Differential Hes1 activation defines neural stem cell lineage commitment and niche maintenance in embryonic and adult mouse cortex

Significance

Here, we explored the functional significance of Notch-independent Hes1 (NIHes1) expression in a subset of neural stem cells (NSCs). Radial glial cells (RGCs), which are well studied, belong to the Notch-dependent Hes1-expressing (NDHes1) subtype and are maintained through Notch-mediated Hes1 activation. In contrast, NIHes1 NSCs maintain Hes1 expression without Notch signaling and hence do not require cell–cell interaction. These NSCs exhibit enhanced stemness, increased proliferation, and are retained in the adult cortex. We also showed that the NIHes1 subclass of Hes1^+ve NSCs can generate intermediate progenitor cells (IPCs) directly, along with RGCs. This work highlights the functional significance of these distinct Hes1-expressing NSC subtypes during development and their role in maintenance of the embryonic and adult neural stem cell niches.

Abstract

Mode of Hes1 activation and its differential expression are crucial for the maintenance of neural stem cells/progenitor cells (NSCs/NPCs) in the embryonic cortex. This differential mode of Hes1 activation has been translated into a heterogeneous population of NSCs comprising Notch-independent Hes1-expressing (NIHes1) NSCs and Notch-dependent Hes1-expressing (NDHes1) radial glial cells (RGCs). Using single-cell transcriptomics and a Nestin-CreERT2;NIHes1^fl/fl conditional knock-out mouse model, we have characterized the NIHes1 NSCs. Our analyses show that NIHes1 NSCs are the ancestral precursor NSCs that generate RGCs and intermediate progenitor cells during development. Loss of NIHes1 expression significantly alters the NSC niche, leading to increased gliogenesis and aberrant migration of projection neurons. NIHes1 NSCs are set aside at embryonic stages as adult neural stem cells and are maintained by NIHes1 expression even at adult stage. Our findings suggest that NIHes1 NSCs are functionally distinct Hes1-expressing NSCs, which are critical for establishing both embryonic and adult NSC niches, thereby contributing to the overall cortical development.

Vertebrate corticogenesis follows a precise sequence of differentiation beginning with neurons, followed by astrocytes and oligodendrocytes (1). These cells arise from the neural stem cell/progenitor niche in the ventricular and subventricular zones (VZ and SVZ). Neuroepithelial cells (NECs) constitute the first cells to emerge in the vertebrate VZ, characterized by symmetric proliferative divisions that amplify the initial pool of NSCs (2, 3). Radial glial cells (RGCs) are the primary subtype of NSCs which are known to produce intermediate progenitor cells (IPCs) and neurons (4). Most studies consider RGCs as the sole NSCs in the VZ. However, the VZ harbors another less-explored distinct pool of progenitor cells: short neural precursors (SNPs) with the cell body residing in the VZ and with a short basal process (2, 5). Further, the SVZ harbors IPCs, which possess a limited potential to divide symmetrically before differentiating into neurons (2, 6). These cells constitute the stem cell/progenitor niche, which generates the differentiated cells of the cortex.

The NSC niche of the VZ has prominently active Notch signaling that plays a pivotal role in the maintenance, proliferation, and differentiation of NSCs/NPCs. In the developing central nervous system (CNS), Notch target gene, Hes1 is the critical regulator of neurogenesis, which represses various proneural genes and maintains NSCs in an undifferentiated and proliferative mode. Even though Notch-dependent Hes1 is well documented in stem cell systems (7–17), there are reports regarding the Notch-independent activation of Notch target genes in the vertebrate nervous system (8, 10, 17–19) and other systems (20–23). It was also observed that Hes1 and Hes3 expressions precede the Notch receptor or ligand expression in NECs (24–26). Common regulatory pathways such as Wnt, Shh, FGF, and MAPK have also been reported to directly activate the expression of various Notch target genes independent of Notch-signaling in a context-dependent manner (8). Our previous report demonstrated the FGF2–JNK–ATF2–Hes1 axis in ES-cell-derived neural progenitors (23). Further, using in utero electroporation (IUE) of the reporter construct that simultaneously marks Notch-independent Hes1 (NIHes1) and Notch-dependent Hes1 (NDHes1)-expressing cells, we have previously identified the differential mode of Hes1 expression in the developing neocortex (11).

We had shown that the developing VZ is composed of NIHes1-expressing NSCs with short apical and basal processes and NDHes1-expressing NSCs which closely resemble the typical RGCs with long basal processes. As corticogenesis proceeds, the NIHes1 NSCs transit into NDHes1-expressing RGCs (NDHes1 NSCs) which generate neurons of layers II and III (11). Further, NIHes1 NSCs were found to be heterogenous with respect to proliferation- having both KI67^+ve and KI67^−ve cells. Even though the pleiotropic mode of Hes1 expression in the developing cortex was characterized previously, how the Notch-independent activation is essential for NSCs is not understood. Further, the functional significance of this unique subpopulation of NIHes1 NSCs in the NSC niche is yet to be explored. A basic understanding of the functional relevance of the dual mode of Hes1 activation and NIHes1 NSCs will have a substantial impact on regulating the proliferation and maintenance of NSC niches in the context of various neurodegenerative diseases.

Here, we have identified and elucidated the functionally distinct gene programs of two Nestin^+ve NSCs (NIHes1 and NDHes1 NSCs) using single-cell transcriptome analyses. Further, we investigated the function of NIHes1 expression in the NSC niche using a Nestin-CreERT2; NIHes1^fl/fl conditional knock-out (cKO) mouse model. Our results demonstrate the vital role of NIHes1 expression in establishing and maintaining the NSC niches in both embryo and adult cortices.

Results

Neural Stem Cell Niche Harbors Two Populations of Hes1-Expressing NSCs with Distinct Functional Signatures.

