Plasticity of iNB in vitro and in vivo

s-aNSC, s-TAP, s-iNB, and s-mNB were sorted from the SVZ of 2-month old C57Bl/6 mice. (A) Population doublings (PD). Data were obtained from 3 independent experiments (n=8). (B) Clonogenicity assay. Results were obtained from 2 independent experiments. The number inside the bars indicated the number of microplate wells analysed (mean ± SEM). (C) Differentiation assay. Representative images of immunofluorescence of freshly sorted iNB cells cultured in oligodendrocyte, astrocyte, or neuronal differentiation medium, and stained for NG2 and CNPase, GFAP and CD133 or βIII-Tubulin and doublecortin (DCX) expressions, respectively. *p<0.05, **p<0.01, ***p<0.001. Scale bar: 20µm. (D) EGFP-positive s-iNB were isolated from β–actin:eGFP mice and transplanted unilaterally at 3 injection points at proximity of the dSVZ/RMS of recipient C57Bl/6J mice. Transplanted brains were analysed five weeks later by immunostaining. (a) Detection of eGFP+ cells in the granule cell layer and the external cell layer of the olfactory bulb of a mouse transplanted. (a1-a2) a2 high magnification of the inset (dotted line in a) showing eGFP+ NeuN+ cells (white arrows). (b-c) Detection (white arrows) of eGFP+ GFAP+ cells in the dorsal (b2) and lateral (c2) SVZ. GCL:granule cell layer, EPL: external plexiform layer, GL: glomerular layer, lSVZ: lateral SVZ, dSVZ: dorsal SVZ, Scale bars= 40µm or 100µm.

Transplantations of eGFP+s-iNB, eGFP−s-iNB and eGFP+s-mNB freshly isolated from β−actin:eGFP mice model

Table recapitulating the immunohistological analyses of the numbers of eGFP+ cells in different regions in recipient C57Bl6/J mice brains, 5 weeks after transplantation.

Transcriptomic analysis of SVZ neural progenitor cells

A) Schematic illustration of our strategy of flow cytometry cell sorting of the five major neurogenic populations (s-qNSC, s-aNSC, s-TAP, s-iNB, s-mNB) of SVZ microdissected from adult mouse brains. (B) Pseudotime analysis showing the lineage progression of sorted cell populations based on the highly dispersed genes between s-qNSC, s-aNSC, s-TAP, s-iNB and s-mNB. (C) Pseudotime analysis after exclusion of s-qNSC based to further discriminate s-aNSC and s-TAP. (E) Heatmap representation of RSR genes differentially expressed among s-aNSC, s-TAP, s-iNB, s-mNB (FDR p-value<0.05).

scRNA-seq analysis of the lateral SVZ

(A) Experimental design. (B) Uniform Manifold Approximation and Projection (UMAP) of the 17343 high-quality sequenced cells annotated with corresponding cell type, combining scRNA-seq datasets of non-irradiated mice and 4Gy irradiated mice. Clustering analysis at resolution 1.2 segregated cells into 33 clusters that were matched with corresponding cell types determined with marker expression in (C). Among them, 14 clusters (dotted lines rectangle) corresponded to the clusters of astrocytes and neurogenic cells (ANC). (C) Violin diagrams illustrating the expression level of known cell markers in the 33 clusters according to the literature. (D) Violin diagrams representing the expression level of selected markers specific to neurogenic cells and astrocyte from ANC clusters at the resolution 1.2. (E) UMAP focusing on the ANC subset. (F) UMAP of the top UCell score for the top 100 highly expressed genes of each neurogenic cell sorted population in the ANC clusters (Supp_data_1). (G) Chord diagram illustrating the correspondence of cell clusters with cell top score. Cluster 10 matched with both s-TAP and s-iNB and clusters 8 and 19 both to s-iNB and to s-mNB. (H) Feature plot of cell cycle scoring (I, J) UMAP representation of ANC clusters in the unirradiated (I) and irradiated (J) samples. (K) Barplot showing the recovery ratios of each ANC cluster in irradiated brains calculated as the number of cells per clusters from irradiated brains/control brains normalized to the respective numbers of s-qNSC (cluster 4). Taking into account UCell score results, s-iNB clusters overlapped the cluster 10 and 8 containing s-TAP and s-mNB, respectively.

Expression of RSR genes in scRNA-seq neurogenic clusters Violin diagrams illustrating the expression of the RSR genes increasing

(A) or decreasing (B) with neurogenesis.

Dcx expression in the neurogenic cell clusters

(A) Feature plot. (B) Barplot representations of the recovery ratios of Dcxlow (left) and DcxHigh (right) in the iNB clusters of the irradiated brains, calculated as in Figure 3K. Violin diagrams illustrating the expression level of RSR genes decreasing (C) and increasing (D) with neurogenesis in Dcxlow and DcxHigh in neurogenic cell clusters.

