
A dual role for Notch signaling in dorsal forebrain oligodendrogenesis
Notch signaling is a major developmental pathway driving brain development, and its main players and mechanistic steps have been identified for years. However, there is an increasing complexity to Notch signaling that lies in its ability to control many distinct characteristics and functions of neural progenitor cells during embryonic brain development, including progenitor self-renewal, cell fate specification, and differentiation (Ohtsuka et al., 1999; Kageyama et al., 2009; Schwanbeck et al., 2011; Guruharsha et al., 2012). For example, Notch transcriptional effectors HES1 and HES5 inhibit neurogenesis by repressing proneural genes such as Neurog2, thereby maintaining a proliferative pool of progenitors (Bertrand et al., 2002; Kageyama et al., 2015). In contrast, several reports implicate Notch signaling in driving gliogenesis from neural progenitors, including the generation of Müller glia in the retina, oligodendrocytes in the spinal cord, and astrocytes in the forebrain (Furukawa et al., 2000; Hojo et al., 2000; Park and Appel, 2003; Namihira et al., 2009; Bansod et al., 2017). Whether Notch signaling also promotes oligodendrogenesis in the dorsal forebrain was previously unknown. We disrupted sequential steps of the Notch pathway to understand its role in regulating oligodendrocyte fate specification in the dorsal forebrain.
Our ex vivo and in vivo studies using DAPT suggest that NICD production is important for generating normal numbers of OPCs from dorsal forebrain progenitors. Interestingly, the number of ventrally derived OPCs that ended up in the dorsal pallium were not affected following DAPT treatment. As ventrally derived OPCs are born earlier than dorsally derived OPCs, it is possible that they have matured past the point at which they would require Notch signaling to maintain an OPC identity (Kessaris et al., 2006).
Inhibiting the Notch pathway with DN-RBPJ similarly prevented dorsal progenitors from generating normal numbers of OPCs, indicating a cell-autonomous role for the NICD-RBPJ complex in driving an oligodendrocyte fate. Together, these data suggest that Notch signaling in neural progenitors, through NICD and RBPJ, is necessary for proper oligodendrocyte production in the dorsal forebrain. Interestingly, RBPJ acts as a transcriptional repressor in the absence of NICD but switches to a transcriptional activator when the Notch pathway is activated (Andersson et al., 2011; Johnson and MacDonald, 2011). In the future, it will be important to identify which transcriptional targets of RBPJ, with and without NICD, are involved in the Notch-mediated neuron–glia switch. A recent study performed such an experiment during neurogenic stages using a targeted biochemical approach (Van Den Ameele et al., 2022), which could be adapted for gliogenic timepoints.
Because Notch activation was required to generate oligodendrocyte lineage cells, we reasoned that overactivating the pathway might promote excess OPC production. However, overexpressing NICD also blocked the production of OPCs, and electroporated cells remained in the ventricular zone. This is consistent with a role for Notch in promoting progenitor cell maintenance (Gaiano et al., 2000; Mizutani et al., 2007). Importantly, Notch activation also prevents neurogenesis by inhibiting the expression of proneural genes (Kageyama et al., 2015). Therefore, it is possible that Notch signaling promotes an oligodendrocyte fate by inhibiting neuronal fates and maintaining a progenitor state throughout neurogenesis. Although previous studies report Notch overactivation can drive oligodendrogenesis in the spinal cord (Park and Appel, 2003), differences in intrinsic and extrinsic signals that converge with Notch in the dorsal forebrain may lead to different cellular outcomes of Notch signaling (Schwanbeck et al., 2011). Apart from activating expression of Hes genes that repress neurogenesis, NICD and the NICD-RBPJ complex are capable of inducing expression of genes related to stem cell maintenance (Li et al., 2012; Van Den Ameele et al., 2022). Therefore, overexpressing NICD may lead to excessive Notch activity that would overcome any pro-OPC factors in favor of progenitor maintenance. Nevertheless, our data indicate that Notch signaling through NICD and RBPJ is required to generate dorsal OPCs. Thus, we propose that Notch signaling plays a dual role in regulating dorsal oligodendrogenesis and must be precisely controlled to properly balance the maintenance of progenitors and the production of OPCs at the right times and in the appropriate numbers (Fig. 12A).
The mechanism by which Notch signaling regulates dorsal oligodendrogenesis
Target genes of NICD-RBPJ following Notch activation include Hes1 and Hes5, which encode transcription factors that inhibit neurogenesis and maintain a proliferative neural progenitor population (Kageyama et al., 2008). Hes1 null mutants exhibit precocious neuronal differentiation and upregulation of proneural genes (Ishibashi et al., 1995; Nakamura et al., 2000). We find that somatically knocking down Hes1 in dorsal progenitors during the neuron–glia switch significantly increased the proportion of OLIG2+ cells produced. This finding suggests that in addition to its role in inhibiting neurogenesis during early forebrain development HES1 also normally inhibits oligodendrogenesis during later stages. In line with this, HES1 overexpression downregulates oligodendrocyte lineage markers (Wu et al., 2003). Interestingly, knocking down both Hes1 and Hes5 simultaneously greatly increased the proportion of OPCs that were made from electroporated dorsal progenitors. These data indicate that Hes1 and Hes5 may be able to partially compensate for the absence of the other in preventing oligodendrocyte production such that the loss of both genes robustly drives oligodendrogenesis.