During mouse neocortical development, Hes1 is expressed in the NSCs/NPCs residing in the VZ, as shown in the coronal brain sections of Hes1-d2EGFP mice (Fig. 1A). Immunohistochemical (IHC) analysis revealed that the expression of NSC marker NES (Nestin) coincides with the HES1-expressing layer in the VZ and SVZ of E18 neocortex (Fig. 1 A and B). IUE with a reporter construct delineating NIHes1 expression with EGFP and NDHes1 expression with mCherry showed the presence of both the NIHes1- and NDHes1-expressing NSCs in the VZ and SVZ (Fig. 1 C–F) (11). Therefore, the HES1-expressing NES^+ve layer comprises NIHes1 and NDHes1 NSCs, which are defined by the differential promoter activation of Hes1.

Fig. 1.

Neural stem cell niche harbors two subtypes of Hes1-expressing NSCs. (A) Coronal section of E18 Hes1-d2EGFP mice cortex showing Hes1 expression. (B) IHC of NSC marker Nestin in E18 cortical sections. (C) Schematic of reporter construct simultaneously reporting NIHes1 (EGFP) and NDHes1 (mCherry) expressions. (D–F) Fluorescent image of E14 coronal section 1-d post-IUE (1DPI) with reporter construct shows NIHes1 (EGFP) and NDHes1 (mCherry) expressions. (G) UMAP of scRNA-seq data showing cell clusters of E14 cortex. (H) Expression plot showing distinct markers of identified clusters. (I) TF activity scores of Notch (NICD), Rbpj, and Hes1 across clusters. (J) Expression plot of DEG across clusters. (K) Cell cycle scoring of clusters. (L) GO-BP analysis of clusters. (Scale bar: 25 µm.)

To further characterize the gene signatures of these NSCs, we analyzed the single-cell RNA sequence (scRNA-seq) data of the embryonic day 14 (E14) stage using the raw data from GSE123335 (27). To identify the NIHes1 and NDHes1 NSCs, the data were clustered with optimum parameters (SI Appendix, Text 2.9) and the clusters of cortices were annotated with marker genes. The cluster with low Hes1 expression and distinct Eomes (Tbr2) expression was identified as IPCs and the clusters with high Rbfox3 (NeuN), Dcx, and Neurod1 expressions were designated as neurons (Fig. 1 G and H) (6, 28, 29). A Hes1 and Nestin-positive NSC cluster was also identified. To differentiate NIHes1 and NDHes1 NSCs within this Hes1-expressing NSC cluster, we subclustered the same with their inherent gene expression differences, using its principal components (PCs) and Louvain algorithm with multilevel refinement (Fig. 1 G and H) (30). Here, we identified two distinct Nestin^+ve Hes1-expressing NSC clusters. The NDHes1 cluster had significantly higher Slc1a3 (GLAST), Fabp7 (Blbp), Tnc, Cdh2, and Vim expressions (RGC markers) than the NIHes1 cluster (31). These markers of RGCs are either regulated by Notch-CBF1 signaling (Fabp7) (32) or relevant to the guidance of neuronal migration which is an exclusive role of NSCs with long basal processes. For instance, Vimentin (Vim) filaments are known to line the long radial fibers of RGCs (33), Tnc is involved in ECM modulation by RGCs (34), and Cdh2 (N-cadherin) has major role in neuronal guidance function of RGCs (35). We assessed the expression of these markers as a gene set module and scored its expression. The NIHes1 cluster had relatively low expression and gene module score of the above RGC markers and hence we speculated this to be the NIHes1 NSCs observed in our IUE experiments (Fig. 1 C–H and SI Appendix, Fig. S1A). To confirm this, we used the scRNA-seq data to identify transcription factors (TFs) and their regulon activity in each cluster (Fig. 1I and Dataset S1). We used the CollecTRI database which has information regarding TFs and their target gene interactions (regulons) weighted based on their mode of regulation (activation or inhibition) (36). Notch1 (NICD) and Rbpj regulons were enriched significantly in NDHes1 NSCs (TF activity score: Notch1 – 1.586 & Rbpj – 1.238) compared to NIHes1 NSCs (Notch1 – 0.734 & Rbpj – 0.779) whereas, Hes1 regulon enrichment was similar in both NSCs (NDHes1 – 0.883, NIHes1 – 0.848). This implies that the Hes1 expression in the NIHes1 cluster is not through Notch-mediated activation and reveals the Notch independent and dependent activations of Hes1 in NIHes1 and NDHes1 NSCs, respectively. Therefore, the second cluster was identified as NIHes1 NSCs.

We further analyzed the differential gene expression (DGE) pattern of these clusters. We observed a similarity in the expression of genes involved in stem cell maintenance, proliferation, and fate specification (Hes1, Id3, Yap1, Gas1, and Zfp36l1) between NIHes1 and NDHes1 NSCs compared to that of the other two clusters (Fig. 1J and Dataset S2) (8, 37–40). Genes involved in RGC/NSC maintenance and proliferation, such as Ednrb and Tnc (RGC marker), were upregulated in NDHes1 NSCs compared to NIHes1 NSCs (41, 42). Further, NDHes1 NSCs had upregulated astrocytic genes (Mlc1, Acsbg1, and Aldh1l1) (43–45). On the contrary, Neurog2, a major proneural gene, had higher expression in NIHes1 NSCs than NDHes1 NSCs (Fig. 1J) (46, 47). Therefore, the two NSCs showed distinct signatures toward different differentiated fates. IPCs showed higher expression of Eomes, Mfap4, and Neurog2 (6). Neurons showed upregulation of genes involved in axonal growth, synapse formation, and plasticity (48–51).