Plasticity of DcxHigh iNB in vitro

(A) Clonogenic potential of FACS-isolated eGFP+iNB and eGFP−iNB, and recombined eGFP+mNB isolated from DCX-CreERT2::CAG-floxed-eGFP mice, 7 days after the first injection of tamoxifen. (B) Population doublings (PD) of FACS-isolated eGFP+iNB and eGFP− iNB, eGFP+mNB cells and total iNB cells (regardless of eGFP status). No statistical difference was found in the growth rate of the different isolated populations. (n = 8) (C) Representative images of immunofluorescence of freshly sorted eGFP+iNB cells cultured in oligodendrocyte,astrocyte, or neuronal differentiation medium, and stained for NG2 and CNPase, GFAP and CD133, βIII-Tubulin and DCX, respectively. Scale: 10µm.

Proposed model of adult mouse SVZ neurogenesis involving major changes in RSR genes expression

eGFP+ s-iNB cells in the rostral migratory stream

Immunofluorescence of mouse brains transplanted unilaterally in 3 injection points near the dSVZ/RMS with eGFP+iNB cells isolated from β-actin:eGFP mice. Transplanted brains were analysed five weeks later. (A) EGFP+iNB cells reached the rostral migratory stream and expressed DCX (insets A2 and A3, white arrow). Insets are high magnification panels. (B) eGFP+ cells persisting at the injection site expressing astrocyte or oligodendrocyte markers. High magnifications of the inset1 (dotted line in A) showing eGFP+ GFAP+ cells (white arrow) (a2) and eGFP+DCX+ cells (white arrow) (a3). High magnifications of the inset2 (dotted line in B) showing eGFP+ CNPase+ (white arrow) (b2). Scale bars= 100 µm.

Functional enrichment analyses of the individual transcriptome signature of neurogenic populations

Comparisons of individual transcriptome signatures with various Gene Ontology gene sets and Biological pathways using the g:Profiler software highlight significant stage-dependant changes. S-qNSCs expressed genes related to Metabolism of lipids, Transport of small molecules. S-aNSC expressed genes involved in Glyoxylate and decarboxylate metabolism. S-TAP significantly displayed genes involved in Mitotic G1 phase and G1/S transition and S Phase. S-iNB specifically expressed Cell Cycle and Mitotic genes in contrast to s-mNB which are characterised by the expression of Axon guidance and Nervous system development genes.

Correspondence between the molecular signatures of sorted neural progenitor populations in our study and available sc-RNA-seq datasets.

Violin diagrams representing the expression of selected clusters from published sc-RNAseq analysis in our Clariom microarray14,17,32. S-qNSC perfectly matched the expression of Glial markers (Class III)32, qNSC17 and Quiescent B cells (clusters 5 and 14)14. In contrast, s-aNSC poorly matched with Lipid biosynthesis (Class III) and Cell cycle (Class IV)32, aNSC17 and Activated B cells (Cluster 13)14. Similarly, s-TAP poorly matched with TAP17 and C cells (Cluster 12)14. S-iNB perfectly fit with Mitosis (Class VI), mitotic TAP (mTAP) (Zywitza et al., 2018) and Mitosis (Cluster 8, 10,16 and 17)14. s-mNB overlapped genes related to Neuron differentiation (Class VII), Late NBs 17 and A cells (clusters 0, 1 and 4).

Quality controls of scRNA-seq on whole SVZ tissues

(A) Violin diagrams of the nCount_RNA, the nFeature_RNA and the mitochondrial percentage (Percent.mito) before applying cut-offs adapted to each non-irradiated and irradiated samples. (B) Proportion of cells from the 4 samples in the 33 clusters.

Annotation of sc-RNAseq clusters

(A) UMAP at the resolution 1.2 combining scRNA-seq datasets of unirradiated and 4Gy irradiated cells segregated into 33 clusters. (B) Violin diagrams illustrating the expression level of known cell markers in the 33 clusters according to the literature. (C) UMAP of top normalized UCellScore Astrocyte- and the B-cell specific genes described by Cebrian-Silla et al 14 for cells from clusters 4 and 13.

Expression of Dcx in scRNA-seq neurogenic clusters

Violin diagram of the expression of Dcx in cells from neurogenic clusters illustrating two populations of iNB: iNB DcHiigh and iNB DcxLowcells.

Expression of RSR genes in scRNA-seq neurogenic clusters in irradiated brains

Violin diagrams illustrating the expression of the RSR genes increasing (A) or decreasing (B) with neurogenesis.

Transplantations of eGFP+s-iNB, eGFP−s-iNB and eGFP+s-mNB freshly isolated from DCX-CreERT2::CAG-floxed-eGFP mice model

Table recapitulating the immunohistological analyses of the numbers of eGFP+ cells in different regions in recipient C57Bl6/J mice brains, 5 weeks after transplantation.