Id4 promotes the elimination of the pro-activation factor Ascl1 to maintain quiescence of adult hippocampal stem cells
- The Francis Crick Institute, United Kingdom
- ICM, Inserm U 1127, CNRS UMR 7225, Sorbonne Université, France
- Erasmus MC, Netherlands
- Institute of Molecular Biotechnology (IMBA), Vienna Biocenter Campus (VBC), Austria
Figures
Figure 1
Ascl1 is transcribed in both quiescent and proliferating hippocampal stem cells.
(A) Immunolabeling for GFP, Ascl1 and GFAP in the subgranular zone (SGZ) of the dentate gyrus (DG) of Ascl1KIGFP reporter mice. White arrows indicate GFP+Ascl1- RGLs; yellow arrows indicate …
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Figure 1—source data 1
Original quantification of Ascl1kiGFP, Ascl1 antibody staining and Ascl1 RNA in active and quiescent RGLs.
- https://doi.org/10.7554/eLife.48561.004
Figure 2 with 1 supplement
Ascl1 is regulated post-translationally in quiescent NSC cultures.
(A) Transcript levels for Ascl1 in DG-derived NSC cultures treated with FGF2 alone (proliferating NSCs) or FGF2 and BMP4 (quiescent NSCs) analyzed by QPCR. Ascl1 mRNA levels are unchanged in …
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Figure 2—source data 1
Original quantification of Ascl1 mRNA and protein in proliferating and quiescent NSCs.
- https://doi.org/10.7554/eLife.48561.007
Figure 2—figure supplement 1
BMP4 induces reversible quiescence of adult hippocampal NSCs.
(A) Scheme of the generation of NSC cultures from the hippocampus of wildtype mice followed by the sequential exposure of NSCs to FGF2 alone and FGF2+BMP4 to induce quiescence. (B) (i) …
Figure 3 with 2 supplements
Id4 is a candidate regulator of Ascl1 protein expression in quiescent hippocampal stem cells.
(A) Transcript levels for the four Id genes (Id1, Id2, Id3, Id4) in FGF2-treated and FGF2+BMP4-treated NSCs cultures analyzed by QPCR. BMP strongly induces the Id genes (Id1 p=0.032; Id2 p=0.074; Id3…
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Figure 3—source data 1
Original quantification of Id4 protein levels in proliferating and quiescent hippocampal NSCs and RGLs.
- https://doi.org/10.7554/eLife.48561.011
Figure 3—figure supplement 1
Dynamics of Id1-4 and Ascl1 expression in proliferating and quiescent NSCs.
(A) Gene expression changes for selected genes between FGF2+BMP4- and FGF2-treated NSCs extracted from the RNA-Seq analysis in Figure 2—figure supplement 1E, F. The number before the gene name …
Figure 3—figure supplement 2
Expression of Id1-4 and Tcf4 in RGLs in the dentate gyrus.
(A) Immunolabeling for Id1, Ki67 and GFAP in the DG. White arrows indicate Ki67+Id1+ RGLs. Scale bar, 30 µm. (B–C) Quantification of the data in (A). Id1 is expressed in around half of all RGLs (B) …
Figure 4 with 1 supplement
Id4 suppresses Ascl1 expression and cell proliferation in NSC cultures.
(A) Model of Ascl1 monomerization and elimination following Id4 overexpression in proliferating NSC cultures. (B) Model of Id protein titration by E47 overexpression in quiescent NSC cultures. (C) …
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Figure 4—source data 1
Original quantification of Ascl1, Ki67 and EdU in Id4- and E47-overexpressing NSCs.
- https://doi.org/10.7554/eLife.48561.014
Figure 4—figure supplement 1
Analysis of Id1-4 overexpression and siRNA knockdown in NSCs.
(A) Quantification of the co-expression of Ascl1 and Ki67 in control and Id4-overexpressing FGF2-treated NSCs. Ascl1 and Ki67 are co-expressed in 15.5 ± 1.1% of control (Id4-negative) and 6.9 ± 1.3% …
Figure 5 with 1 supplement
Id4 regulation of Ascl1 targets contributes to BMP-induced NSC quiescence.
(A) Volcano plot displaying gene expression changes between control and Id4-overexpressing FGF2-treated NSCs analyzed by RNA-Seq. (B) Venn diagrams indicating the number of genes up- and …
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Figure 5—source data 1
Significantly regulated genes and gene ontology analysis, and CPM values for Ascl1 target genes in Id4-overexpressing NSCs.
- https://doi.org/10.7554/eLife.48561.017
Figure 5—figure supplement 1
RNAseq analysis of proliferating NSCs overexpressing Id4.
(A) Normalized transcript numbers from RNA-seq data (counts per million) of Ascl1, Tcf3, Tcf4, Tcf12, Hes1, Hes5 and Hey1 are unchanged between FGF2-treated NSCs overexpressing GFP or Id4. n = 3. (B)…
Figure 6 with 1 supplement
Loss of Id4 results in activation of quiescent RGLs in the adult hippocampus.
(A) Design of the experiment for acute and long-term deletion of Id4 from RGLs of the adult hippocampus using Id4cKO mice. (B) Immunolabeling for GFAP, tdTomato, Ascl1 and DAPI staining in control …
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Figure 6—source data 1
Original quantification of Ascl1, Ki67, Id1 and Id3 protein levels in RGLs following Id4 deletion.
- https://doi.org/10.7554/eLife.48561.020
Figure 6—figure supplement 1
Expression of Id4, Ascl1, Ki67, Id1 and Id3 in RGLs following loss of Id4.
(A) Structure of the Id4flox allele. (B) Immunolabeling for GFAP, tdTomato, Id4 and DAPI staining in control and Id4cKO mice demonstrating Id4 elimination in Id4cKO mice after 5 days of tamoxifen …
Author response image 1
Id4 expression is absent in Id4floxed NSCs.
(A-B) Immunolabeling for Id4, Ascl1 and DAPI staining in Id4fl/fl NSCs in FGF2+BMP4 conditions, infected with either GFP-expressing or Cre-expressing adenovirus in order to delete Id4. No Id4 …
Additional files
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Source data 1
Transparent reporting summary statistics.
- https://cdn.elifesciences.org/articles/48561/elife-48561-data1-v2.pdf
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Supplementary file 1
Key resources table.
- https://doi.org/10.7554/eLife.48561.021
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Transparent reporting form
- https://doi.org/10.7554/eLife.48561.024