Further, cell cycle analysis of the clusters was performed based on cell cycle phase scoring on these cell types to confirm their proliferative signatures. It was observed that NIHes1 NSC exhibited higher G2/M scores, indicating that these cells were rapidly dividing in nature compared to NDHes1 NSCs (Fig. 1K). However, we previously reported the NDHes1 NSCs to be more proliferative (11). We also showed the presence of the two subpopulations – rapidly dividing (KI67^+ve) and nondividing (KI67^−ve) NIHes1 NSCs (11). To clear this discrepancy, we subclustered the NIHes1 NSC cluster using “FindSubCluster” function of Seurat (v5.3). We obtained two Hes1^+ve and Nes^+ve subclusters, of which one was negative for Ki67 and Aurkb (SI Appendix, Fig. S1 B and C). Therefore, we identified this cluster as a nonproliferative subtype of NIHes1 NSCs – “NIHes1_npNSC” that we observed in our earlier reports. The other cluster was Ki67^+ve, Aurkb^+ve and identified as proliferative NIHes1 NSCs – “NIHes1_pNSCs.” We could not ascertain whether NIHes1_npNSC are quiescent NSCs due to lack of robust markers for embryonic quiescent NSCs. DGE between NIHes1_pNSC and NIHes1_npNSC revealed higher expression of differentiation-related genes such as Neurog2 and Eomes in NIHes1_npNSC cluster (SI Appendix, Fig. S1D and Dataset S3). Therefore, this population could be the intermediate/transitory population between Hes1^+ve NSCs and Hes1^−ve Eomes^+ve IPCs as described by Shimojo et al. (46). This confirmed that although NIHes1 NSCs show a higher division rate than NDHes1 NSCs, they are indeed composed of two subpopulations with different proliferative potential. In our earlier report, we may have observed a reduced proliferation of NIHes1 NSCs compared to NDHes1 NSCs since these cells were identified using IUE of reporter plasmids which will only report a small number of electroporated cells in the VZ, thereby limiting the interpretation. Cell cycle analysis of NDHes1 NSC displayed a reduced G2/M score, suggesting lower proliferation potential favoring differentiation (Fig. 1K). On the other hand, the IPCs showed more G1 phase markers along with less S and G2/M scores, whereas the neurons were arrested in the G1 phase. In addition, Gene Ontology (GO-BP) analysis revealed enrichment of cell proliferation, stem cell maintenance, and negative regulation of differentiation in NIHes1 NSCs (Fig. 1L) (52). At the same time, NIHes1 and NDHes1 NSCs were enriched with gliogenesis-related genes and negative Wnt signaling regulation. While IPCs showed enrichment of morphogenesis, neurons showed upregulation of genes regulating synaptic signaling and neurotransmitter transport.

Next, we sought to understand the active gene programs in the different cell types. The differences in gene programs were summarized by TF activity, pathway enrichment, and ligand–receptor interactions. The TF regulon activity showed higher enrichment of Pou3f1 and Sox10 regulons, in NDHes1 NSCs that can trigger fate specification toward neurons and oligodendrocytes, respectively (Fig. 2A and Dataset S1) (53, 54). NIHes1 NSCs, however, have higher enrichment of Ctnnb1 (β-catenin) which indicates active Wnt signaling and have enrichment of Myc regulon which specifies the fate of NSCs to IPCs (55). IPCs show higher enrichment of Neurog2 regulon, confirming a neuronal fate (46). Additionally, both NSCs show TF regulon enrichments of Sox2, Gli2, Prrx1, and Nr2f1, regulators of NSC maintenance and proliferation (56–58). Neurons showed involvement of TF regulons related to chromatin modifications (Hdac1), neuronal differentiation, migration, and axonal pathfinding (Neurod2) (Fig. 2A) (59, 60). To understand the major signaling pathways in the NSC subtypes, we performed pathway enrichment analysis using KEGG and WikiPathways databases (SI Appendix, Fig. S2A) (61–63). We showed that NIHes1 and NDHes1 NSCs had enrichment of genes associated with cell cycle, stemness, Hippo, and Wnt pathways, but to a higher degree in NIHes1 NSCs. Meanwhile, NDHes1 NSCs had higher activity in Delta-Notch, PI3K-Akt signaling, and axon guidance pathways (64). The IPCs were enriched with genes related to neurogenesis, while the neurons were enriched with synaptic vesicle signaling, endocytosis, G protein, and cAMP signaling (SI Appendix, Fig. S2A). We also analyzed the intercellular interactions of the four clusters using the LIANA framework and identified active Notch signaling and possible negative regulation of Wnt signaling in NDHes1 NSCs (SI Appendix, Fig. S2B and Text 1.1).

Fig. 2.

NIHes1 and NDHes1 NSCs differ by functional signatures and lineage commitment. (A) TF activity of clusters analyzed using the CollecTRI database. (B) Gene module scoring for gene sets – “Mechanisms associated with pluripotency” from WikiPathways (WP1763) and “Stem cell proliferation” from Gene Ontology (GO:0072089). (C) Normalized cell proportions in E10-P4 cortices. (D) Normalized cell proportions of stem cells and progenitors in E10-P4 cortices. (E) Lineages of E14 cell clusters using pseudotime trajectory analysis. Lineage 1 showed a trajectory from NIHes1 NSCs to IPCs and then to neurons. Lineage 2 showed a trajectory from NIHes1 NSCs to NDHes1 NSCs.

These analyses reveal a distinct cluster of NIHes1-expressing NSCs with active Hes1 expression devoid of Notch/CBF1 interaction. These NSCs may also exhibit active Wnt signaling, that needs further confirmation. Further, the NIHes1 NSCs are imprinted with neuronal signatures, while NDHes1 NSCs have more glial signatures. Moreover, the guidance function of NDHes1 NSCs (RGCs) in neuronal migration is also evident from the above data.

NIHes1 NSCs Are Multipotent and Act As the Precursors of Other Stem Cell Subpopulations.

