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. 2022 Sep 13;25(10):105102.
doi: 10.1016/j.isci.2022.105102. eCollection 2022 Oct 21.

Neuroblasts contribute to oligodendrocytes generation upon demyelination in the adult mouse brain

Bilal El Waly  1 , Claire Bertet  1 , Mathilde Paris  1   2 , Marie Falque  1 , Pierre Milpied  3 , Karine Magalon  1 , Myriam Cayre  1 , Pascale Durbec  1
Affiliations

Neuroblasts contribute to oligodendrocytes generation upon demyelination in the adult mouse brain

Bilal El Waly et al. iScience. .

Abstract

After demyelinating insult, the neuronal progenitors of the adult mouse sub-ventricular zone (SVZ) called neuroblasts convert into oligodendrocytes that participate to the remyelination process. We use this rare example of spontaneous fate conversion to identify the molecular mechanisms governing these processes. Using in vivo cell lineage and single cell RNA-sequencing, we demonstrate that SVZ neuroblasts fate conversion proceeds through formation of a non-proliferating transient cellular state co-expressing markers of both neuronal and oligodendrocyte identities. Transition between the two identities starts immediately after demyelination and occurs gradually, by a stepwise upregulation/downregulation of key TFs and chromatin modifiers. Each step of this fate conversion involves fine adjustments of the transcription and translation machineries as well as tight regulation of metabolism and migratory behaviors. Together, these data constitute the first in-depth analysis of a spontaneous cell fate conversion in the adult mammalian CNS.

