Transcriptional regulation of neural stem cell expansion in the adult hippocampus
- Center for Regenerative Medicine, Massachusetts General Hospital, United States
- Harvard Stem Cell Institute, United States
- Department of Psychiatry, Massachusetts General Hospital, Harvard Medical School, United States
- BROAD Institute of Harvard and MIT, United States
- Ruth L. and David S. Gottesman Institute for Stem Cell Biology and Regenerative Medicine; Dominick Purpura Department of Neuroscience, Albert Einstein College of Medicine, United States
- Department of Molecular Biology, Massachusetts General Hospital, Harvard Medical School, United States
Figures
Figure 1 with 3 supplements
Klf9 is elevated in nondividing radial-glial neural stem cells (RGLs) and loss of Kruppel-like factor 9 (Klf9) promotes RGL activation.
(A, B) Klf9 expression inferred from LacZ expression intensity in quiescent RGLs, qRGL (GFP+ MCM2 with radial process, arrows), activated RGL, aRGL (GFP+ MCM2+ with radial process, arrowheads) and activated neural progenitors, aNPCs (GFP+ MCM2+ without a radial process) in Klf9LacZ/+;Nestin GFP transgenic. qRGLs exhibit higher Klf9 expression than aRGLs and aNPCs. n = 3 mice/group. (C–E) Fluorescence in situ hybridization using a Klf9-specific riboprobe and immunohistochemistry for GFP and BrdU on adult hippocampal sections obtained from Klf9LacZ/+ or LacZ/LacZ;Nestin GFP transgenic mice. (D) Specificity of riboprobe established by detection of Klf9 expression in dentate gyrus of Klf9+/+ but not in Klf9LacZ/LacZ mice. (C, E) Klf9 is expressed in qRGLs but not in dividing (BrdU+) RGLs or aNPCs. n = 3 mice/group. (F, G) Inducible deletion of Klf9 in Gli1+ RGLs in adult mice (Gli1CreERT2:Klf9+/+:Ai14 vs. Gli1CreERT2:Klf9f/f:Ai14) results in increased RGL activation (percentage of MCM2+ tdTomato + Nestin+ RGLs). n = 3 and 4 mice/group. Data are represented as mean ± standard error of the mean (SEM). *p < 0.05, **p < 0.01, ****p < 0.0001. Scale bar: B, F, 50 μm; D, 250 μm; E, 20 μm.
Figure 1—figure supplement 1
Generation and characterization of Kruppel-like factor 9 (Klf9) conditional mutant mouse line.
(A) Schematic of wild-type and modified Klf9 alleles. (B) PCR on tail DNA showing expected bands conveying wild-type and conditional alleles. (C) Left: Klf9 in situ hybridization on hippocampal sections from 4 months old POMC Cre: Klf9+/+ or f/f mice showing expected salt and pepper pattern of recombination in dentate gyrus that is characteristic of POMC Cre recombination pattern in dentate gyrus. Right: Klf9 in situ hybridization on hippocampal sections from adult Klf9−/− mice conveying specificity of Klf9 riboprobe. Scale bar: 500 µm.
Figure 1—figure supplement 2
Estimation of Kruppel-like factor 9 (Klf9) recombination frequency in Gli1-positive tdTomato-labeled radial-glial neural stem cells (RGLs).
(A) Representative low magnification images of Klf9 FISH signal (Klf9 transcripts) in dentate gyrus sections obtained from Gli1CreERT2:Klf9+/+:Ai14 and Gli1CreERT2:Klf9f/f:Ai14 mice following TAM-mediated induction of Klf9 recombination in Gli1-positive RGLs. (B) High magnification images of Klf9 FISH signal (Klf9 transcripts) in Gli1-positive tdTomato-labeled RGLs in Gli1CreERT2:Klf9+/+:Ai14 and Gli1CreERT2:Klf9f/f:Ai14 mice. (C) Quantification of Klf9 transcript-associated fluorescence intensity in Gli1-positive RGLs in Gli1CreERT2:Klf9+/+:Ai14 and Gli1CreERT2:Klf9f/f:Ai14 mice following TAM-mediated induction of recombination. RGLs (n = 23 Klf9+/+, n = 24 Klf9f/f) were analyzed from 2 mice/group. Scale bar: 100 µm (A), 20 µm (B). Data are represented as mean ± standard error of the mean (SEM). *p < 0.05.