Since we now know that NIHes1 and NDHes1 NSCs are distinct and differ by gene programs, we were curious to know the differences in potency and lineage potential of these NSCs. Interestingly, we found higher gene set enrichment of “Mechanisms associated with pluripotency,” which comprises genes involved in stemness, with pathway enrichment analysis and a higher enrichment of “stem cell proliferation” in GO analysis in NIHes1 NSCs (Fig. 2B and SI Appendix, Fig. S3). To further understand the stemness and the precursor of the NSC niche, we estimated the cell proportions of each cell type at each developmental time point. We reanalyzed the scRNA-seq raw data of 11 developmental stages (E10-P4) downloaded from the GEO database (GSE153164) (65). The differentially expressed gene (DEG) signatures of NIHes1 and NDHes1 NSCs from the E14 stage were used to enrich and identify clusters at different developmental time points (Dataset S4). The identified data were plotted, representing the proportion of cortical cell types (Fig. 2C) and NSC/NPC of the VZ and SVZ (Fig. 2D). As observed earlier, the Nes^+ve stem cell pool gets reduced throughout development and generates other cell types, including the later-stage stem/progenitor cells. We found a higher proportion of NIHes1 NSCs at the earlier embryonic stages than NDHes1 NSCs and IPCs. This is expected as NIHes1 NSCs are known to produce NDHes1 NSCs (RGCs) (11). The IPCs peak toward the E14-E15 stages, coinciding with a reduction in NIHes1 NSCs. The proportion of NDHes1 NSCs fluctuates but remains mostly the same until the postnatal stages, before it starts peaking. We also observed a drastic and rapid increase in the number of neurons at the post–E13 stage, which is supported by the increase in IPCs and its subsequent reduction as differentiation increases. Further, the astrocytes start appearing by the E17 stage and gradually increase toward the postnatal stages. The oligodendrocyte precursor cells (OPCs) and oligodendrocytes start appearing toward the E16 and E18 stages (Fig. 2 C and D).

Next, to analyze the lineage trajectories between clusters, we performed a slingshot “pseudotime” analysis for constructing cellular differentiation trajectory (66). In E14 scRNA-seq data, the slingshot pseudotime trajectory inference analysis showed two lineages (Fig. 2E). The lineage 1 showed a trajectory from NIHes1 NSCs to IPCs and then to neurons. In the case of lineage 2, the trajectory is from NIHes1 NSCs to NDHes1 NSCs. These results suggested that the NIHes1 NSCs transit toward the NDHes1 state, reflecting the similar transition from NIHes1 to NDHes1 cells observed in vivo (11). Further, we speculated that the neurogenic IPCs could be another direct progeny of the NIHes1 NSCs. Together, these data point to a unique gene program in the NIHes1 NSCs with higher stemness potential and possibly the potency to generate primary component cells of the early embryonic stem cell niche.

Knockout of NIHes1 Expression in the Neocortex Leads to Depletion of Proliferating NSCs.

Next, to validate and understand the significance of NIHes1 expression, a Nestin-CreERT2;NIHes1^fl/fl mouse model was generated where the NIHes1 promoter region can be conditionally knocked out (at the desired developmental time point) in Nestin-expressing cells (Fig. 3A and SI Appendix, Text 1.2). We observed no apparent differences in the morphology of control and KO embryos or their brains following NIHes1 knockout (KO) at E14 and analysis at E18 (Fig. 3 B and C). Although insignificant, we observed a decrease in Hes1 expression in real-time PCR analysis of the control and KO neocortex which could be attributed to the unaffected NDHes1 expression (Fig. 3D).

Fig. 3.

NIHes1 expression maintains the proliferating neural stem cells. (A) Schematic representing generation of NestinCreERT2; NIHes1^fl/fl cKO mouse model. (B and C) Representative image of NIHes1^+/+ control and NIHes1^−/− KO E18 embryo and brain. (D) Relative gene expression analysis of Hes1 mRNA in NIHes1^+/+ and NIHes1^−/− cortices, (N = 3). (E and F) Representative image of BrdU staining of NIHes1^+/+ (E and e’) and NIHes1^−/− (F and f’) E18 cortical sections. (G) Graph showing the number of BrdU^+ve cells (cells/mm²) in the VZ of NIHes1^+/+ and NIHes1^−/− neocortices, (N = 6). (H–M) Section of E18 NIHes1^+/+ (H–J) and NIHes1^−/− cortex (K–M) coimmunostained for AURKB and KI67. (N and O) Graph representing the number of AURKB- and KI67-expressing cells in the VZ of E18 NIHes1^+/+ control and NIHes1^−/− KO neocortices (AURKB, N = 4; KI67, N = 5) (P) Immunoblots showing AURKB and CYCLIN A2 protein expressions in NIHes1^+/+ control and NIHes1^−/− KO whole cortices. β-ACTIN was used as internal control. (Q and R) Quantification of AURKB and CYCLIN A2 protein expression in NIHes1^+/+ control and NIHes1^−/− KO whole cortices. The expressions were normalized with respective expressions of β-ACTIN (internal control). (AURKB, N = 5; CYCLIN A2, N = 5). (S–X) Cortical section of E14 Hes1-d2EGFP mouse brain (S–U) and E14 Hes1-d2EGFP mouse treated with 6 h DAPT treatment (V–X) immunostained for BrdU, and d2EGFP to mark HES1 expression. (Y) Graph showing the number of BrdU^+ve and d2EGFP^+ve cells in the VZ of control and DAPT treated mouse brain. (N = 6). P-value: *(P ≤ 0.05), **(P ≤ 0.01), and ****(P ≤ 0.0001). Results are shown as mean ± SD. [Scale bar: (E and F) 50 µm, (H–M and S–X) 25 µm.]

We investigated the effect of knocking out NIHes1 expression on the proliferation of the NIHes1-expressing NSCs. We tracked BrdU incorporation upon KO after a pulse of 6 h and confirmed a reduction in proliferating NSCs in NIHes1 KO. We observed clusters of BrdU^+ve cells in the VZ in NIHes1 KO compared to the evenly distributed BrdU^+ve NSCs in control (Fig. 3 E–G). Further, immunostaining with the proliferation markers KI67 and AURKB showed a significant reduction in the number of proliferating NSCs in the VZ of NIHes1 KO compared to control (Fig. 3 H–O). The decline in proliferation was confirmed with western blot analysis of AURKB and CYCLINA2 expression (Fig. 3 P–R). This observation corroborates the findings of E14 scRNA-seq analyses that NIHes1 NSCs bear higher proliferation potential (Fig. 1K). Altogether, our data conclude that NIHes1 expression has a significant role in proliferating NSCs.