We further found that loss of Hes1 and Hes5 increased expression of the pro-OPC transcription factor ASCL1, raising the possibility that the mechanism by which HES1 and 5 inhibit dorsal oligodendrogenesis is through the inhibition of Ascl1. In line with this model, HES1 and 5 bind to the promoter and repress Ascl1 expression (Kageyama et al., 2007). HES1 and 5 also repress their own expression by binding to their own promoters, thereby driving both HES1/5 and ASCL1 oscillations in neural progenitors (Kageyama et al., 2007). Thus, the balance between HES1/5 and ASCL1 is important for progenitor maintenance and proliferation (Imayoshi et al., 2013). Loss of HES1/5 may therefore allow higher expression of ASCL1 and instruct progenitors to differentiate toward the oligodendrocyte lineage. Consistent with this, overexpression of ASCL1 was sufficient to increase the proportion of cells that expressed OLIG2. Importantly, we observed a greater increase in OLIG2+ cells and OLIG2+ PDGFR α+ OPCs when we electroporated both NICD and ASCL1 together, suggesting that Notch signaling and ASCL1 act synergistically to promote an OPC fate. This idea is further supported by a previous study showing that ASCL1+ progenitors in the dorsal forebrain, which are biased toward an oligodendrocyte fate, have higher levels of NICD than their NEUROG2+ neurogenic counterparts (Han et al., 2021). Altogether, our studies point to a model in which Notch signaling promotes oligodendrogenesis through NICD and RBPJ, but this effect is counterbalanced by Notch-mediated HES1/5 repressing ASCL1 (Fig. 12B).
Future studies are aimed at uncovering the regulatory mechanisms that fine-tune the output of the Notch pathway to determine whether individual cells remain progenitors or differentiate into OPCs. One possible mechanism is through a decrease in HES1/5, thereby allowing increased ASCL1 expression. We showed previously that Ascl1 expression is initially low in the dorsal forebrain but increases as oligodendrogenesis peaks (Winkler et al., 2020). Thus, HES1/5 may keep ASCL1 levels lower during neurogenesis, and relieving this inhibition could trigger gliogenesis. Another possibility is that ASCL1 expression is increased by a different mechanism that can overcome inhibition by HES1/5. A low-affinity consensus binding sequence has been identified at the promoter of Ascl1, at which RBPJ can bind and induce Ascl1 transcription (Shi et al., 2012). These findings raise the intriguing possibility that the NICD-RBPJ complex promotes ASCL1 expression directly during oligodendrogenesis (Fig. 12B). However, we found that overexpression of ASCL1 did not rescue the inhibitory effects of DN-RBPJ on oligodendrogenesis, suggesting that the NICD-RBPJ complex is likely also influencing pathways parallel to ASCL1 to promote an oligodendrocyte fate (Fig. 12B,C). In line with this idea, we found that ASCL1 overexpression alone increased production of OLIG2+ cells, but not OLIG2+ PDGFRα+ OPCs, whereas ASCL1 plus NICD together increased OPC production. Together these data indicate that ASCL1 and NICD-RBPJ work through separate pathways to promote OPC genesis cooperatively (Fig. 12B,C). Further investigations into these and other molecular mechanisms that control oligodendrocyte specification will be critical for better understanding the neuron–glia switch that is fundamental to CNS development.
Footnotes
This work was supported by National Institutes of Health (NIH)–National Institute of Neurological Disorders and Stroke Grants R01 NS109239 (S.J.F.) and R01 NS124166 (S.J.F.), NIH–National Institute of General Medical Sciences Grant T32 GM136444 (L.N.T.), and the Gates Summer Internship Program through the Gates Institute (S.K.L.). DNA sequencing was performed in the Molecular Biology Core at the University of Colorado Diabetes Research Center, supported by NIH/NIDDK P30 DK116073. We thank Lester Acosta for help with FACSorting performed in the Flow Cytometry Core, supported by Cancer Center Support Grant NIH/NCI P30 CA046934. We also thank Dr. Michael Kaufman and Dr. Joseph Brzezinski for providing the CRISPR plasmid backbones, Dr. Santiago Fregoso for the PB-CAG-IRES-GFP plasmid, Madalynn Welch and Kristen Schuster for help cloning PB-CAG-DN-RBPJ-IRES-mTagBFP2, and Sylvia Nunez for help cloning the ASCL1 overexpression plasmid; Aiman Sabaawy for technical assistance; and Aleezah Balolia, Christina Como, Madisen Mason, Kristen Schuster, Madalynn Welch for discussions and critical reading of the manuscript.
The authors declare no competing financial interests.
- Correspondence should be addressed to Santos J. Franco at Santos.Franco{at}CUAnschutz.edu