Keywords: Cellular neuroscience; Developmental neuroscience; Molecular neuroscience.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Lineage plasticity of SVZ neuroblasts after LPC induced demyelination (A–C) LPC-induced demyelination of GAD67-GFP mouse, 6 days after injection. (A) GFP+ NB (green) ectopically migrate from the RMS toward the lesion site in the CC (dashed line). Higher magnification: GFP+ NB exiting the RMS. (B) Some GFP+ NB start expressing oligodendrocyte markers such as Olig2 (red). (C) Density of GAD67-GFP+ Olig2+ cells in the CC and SVZ, 3 and 6 dpi. Error bars: mean ± SEM ∗p < 0.05, ∗∗p < 0.01; Mann-Whitney test (n= 5 mice). (D and E) LPC-induced demyelination of DCX-CreERT2:YFP mouse. 7 days after LPC injection, YFP+ NB are identified in the lesion (dashed line) and some of them express the oligodendrocyte marker Olig2 (red). (F–H) LPC-induced demyelination of DCX-CreERT2:mTmG mouse. 3 weeks after LPC injection at the site of the lesion, GFP+ cells adopt a complex morphology and express the mature oligodendrocyte marker MBP (red in F) and GFP+ segments are flanked by Caspr staining indicating paranodal organization. (H) Electron micrograph showing GFP immunogold-labeled myelin sheaths. The presence of gold beads within compact myelin sheaths surrounding axons an attests for the differentiation of GFP+ NB into myelinating oligodendrocytes. Scale bars: 200 μm in A and D, 20 μm in B, 50 μm in E to G and 150 nm in H. SVZ: sub-ventricular zone, CC: corpus callosum, CX: cortex, LV: lateral ventricle.
Figure 2
Figure 2
Single cell RNA-seq of 155 cells from five populations representing the continuum from neuroblast to oligodendrocyte during demyelination-induced fate conversion (A) FACS strategy to isolate the 5 cell populations. All cells derive from GAD67-GFP:Olig2-tdTomato mice. Neuroblasts (GFP+, referred to NB and NBL in control and lesion mice respectively) and oligodendrocytes (Tomato+, referred to O and OL in control and lesioned mice, respectively) were purified from the SVZ and CC (n = 5 mice per group). In lesioned mice (6 days after LPC injection), a double-positive population (GFP+ Tomato+, referred to OligNBL) appeared and was also purified for RNA-sequencing. (B) PCA of 155 high-quality single cell RNA-seq transcriptomes separated the cells into 4 clusters corresponding to their population of origin: #1 NB, #2 NBL, #3 OligNBL and #4 O + OL. (C) Projection of gene coordinates on the PCA. As expected, NB (cluster #1) and O + OL (cluster #4) are associated with expression of neuronal (green dots) and oligodendrocyte genes (red dots), respectively. (D) Biclustering using the top 100 genes specifically enriched in each cell population cluster. Cells are shown in columns and genes in rows.
Figure 3
Figure 3
SVZ neuroblast fate conversion does not involve a dedifferentiation step (A) SVZ NB fate conversion into O could either involve a dedifferentiation step into aNSC/NPC or could proceed through formation of an intermediate cell type. (B) PCA on a meta-dataset of single cell RNA-seq experiments comprising our data (triangles) and datasets from Dulken et al. (Dulken et al., 2017) (diamond shapes) and Marques et al. (Marques et al., 2018) (dots), using the top 100 genes defining our 4 cell populations according to biclustering analyses. NBL (light green triangles) and OligNBL (yellow triangles) cluster between aNSC/NPC (blue diamond shapes) and OPC (red dots). O (red triangles) and OL (dark red triangles) cluster with mature oligodendrocytes. (C) PCA using only immature cells from the meta-dataset shows that NBL and OligNBL cluster away from aNSC and NPC. (D) Expression levels (TPM) of relevant markers for aNSC/NPC (GLAST/Slc1a3, Rpl32, Egfr, Cdk1) and the OSKM reprogramming set (Pou5f1/Oct4, Sox2, Klf4, Myc) in various cell populations. NBL and OligNBL do not express significant levels of these markers but express the oligodendrocyte marker Tcf4 (positive control). (E) OligNBL harbors a unique molecular identity, distinct from pri-OPC. Dot plot of expression of select genes in 11 cell populations from a meta-dataset comprised 152 aNSCs and 29 NPCs from Dulken et al. (2017); our 37 NB, 27 NBL and 40 OligNBL, 1070 NB, 143 pri-OPCs-GFAP, 738 pri-OPCs-PDGFR, 90 OPCs-GFAP and 340 OPCs-PDGFR from Weng et al. (2019) and 201 OPCs from Marques et al., (2018) for a total of 2877 cells.
Figure 4
Figure 4
SVZ neuroblast fate conversion proceeds through formation of an intermediate cell type carrying both NB and O identities (A) Pseudo-temporal ordering of the 5 cell populations based on the expression of 396 genes identified by Monocle as differentially expressed between NB and O. The trajectory of each cell population is illustrated below the main graph. NBL and OligNBL align between NB and O suggesting that their transcriptome is intermediate between these 2 cell types. (B)Number of genes characterizing the NB (489 genes) versus O (344 genes) identities (as defined by SCDE) in each cell population. A gene is considered as expressed in a given population if its abundance is >1TPM in at least 25% of the cells. NB-identity genes are progressively turned down whereas O-identity genes are progressively activated during the NB to O conversion. (C) Immunolabeling for neuronal (PSA-NCAM, Tuj1) and oligodendrocyte (Olig2, Cspg4, Pdgfra, Sox10) genes in the SVZ and CC of GAD67-GFP mice 6 days after LPC injection confirm that OligNBL have a dual NB/O identity. (D) Percentage of Edu+ cells in NBL (GFP+), OL (Tomato+), OligNBL (GFP+, Tomato+) and GFP− Tomato− cell populations deriving from the SVZ and CC of GAD67-GFP:Olig2-tdTomato mice 6 days after LPC injection.
Figure 5
Figure 5
LPC-induced demyelination modifies the transcriptional state of SVZ neuroblasts (A) GO-term analyses of the 5 cell populations show that their gene-signatures are enriched for functions controlling transcription and translation (purple), cell adhesion and cytoskeleton (blue), cell division (black), respiratory chain (green) and cell trafficking (orange). (B) Heatmap of mRNA levels for transcription and translation genes in the 5 cell populations. GO terms: transcription (GO: 0,006,351), mRNA processing (GO: 000,639) and ribosome composition (GO: 0,005,840). Red rectangles highlight the set of genes enriched in the gene-signatures of the different populations for each GO-term. A gene is considered as enriched in a cell population if it is differentially expressed between this cell population and at least one other population. Cells are shown in columns and genes in rows. (C) Summary of characteristic neuronal and oligodendrocyte genes whose expression switches during the various transitions of the fate conversion process. Detailed mRNA levels are given for some oligodendrocyte (Nfib, Tcf4, Nr2f1, PDGFRa, Cspg4, Plp1) and neuronal (Sp8, Cbx5, Dlx6, Slc29a4, Ercc1, Tubb2b) genes.
Figure 6
Figure 6
Changes in genes associated with cell migration occurring during SVZ neuroblast fate conversion (A) Detailed mRNA levels for genes involved in cell adhesion and migration, which are enriched in specific cell populations. Map2 (GO:0,005,874∼microtubule) is enriched in NB; Picalm (GO:0,005,913∼cell-cell adherens junction) in NBL; Atat1 (GO:0,005,925∼focal adhesion) in NBL and OligNBL; Actg1 (GO:0,005,925∼focal adhesion and GO:0,031,012∼extracellular matrix) in NBL and OligNBL; Palm (GO:0,030,175∼filopodium) in OligNBL. (B) Detailed mRNA levels for genes involved in EMT (Srf, Zeb1, Scrt1), which are specifically upregulated in NBL according to SCDE.
Figure 7
Figure 7
Metabolic changes associated with SVZ NB fate conversion (A) Number of genes involved in glycolysis (green), TCA cycle (blue) and mitochondrial respiratory chain (red) in each cell population. A gene is considered as expressed in a given population if its abundance is >1TPM in at least 25% of the cells. (B) Schematic representing the glycolysis pathway and TCA cycle. Genes enriched in specific cell populations are highlighted with the following color-code: green if enriched in NB, light green for NBL, yellow for OligNBL, red for O and dark red for OL. A gene is considered as enriched in a cell population if it is differentially expressed between this cell population and at least one other population. Detailed mRNA levels are given for some glycolysis (Slc2a3, Hk1, Eno1, PKM) and TCA cycle (Idh1, Mdh2) genes. (C and D) SVZ cells from GAD67-GFP:Olig2-tdTomato neonate mice were cultured in presence or absence of 2DG. GFP+ Tomato+ cells among GFP+ cells were observed (C) and quantified (D) in each condition. Scale bars: 50 μm.
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