These findings prompted us to also investigate the role of NDHes1 expression in NSCs. We used a γ-secretase inhibitor, DAPT (100 mg/kg), which was previously proven to abolish NDHes1 expression in the NPCs residing in the VZ of the neocortex (11, 23). A 6-h BrdU pulse was given to Hes1-d2EGFP mice to label proliferative cells. In contrast to NIHes1 KO, an evident and significant increase in proliferating NSCs was observed in the DAPT-treated group. The change in the cell cycle profile was confirmed using BrdU (Fig. 3 S–Y) and KI67 (SI Appendix, Fig. S4 C–K). We also found a proliferating group of cells negative for HES1 expression but positive for BrdU and KI67 proliferation markers, which could be the IPCs. Our observation suggests that ablation of NIHes1 expression leads to the reduction in proliferating NSCs.

Knockout of NIHes1 Expression Results in Increased Number of RGCs Leading to Enhanced Differentiation.

The observed reduction in proliferation in NIHes1 KO could result from one of the three possibilities: a) cell death of NIHes1 NSCs, b) a direct effect of KO on proliferative capacity of NIHes1 NSCs, or c) transition of NIHes1 NSCs into NDHes1 NSCs/IPCs. To delineate these possibilities, we first measured the number of cells and width of the VZ. We observed an overall reduction in the DAPI^+ve cells (Fig. 4A) and a consequent reduction in the thickness of the VZ in NIHes1 KO (Fig. 4B). However, immunostaining of E14 NIHes1 KO tissue at E18 for cleaved Caspase-3 did not show any significant difference between control and KO. This rules out the possibility of cell death in KO NIHes1 NSCs even though cell death prior to this stage could not be accounted for as effector caspase expression is transient (SI Appendix, Fig. S5).

Fig. 4.

NIHes1 KO leads to increase in RGCs and premature differentiation to astrocytes. (A) Graph showing number of DAPI^+ve cells in the VZ of NIHes1^+/+ control and NIHes1^−/− KO neocortex. (N = 4) (B) Graph showing VZ thickness between NIHes1^+/+ and NIHes1^−/− neocortex. (N = 3) (C) Schematic of wild type Hes1 promoter (WT-Hes1) driven EGFP reporter construct. (D and E) IUE of E14.5 cortex with WT-Hes1-d2EGFP construct and analyzed at 0.5 DPI shows two morphologically distinct HES1-expressing populations in the VZ of the neocortex. The arrow head marks radial fibers of RGCs. (F–I) IHC of NES expression in the coronal sections of E18 NIHes1^+/+ (H and h’) and NIHes1^−/− (I and i’) neocortex counterstained with DAPI (F and G) revealed an enhanced RGC like morphology (arrowheads) upon knocking out NIHes1 compared to NIHes1^+/+. (J) Relative gene expression of Hes5 mRNA in NIHes1^+/+ and NIHes1^−/− cortices. (N = 3). (K–P) IHC of GFAP shows increased expression in NIHes1^+/+ neocortex (L, M, and m’) compared to NIHes1^−/− KO neocortex (O, P, and p’). P-value: ns (P > 0.05), *(P ≤ 0.05), and **(P ≤ 0.01). Results are shown as mean ± SD. [Scale bar: (C–F, H, and I) 50 µm, (K–P) 100 µm.]

Given the overall reduction in proliferative cells within the VZ, a corresponding decrease in differentiating cells would be expected if NIHes1 knockout solely impairs the proliferative capacity of NIHes1 NSCs. Alternatively; the decline in proliferating cells may reflect a premature transition toward less proliferative NDHes1 NSCs, and IPCs, leading to a subsequent increase in differentiation. Therefore, we analyzed the NDHes1 NSCs and IPCs in NIHes1 KO embryos. IUE with the WT-Hes1-EGFP construct revealed morphologically distinct cells with short apical and basal processes within the VZ and cells with short apical and long basal processes reaching the pial surface (Fig. 4 C–E). We previously showed that NDHes1-expressing cells are the RGCs with long basal processes, whereas NIHes1 cells have short basal processes (11). IHC analysis of NES in NIHes1 KO showed a pronounced increase of NDHes1 NSCs (RGCs) with long basal processes (Fig. 4 F–I, h’, and i’). We also observed a higher mRNA expression of Hes5, a Notch-dependent gene, in NIHes1 KO (Fig. 4J). Further, immunostaining for GFAP, showed a significant increase of GFAP^+ve cells in NIHes1 KO (Fig. 4 K–P). The GFAP^+ve cells in the VZ are RGCs (with astrocyte fate) and the GFAP^+ve cells in other layers are astrocytes (Fig. 4 m’ and p’). We also observed a corresponding significant decrease of IPC marker, TBR2^+ve (P < 0.05) and KI67^+ve cells (P < 0.05) in the SVZ of NIHes1 KO (Fig. 5 A–H). These findings indicate that NIHes1 KO does not impair the proliferative potential of NIHes1 NSCs, instead, it disrupts their maintenance. NIHes1 NSCs, upon KO, preferentially transit into NDHes1 NSCs, prematurely exhausting the pool of NIHes1 NSCs which could have also generated IPCs. This highlights the key role of NIHes1 in generating and maintaining NSC niche heterogeneity, consistent with the multipotent nature of NIHes1 NSCs seen in the scRNA-seq data (Fig. 2E).

Fig. 5.