Figure 1—figure supplement 3
Inducible overexpression of Kruppel-like factor 9 (Klf9) in activated neural stem and progenitors promotes quiescence.
(A, B) Two cohorts of adult Sox1tTA: tetO Klf9 mice were used. Klf9 induction in neural stem and progenitors following 3 weeks off Dox significantly reduced the fraction of activated radial-glial neural stem cells (RGLs) (%MCM2+ Nestin+ RGLS, n = 6 and 4 mice/group). Representative images shown in bottom panel. (C, D) A second cohort of mice was given BrdU pulses during the Off Dox window when Klf9 is upregulated. Analysis of BrdU+ Nestin+ RGLs (n = 3 mice/group) revealed a significant reduction in total numbers of dividing RGLs. Representative images shown here. Unpaired t-tests, Panel B: p = 0.0003, Panel D: p = 0.005. Data are represented as mean ± standard error of the mean (SEM). **p < 0.01, ***p < 0.001. Scale bar: 100 µm (top), 50 µm (C).
Figure 2 with 9 supplements
Kruppel-like factor 9 (Klf9) deletion in radial-glial neural stem cells (RGLs) produces supernumerary RGL clones.
(A–D) Clonal analysis of sparsely labeled Gli1+ RGLs in adult Gli1CreERT2:Klf9+/+or f/f:Ai14 mice at 7 dpi. (A, C) Representative images of labeled RGL clones and descendants. For example, A top: single RGL (white arrow), A bottom: 2 RGLs. Identification was based on tdTomato+ morphology and GFAP immunohistochemistry. (B) Statistical representation of clones for specified compositions for both genotypes expressed as fraction of total clones quantified. (D) Breakdown of clones into 2 RGLs+ (two or more RGL containing clones and progeny) and single RGL+ clones (clones containing only 1 RGL and progeny). Loss of Klf9 in Gli1+ RGLs results in statistically significant overrepresentation of two or more RGL containing clones and significant reduction in ‘1 RGL containing clones’ suggestive of Klf9 repressing RGL expansion. n = 4 mice/group. (E, F) Clonal analysis of sparsely labeled Gli1+ RGLs (white arrow) in adult Gli1CreERT2:Klf9+/+or f/f:mTmG mice at 7 dpi. Inducible deletion of Klf9 in Gli1+ RGLs results in statistically significant overrepresentation of multi-RGL containing clones (two or more RGLs, 2 RGLs+) and a significant reduction in single RGL containing clones (1 RGL+). Identification was based on GFP+ morphology and GFAP immunohistochemistry. Representative images (E) and corresponding quantification in (F). n = 4 and 5 mice/group. P: rogenitor(s), A: astrocyte. Data are represented as mean ± standard error of the mean (SEM). *p < 0.05, ***p < 0.001, ****p < 0.0001. Scale bar: A, C, E, 20 μm.
Figure 2—figure supplement 1
Analysis of clonal composition in Figure 2C.
Representative images of labeled radial-glial neural stem cell (RGL) clones and descendants. Identification was based on tdTomato+ morphology and GFAP immunohistochemistry. Z-series of confocal images in Figure 2C were processed using Imaris software. Scale bar: 15 µm.
Figure 2—video 1
Three-dimensional (3D) images of representative radial-glial neural stem cell (RGL) clonal compositions depicted in Figure 2C 1 RGL.
Figure 2—video 2
Three-dimensional (3D) images of representative radial-glial neural stem cell (RGL) clonal compositions depicted in Figure 2C 1 RGL.
Figure 2—video 3
Three-dimensional (3D) images of representative radial-glial neural stem cell (RGL) clonal compositions depicted in Figure 2C 1 RGL+ P + A.
Figure 2—video 4
Three-dimensional (3D) images of representative radial-glial neural stem cell (RGL) clonal compositions depicted in Figure 2C 1 RGL+ A.
Figure 2—video 5
Three-dimensional (3D) images of representative radial-glial neural stem cell (RGL) clonal compositions depicted in Figure 2C 1 RGL+ P.
Figure 2—video 6
Three-dimensional (3D) images of representative radial-glial neural stem cell (RGL) clonal compositions depicted in Figure 2C 2 RGLs.