NIHes1 expression reduces IPCs and alters cortical lamination. (A–F) Sections of E18 NIHes1^+/+ control (A–C) and NIHes1^−/− KO cortices (D–F) immunostained for TBR2 and KI67. (G and H) Quantitative spatial distribution graph of the TBR2^+ve (red) and KI67^+ve (green) cells in the VZ and SVZ of E18 NIHes1^+/+ (G) and NIHes1^−/− cortex (H). P-value for both TBR2 and KI67 is P < 0.05. (I–L) Sections of E18 NIHes1^+/+ and NIHes1^−/− neocortices immunostained for the cortical layer markers [Layer I - REELIN, layer II & III - BRN2 (I and J), layer IV-V - CTIP2 and Layer VI - TBR1 (K and L)]. (M) Schematic of aberrant cortical layer distribution. [Scale bar: (A–F) 25 µm, (I–L) 100 µm.]

We also looked at the changes in differentiated cortical layers using IHC. We could observe evident defects in neuronal positioning of BRN2 (layer II, III) expressing upper cortical layer neurons, but there were no differences in RELN-expressing layer I (Fig. 5 I and J). The cellular distribution of subcerebral projection neurons (layer IV) labeled with CTIP2 expression increased and spread to deep layer VI, which expresses TBR1 (Fig. 5 K and L). These results indicated that neurons destined for layers II, III, and IV have not reached their destined positions upon NIHes1 KO, indicating excessive differentiation and altered neuronal migration in the absence of NIHes1 expression (Fig. 5M).

To confirm the above findings, a transcriptomic analysis (bulk RNA sequencing analysis) was performed using dissociated whole cortex cells of control and KO at E18 after tamoxifen administration at E14 stage. We observed similar outcome in terms of marker genes, and cell type signatures (SI Appendix, Figs. S6 and S7 and Text 1.3). Overall, the NIHes1 KO in vivo studies and RNA-seq analyses revealed an increase in transition of NIHes1 NSCs into RGCs, a depletion of proliferating stem cell pool and an imbalance in the composition of the NSC niche leading to precocious and increased differentiation, mainly to astrocytes. It also confirms that NIHes1 expression is essential to maintain the NIHes1 NSCs.

Adult Quiescent Neural Stem Cells Originating from Embryonic NSCs Have NIHes1 Expression.

Upon analysis of the proportion of cell types across developmental stages, we observed that a small number of NIHes1 NSCs persisted until P4 (Fig. 2 C and D). According to the “set aside” model of adult stem cell origin, embryonic NSCs are set aside to generate an adult stem cell pool around E13.5 to 15.5 (67, 68). These set aside stem cells remain quiescent until adulthood and are then reactivated. In mice, adult NSCs generate different subtypes of neurons through neuroblasts which migrate to the olfactory bulb (OB) through rostral migratory stream (RMS) (69). Hence, we were intrigued about the fate of these persisting NIHes1 NSCs in the adult brain.

To understand whether NIHes1 NSCs are the set aside quiescent NSCs (qNSCs), we adopted a long-term BrdU pulse–chase (en rule) strategy, wherein a single shot of BrdU is administered to E14 embryos and electroporated with the pmtCBF1-Hes1-d2EGFP construct (labeling NIHes1 expression with d2EGFP) at adult stage (Fig. 6A). We could observe that unfragmented BrdU retaining cells associated with d2EGFP expression affirming the presence of NIHes1 expression in the adult SVZ in the embryonically set aside qNSCs (Fig. 6 B–D). Besides, there were cells with d2EGFP alone and no BrdU, as well as d2EGFP with fragmented BrdU. We understand that the efficiency of electroporation and the timing of a single shot of BrdU within the developmental window of quiescence entry might limit the marking of all the cells set aside. Thus, the occurrence of d2EGFP^+ve BrdU^–ve cells could indicate the cells set aside earlier to BrdU pulse at E14. Whereas, d2EGFP cells with fragmented BrdU might suggest quiescence entry toward late E15 after completing a few rounds of division, and/or activation of qNSCs at adult stages. Thus, we present evidence for the existence of NIHes1-expressing qNSCs in the adult SVZ which are embryonically set aside and could be primed toward active NSC (aNSC) fate.

Fig. 6.

NIHes1 expression maintains adult aNSCs, and its KO leads to enhanced differentiation to OB neurons. (A) Schematic showing timeline for BrdU injection and in vivo electroporation with reporter construct pmtCBF1-Hes1-d2EGFP and tissue collection. (B and C) Coexpression of unfragmented BrdU and d2EGFP in adult NIHes1 cells indicate that these NSCs underwent a minimum number of divisions during transition from embryonic to adult stage. (D) Graph showing number of d2EGFP cells that colocalized with cells having unfragmented BrdU, fragmented BrdU, and without BrdU in the adult mouse SVZ (N = 3). (E) Schematic showing the timeline of tamoxifen administration, BrdU injection, and tissue collection in the NIHes1^fl/fl mouse model. (F and G) BrdU immunostaining showing NIHes1 KO leads to reduction in aNSCs in the VZ. (H) Graph showing the number of BrdU^+ve cells in NIHes1^−/− KO and NIHes1^+/+ control (N = 3). (I) UMAP plot shows cell clusters of P30 adult SVZ stem cell niche (70). Cell cycle scoring is shown as a pie chart. (J-K) UMAP plot of Nes (J) and Hes1 (K) gene expressions in the scRNA-seq dataset of P30 adult SVZ. (L) TF activity of each cluster. (M and N) Immunostaining of control (M) and KO (N) adult OB for DCX (neuroblast marker). In control, tdT^+ve cells are progeny of Nes^+ve cells and in KO, tdT^+ve cells are progeny of NIHes1 knocked out Nes^+ve cells. (O) Quantification of tdT^+ve and DCX^+ve cells in control and KO adult OB. (N = 5). (P and Q) Immunostaining of control (P) and KO (Q) adult OB for NEUN (neuronal marker). In control, tdT^+ve cells are progeny of Nes^+ve cells and in KO, tdT^+ve cells are progeny of NIHes1 knocked out Nes^+ve cells. (R) Quantification of no. of tdT^+ve and NEUN^+ve cells in control and KO adult OB. (N = 5). (S) Panel depicting the model of embryonic and adult stem cell niche with all the subtypes of cells classified in this study. P-value: *(P ≤ 0.05), **(P ≤ 0.01), ***(P ≤ 0.001) and ****(P ≤ 0.0001). Results are shown as mean±SD. [Scale bar: (B) 50 µm; (C) 25 µm; (F and G) 200 µm; (M, N, P, and Q) 100 µm.]