Figure 2—video 7
Three-dimensional (3D) images of representative radial-glial neural stem cell (RGL) clonal compositions depicted in Figure 2C 2 RGLs+ P + A.
Figure 2—video 8
Three-dimensional (3D) images of representative radial-glial neural stem cell (RGL) clonal compositions depicted in Figure 2C 2 RGLs+ P.
Figure 3 with 5 supplements
Kruppel-like factor 9 (Klf9) functions as a brake on symmetric self-renewal of radial-glial neural stem cells (RGLs).
(A) Diagram of experimental design for in vivo two-photon imaging experiments. Inset is a high magnification image of a sparsely labeled single RGL in an adult Gli1CreERT2:Klf9+/+:Ai14 mouse. (B) Representative series of longitudinal imaging from four fields of view showing RGL symmetric and asymmetric divisions. Row 2: control. Rows 1, 3, and 4: experimental. Arrows point to mother cell and arrowheads point to daughter cells. Scale bar: 20 µm. (C) Quantification of RGL symmetric and asymmetric divisions showing an increase in symmetric divisions in Gli1CreERT2:Klf9f/f:Ai14 mice. n = 8 Gli1CreERT2:Klf9+/+:Ai14 mice, 65 divisions; n = 10 Gli1CreERT2:Klf9f/f:Ai14 mice, 77 divisions. Odds of symmetric division are 2.7× higher in Gli1CreERT2:Klf9f/f:Ai14 mice, p = 0.015 likelihood-ratio test., *(D) Similar number of divisions was recorded for each group to avoid biased assessment of division mode (n = 8 and 10 mice/group).
Figure 3—figure supplement 1
Representative images of radial-glial neural stem cell (RGL) divisions captured using two-photon imaging in vivo.
(A) Representative two-photon images of RGL cells R1 and R2 in vivo and their respective post hoc fluorescence image. (B) Confocal immunofluorescence images of the same GFAP+/tdTomato+ cells at different depths, confirming their RGL identity. (C) Imaris deconvolution of tdTomato-labeled RGLs in B. Scale bar: 20 µm.
Figure 3—video 1
In vivo two-photon imaging of Gli1-postive radial-glial neural stem cells (RGLs).
Narrated example of longitudinal imaging of asymmetric neural stem cell (NSC) divisions. Two-photon imaging across days showing two examples of asymmetric division of NSCs (red arrows).
Figure 3—video 2
In vivo two-photon imaging of Gli1-postive radial-glial neural stem cells (RGLs).
Narrated example of longitudinal imaging of symmetric cell divisions. Two-photon imaging across days showing two examples of symmetric division of neural stem cells (NSCs; blue arrows).
Figure 3—video 3
In vivo two-photon imaging of Gli1-postive radial-glial neural stem cells (RGLs).
Three-dimensional reconstruction of RGL cells imaged in vivo before undergoing symmetric division. Field of view corresponds to second row of Figure 3B at 18 dpi.
Figure 3—video 4
In vivo two-photon imaging of Gli1-postive radial-glial neural stem cells (RGLs).
Three-dimensional reconstruction of RGL cells imaged in vivo after undergoing symmetric division. Field of view corresponds to second row of Figure 3B at 30 dpi.
Figure 4 with 1 supplement
Kruppel-like factor 9 (Klf9) regulates genetic programs underlying radial-glial neural stem cell (RGL) expansion.
(A) Schematic of experimental workflow to biochemically isolate and sequence translated mRNAs from Gli1+ RGLs (Gli1CreERT2:Rpl22HAf/+:Klf9f/f or +/+ mice). n = 3 mice, 6 dentate gyri/sample, 3 samples per group. (B) Principal component analysis (PCA) plot of translational profiles of Gli1CreERT2-targeted Klf9+/+ or f/f RGLs. First two principal components are shown with the corresponding fractions of variance. (C) Left: heatmap of expression values for differentially expressed genes. Middle: volcano plot of statistical significance (−log10 p value) vs. magnitude of change (log2 of fold change) of gene expression. Differentially expressed genes are marked in red. Upregulated genes in Klf9f/f RGLs are on the right and downregulated genes in Klf9f/f RGLs are on the left. Right: pie chart of numbers of upregulated and downregulated genes in Gli1CreERT2-targeted Klf9f/f RGLs. (D) qRT-PCR on biochemically isolated mRNAs from Gli1CreERT2:Rpl22HAf/+:Klf9f/f or +/+ mice validating candidate differentially expressed genes. n = 3 samples, 6 dentate gyri/sample, 3 samples per group. (E) Immunostaining and quantification of Notch1 intracellular domain (NICD) in RGLs of Gli1CreERT2: Klf9f/f or +/+ mice. Deletion of Klf9 results in increased NICD levels in RGLs consistent with Lnfg-dependent potentiation of Notch1 signaling (Cartoon, top left). n = 3 mice/group. Data are represented as mean ± standard error of the mean (SEM). *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar: 10 μm.