To further investigate the role of NIHes1 expression, we knocked out the NIHes1 promoter region in the Nestin-CreERT2;NIHes1^fl/fl mice using tamoxifen at PN45. Cre-negative NIHes1^fl/fl mice and vehicle injected Cre-positive Nestin-CreERT2;NIHes1^fl/fl mice were used as controls. Subsequently, five short pulses of BrdU were given to control and KO 20 d after tamoxifen (20 DAT) at 3 h-intervals to mark all actively dividing cells (Fig. 6E). The tissues were collected 1 h after last BrdU injection and the number of BrdU^+ve cells in the adult SVZ were counted (Fig. 6 F–H). The SVZ harbors aNSCs, and a transient state between aNSC and neuroblasts called transit amplifying cells, as dividing cells. Since the transit-amplifying cells (Nes^−ve) are short-lived, the BrdU^+ve cells must be predominantly aNSCs (71, 72). Therefore, we observed a significant reduction in the number of aNSCs in KO (Fig. 6 F–H).

NSCs in the adult mouse SVZ undergo differentiation to neuroblasts, which migrate through the RMS toward the OB and generate neurons. Therefore, to analyze the fate of aNSCs upon NIHes1 KO, we generated a triple mutant conditional knock-out mouse line, Nestin-CreERT2;NIHes1^fl/fl;CAG-Stop^fl/fl-tdTomato (NIHes1 cKO), by crossing the Nestin-CreERT2;NIHes1^fl/fl and Nestin-CreERT2;CAG-Stop^fl/fl-tdTomato lines (SI Appendix, Fig. S8 A and B). Upon tamoxifen injection, the floxed NIHes1 promoter region will be knocked out in Nes^+ve cells. The knocked out NSCs and subsequent differentiated cells post–Cre recombination will be marked by fluorescent tdTomato (tdT) expression. Tamoxifen administered Nestin-CreERT2;CAG-Stop^fl/fl-tdTomato mice were used as controls. Using these strains, we immunostained the cells in the SVZ of control and KO cortices for DCX (neuroblast marker). The KO cells of the VZ, labeled with tdT expression, colocalized with DCX expression and increased in number (SI Appendix, Fig. S9 A–G). This shows that the knocked out cells predominantly differentiate into a neuroblast fate. To understand which NSCs are perturbed by NIHes1 KO in the adult cortex SVZ, we accessed and reanalyzed the scRNA-seq data of adult (8 to 12 wk old) mouse cortices (GSE67833) (Fig. 6I and SI Appendix, Text 1.4) (70). The qNSC clusters bear very few Nes^+ve cells while aNSC clusters were predominantly Nes^+ve (Fig. 6J). Whereas, both aNSCs and qNSCs expressed Hes1 (Fig. 6K). This indicates that the NIHes1 KO has happened exclusively in the aNSCs and corroborates the reduction of aNSCs upon KO (Fig. 6 F–H). We also investigated the TF activity of these NSCs and observed that even though qNSCs and aNSCs have NIHes1 expression, the overall gene programs in these NSCs appear to be different (Fig. 6L and SI Appendix, Text 1.4).

To check the fate of neuroblasts migrating from the SVZ to the OB, we knocked out NIHes1 expression at PN45 and analyzed the number of tdT^+ve newborn neurons in the OB 20 DAT. We observed a significant increase in the number of tdT^+ve cells in the KO (SI Appendix, Fig. S9 H–J). To understand the fate of the KO neuroblasts in the OB, we performed immunostaining of these tdT^+ve cells for DCX, NEUN (neuronal marker) and GFAP (astrocyte marker). We found colocalization of tdT^+ve cells with DCX and NEUN (Fig. 6 M–R). However, we found a reduction of DCX^+ve neuroblasts and an increase of NEUN^+ve neurons upon KO. We did not find a significant colocalization of GFAP^+ve and tdT^+ve cells in control and KO (SI Appendix, Fig. S10 A–C). These results indicated that, upon NIHes1 KO, the neuroblasts arising from the SVZ rapidly differentiate into neurons and get depleted at the OB. The increased number of tdT cells is confirmed to be neurons and not astrocytes. Thus, NIHes1 KO promotes differentiation and reduces aNSCs, resulting in an increase in the number of neurons in the OB. These results suggest that NIHes1 expression is required to maintain aNSCs in the adult SVZ as well.

Discussion

Our study reveals that Nes^+ve Hes1^+ve NSCs in the VZ are more heterogeneous than previously appreciated, with distinct subclasses defined by mode of Hes1 expression (11). While NDHes1 expression characterizes RGCs, the NIHes1 expression labels cells with short apical and basal processes (likely SNPs) (11). The NIHes1 NSCs are unique since they are not dependent on cell–cell interaction for Hes1 expression. Therefore, it is essential to characterize NIHes1 NSCs, which will have profound implications regarding maintenance and expansion of cortical NSCs.