Figure 4—figure supplement 1
Annotation of upregulated and downregulated differentially expressed genes (DEGs) in Gli1+ radial-glial neural stem cells (RGLs) following Kruppel-like factor 9 (Klf9) deletion.
Gene ontology annotation (gGOSt, https://biit.cs.ut.ee/gprofiler/gost) of DEGs in Gli1+ RGLs following Klf9 deletion.
Figure 5
Summary schematic conveying Kruppel-like factor 9 (Klf9) functions in radial-glial neural stem cell (RGL) activation and self-renewal RGLs integrate extracellular, experiential signals to exit quiescence, the dominant state, and become activated.
Klf9 expression is elevated in quiescent RGLs. Low levels of Klf9 in RGLs are associated with increased activation. Once activated, RGLs lacking Klf9 are biased toward symmetric self-renewal and RGL expansion. Translational profiling of RGLs reveals how loss of Klf9 results in downregulation of a program of quiescence and activation of genetic (mitogen, notch) and metabolic (fatty acid oxidation and lipid signaling) programs underlying RGL symmetric self-renewal. Candidate differentially expressed upregulated (orange) and downregulated genes (blue) in RGLs following Klf9 deletion are shown here. Genes in bold indicate validation by qRT-PCR.
Author response image 1
Inducible deletion of Klf9 in Ascl1+ RGLs in adult mice (Ascl1 CreERT2:Klf9+/+:Ai14 vs. Ascl1 CreERT2:Klf9f/f:Ai14) results in increased RGL activation (percentage of MCM2+tdTomato+Nestin+RGLs) n=4, 5 mice/group.
Data are represented as mean ± SEM. * p=0.02, Scale Bar 50 µm.
Author response image 2
A. Inducible deletion of Klf9 in Gli1+RGLs in adult mice (Gli1CreERT2:Klf9+/+:Ai14 vs. Gli1 CreERT2:Klf9f/f:Ai14) results in expansion of RGLs as assessed at 7dpi. Representative images (A-B) in and corresponding quantification (bottom) 7 dpi: n=4 and 5 mice/group. Data are represented as mean ± SEM. ** p<0.01. White arrowheads indicate RGLs. Note the dramatic expansion in RGL numbers throughout DG in B. Scale Bar is 20 µm.
Additional files
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Supplementary file 1
Complete lists of differentially expressed genes (DEGs) in Gli1+ radial-glial neural stem cells (RGLs) following Kruppel-like factor 9 (Klf9) deletion.
DEGs were defined by at least 1.2-fold change with FDR < 0.05.
- https://cdn.elifesciences.org/articles/72195/elife-72195-supp1-v2.xls
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Supplementary file 2
Gene ontology annotation (gGOSt, https://biit.cs.ut.ee/gprofiler/gost) of differentially upregulated genes in Gli1+ radial-glial neural stem cells (RGLs) following Kruppel-like factor 9 (Klf9) deletion.
- https://cdn.elifesciences.org/articles/72195/elife-72195-supp2-v2.xlsx
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Supplementary file 3
Gene ontology annotation (gGOSt, https://biit.cs.ut.ee/gprofiler/gost) of differentially downregulated genes in Gli1+ radial-glial neural stem cells (RGLs) following Kruppel-like factor 9 (Klf9) deletion.
- https://cdn.elifesciences.org/articles/72195/elife-72195-supp3-v2.xlsx
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Supplementary file 4
Statistical Analysis.
- https://cdn.elifesciences.org/articles/72195/elife-72195-supp4-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/72195/elife-72195-transrepform1-v2.pdf
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Transcriptional regulation of neural stem cell expansion in the adult hippocampus
eLife 11:e72195.
https://doi.org/10.7554/eLife.72195