In the NIHes1^fl/fl mice, KO in Nes^+ve cells affect only the NIHes1 NSCs, sparing IPCs and other cell types. Here, NIHes1 KO shows that NIHes1 expression is dispensable for NSC proliferation (Fig. 3 E and F), but it is indispensable for NIHes1 NSC identity, as evidenced from increased transition to NDHes1 RGCs. A significant upregulation of the classical astrocyte markers such as Gfap, S100b, and Ndrg2 was also observed upon NIHes1 KO, which could indicate the demethylation of the gliogenic genes in the progenitors (SI Appendix, Fig. S6B and Dataset S5) (26, 73). Further, scRNA-seq analysis shows that NDHes1 NSCs in KO window corresponds to gliogenic RGCs (Gfap^+ve, Nes^+ve, and Hes^+ve cells) (SI Appendix, Figs. S11 and S12). This could lead to acceptance of exogenous astroglial signals like Stat3 and hence, the promotion of astroglial differentiation (Fig. 4 K–P) (1, 74).

Beyond their role in RGC fate, NIHes1 NSCs could contribute directly to neurogenic IPC fate. During corticogenesis, IPCs arise at two different stages. First, as unipotent pyramidal neuron-IPCs (PyN-IPCs) which generate PyNs (E11.5 to E16.5) (6, 75) and as gliogenic IPCs generating astrocytes and oligodendrocytes (after E16.5) (76, 77). This highlights a high degree of intrinsic heterogeneity within primary NSCs. Previous literature broadly described the Nes^+ve primary NSCs arising from NECs as “RGCs”. However, pseudotime analysis of E14 scRNA-seq data places NIHes1 NSCs as the primitive NSCs acting as a branching point between IPCs and NDHes1 NSCs (RGCs, Fig. 2E). Further, Shimojo et al. (46) identified Neurog2 mechanistically driving the transition of Hes1^+ve NSCs to Hes1^−ve, Tbr2^+ve (Eomes^+ve) IPCs. ScRNA-seq analyses showed higher Neurog2 expression in NIHes1 NSCs than NDHes1 NSCs (Fig. 1J). Further, NIHes1 KO reduces IPCs, while DAPT treatment increases IPCs as shown by Kawaguchi et. al. (78), and the proliferating Hes1^−ve cells in the SVZ (Fig. 3 S–X). The impairment of laminar positioning of neurons is a phenotype of IPC depletion (Fig. 5 I–M) (79). Taken together, our findings show that Hes1^−ve Tbr2^+ve early neurogenic IPCs potentially arise from the NIHes1 NSCs, and not from NDHes1 NSCs (Fig. 2E). Therefore, NIHes1 expression is indispensable in deciding the fate of NIHes1 NSCs and thereby in establishing the NSC niche. Further in vivo studies are needed to confirm this.

Another critical finding of this study was NIHes1-expressing adult qNSCs and aNSCs of which the latter is maintained by NIHes1 expression. BrdU incorporation experiments showed that these qNSCs are a “set-aside” population of embryonic NSCs. NIHes1 expression maintains the stemness of aNSCs and its KO induces differentiation toward neuroblasts and neurons. The role of NIHes1 expression in the qNSCs and the mechanism of maintenance of aNSCs need to be explored. However, it is clear that the roles of NIHes1 expression and NIHes1 NSCs transcend beyond embryonic stages. Altogether, within the neural stem cell niche, the Hes1^+ve NSCs are more heterogeneous than previously understood. The Hes1^+ve NSCs have been studied as a homogenous pool of NSCs (as “RGCs”) and therefore, characteristics and functions attributed to RGCs may not be entirely of RGCs but the NIHes1 NSCs as well. Our study demonstrates that NIHes1 cells are precursor NSCs. The NSC niche initially has a proliferative phase of NIHes1 NSCs (E10-E12), possibly producing a peak of neurogenesis through IPCs (E14, Fig. 2 C and D). This is followed by transition to RGCs (E15-E17) and subsequent gliogenesis (Fig. 2C) (11). The NIHes1 NSCs are retained beyond the prenatal stages (E18-P4) as adult quiescent stem cells (Fig. 2D) and are activated to produce neuroblasts (SI Appendix, Fig. S13) that migrate to the olfactory lobes to generate neurons. Thus, NIHes1 NSCs and NIHes1 expression are pivotal for embryonic and adult cortical development (Fig. 6S).

Materials and Methods

Animal Models.

All animal experiments were carried out with approval from the Institutional Animal Ethics Committee (IAEC) of the BRIC-Rajiv Gandhi Centre for Biotechnology (BRIC-RGCB) (IAEC/179/JAC/2012, IAEC/728/JAC/2017, and IAEC/774/JAC/2019). The animals were housed in IVC cages under standard temperature, humidity, and light cycle and were provided with standard feed and water ad libitum. Mouse lines used in our study were C57BL/6-Tg(Nes-cre/ERT2)KEisc/J (IMSR_JAX:016261 – Nestin-CreERT2), NIHes1^fl/fl, B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J (IMSR_JAX:007914 – CAG-Stop^fl/fl-tdTomato) and Hes1-d2EGFP mice. The Hes1-d2EGFP mouse, which marks all the HES1-expressing cells with d2EGFP, was a kind gift from Dr. Ryochiro Kageyama, Kyoto University, Japan. Nestin-CreERT2 and CAG-Stop^fl/fl-tdTomato mice were procured from JAX. NIHes1^fl/fl mice were custom-generated through Cyagen (Cyagen, 2255 Martin Avenue, Santa Clara, CA 95050-2709, US). Details regarding experimental animal breeding, genotyping, plasmids, electroporations, IHC, BrdU experiments, PCR, Western blotting, RNA-seq analyses, image and statistical analyses are mentioned in SI Appendix.

Supplementary Material

Appendix 01 (PDF)

Dataset S01 (XLSX)

Dataset S02 (XLSX)

Dataset S03 (XLSX)

Dataset S04 (XLSX)

Dataset S05 (XLSX)

Data, Materials, and Software Availability

RNA sequencing raw data (nucleic acid sequences dataset) and original computer codes data have been deposited in NCBI Sequence Read Archive (SRA) and Zenodo [PRJNA1256699 (80) and 10.5281/zenodo.15308921 (81)]. Previously published data were used for this work (27, 65, 70).