Neuroscience

Chromatin regulator Kdm6b is required for the establishment and maintenance of neural stem cells in mouse hippocampus

Eugene Gil
Sung Jun Hong
David Wu
Dae Hwi Park
Ryan N. Delgado
Martina Malatesta
Sajad Hamid Ahanger
Karin Lin
Saul Villeda
Daniel A. Lim author has email address

Department of Neurological Surgery, University of California, San Francisco, San Francisco, CA 94143, USA
Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA 94143, USA
Developmental and Stem Cell Biology Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA
BTIG, Garden City, NY 11530, USA
Medical Scientist Training Program, Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA
GC Cell, Yongin-Si, Gyeonggi-do, South Korea
Departments of Genetics and Ophthalmology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA
Bristol Myers Squibb, San Francisco, CA 94158
Department of Anatomy, University of California, San Francisco, San Francisco, CA 94143, USA
Neuroscience Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA
Calico Life Sciences, South San Francisco, CA 94080
San Francisco Veterans Affairs Medical Center, San Francisco, CA 94121, USA

https://doi.org/10.7554/eLife.97262.1

Open access
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Figures and data

Postnatal DG development is impaired with Kdm6b-deletion
A, Schematic coronal section showing the dentate gyrus of the hippocampus; red box indicates regions shown in (B). B, Schematic illustration of neurogenesis in the DG. C, KDM6B (green) expression in the DG of P21 mouse brains. Immunohistochemistry (IHC) is shown for SOX2 and NESTIN. D-E, IHC for DAPI (white) in coronal DG sections of P60 control (D) and hGFAP-Cre;Kdm6b^F/F mice (E). F, Quantification of length of the DG in control and hGFAP-Cre;Kdm6b^F/F mice from P0 and P21 (n = 3 each), two-tailed unpaired t test (* = p < 0.05, *** = p < 0.0005, **** = p < 0.0001, NS = not significant).

Adult hGFAP-Cre;Kdm6b^F/F mice display defective hippocampal-dependent behavior
A, Baseline freezing during fear conditioning training. B, Hippocampal-dependent learning and memory assessed in control and hGFAP-Cre;Kdm6b^F/F mice by contextual memory testing. C-D, Non-hippocampal dependent learning and memory assessed in control and hGFAP-Cre;Kdm6b^F/F mice by cued memory test. Two-tailed unpaired t test (** = p < 0.01)

Reduction of NSCs in the developing DG of hGFAP-Cre;Kdm6b^F/F mice.
A, IHC for PAX6 (green), SOX2 (purple), and DAPI (blue) in coronal sections of developing DG in control and hGFAP-Cre;Kdm6b^F/Fmice at P0 and P7. B, Quantification of DG neural precursor/NSCs in control and hGFAP-Cre;Kdm6b^F/F mice at P0 and P7 (n = 3 each), two-tailed unpaired t test (* = p < 0.05, ** = p < 0.01). C-H, IHC for NESTIN (green), GFAP (red), and DAPI (blue) in coronal DG sections of P60 control (C-E) and hGFAP-Cre;Kdm6b^F/Fmice (F-H). I-J, IHC for DCX (red) and DAPI (blue) in coronal DG sections of P60 control (I) and hGFAP-Cre;Kdm6b^F/F mice (J).

Kdm6b-deletion leads to premature differentiation of embryonic DG precursor cells
A, Schematic experimental design and coronal section showing the developing dentate gyrus; boxes indicate regions of quantification for (C, D). B, IHC for BrdU (green), TBR2 (white), and SOX2 (purple) with DAPI (blue) in coronal sections of DG in control and hGFAP-Cre;Kdm6b^F/F mice at E17.5. C, Quantification of BrdU+ TBR2+ cells in dentate migratory stream (DMS) of control and hGFAP-Cre;Kdm6b^F/F mice, two-tailed unpaired t test. D, Quantification of BrdU+ TBR2+ and BrdU+ SOX2+ cells in developing DG blades of control and hGFAP-Cre;Kdm6b^F/F mice, two-tailed unpaired t test (* = p < 0.05).

Excess IPCs in Kd6mb-deleted mice further mature into DG neurons
A, Schematic experimental design. B, IHC for BrdU (green), PROX1 (red), TBR2 (white), and DAPI (blue) in coronal sections of DG in control and hGFAP-Cre;Kdm6b^F/Fmice at P0.5. C, Quantification of (B), two-tailed paired t test (* = p < 0.05). D. IHC for BrdU (green), SOX2 (purple), and DAPI (blue) in coronal sections of DG in control and hGFAP-Cre;Kdm6b^F/F mice at P0.5. E, Quantification of (D), two-tailed paired t test (* = p < 0.05).

Single cell RNA-seq reveals disrupted stem cell maintenance gene signature in Kdm6b-deleted NSCs.
A, Schematic of single cell RNA-seq experiment design. B, t-SNE plot of DG cells from hGFAP-Cre;Kdm6b^F/F mice and control mice labeled with corresponding cell type identity. C, t-SNE plot of DG cells with specific marker expression. D, Volcano plot of differentially expressed genes in NSCs.

Kdm6b-deletion increases H3K27me3 levels at regulatory elements
A, Schematic of CUT&RUN experiment design. B, Correlation plot of H3K27me3 enrichment and gene expression in control mice. C, Bar on left depicts the overlap between genes downregulated in stem cells and in all cells pooled together. Bar on right depicts the overlap between NSC maintenance genes and genes downregulated in all cells pooled. D, Bars depict changes in H3K27me3 enrichment for overlapping genes shown by the left and right bars in (C), respectively. E-F, H3K27me3 signal over the Id2 locus (E) and Id3 locus (F) in cells from control and Kdm6b-deleted DG. Yellow column indicates +/1kb of the transcriptional start site (TSS). Black bar indicates regions with statistically significant differences in H3K27me3 enrichment. Two biological replicates are shown.

Kdm6b is required for maintenance of adult SGZ NSCs
A, Schematic of experimental design. B, Quantification of number of tdTomato+ cells at the end of TAM administration comparing Nestin-Cre^ERT2;Kdm6b^F/F;Ai14 to control. Two-tailed unpaired t test (NS = not significant). C, IHC for tdTomato (red), TBR2 (green), DCX (white), and DAPI (blue) in coronal sections of DG in control and Nestin-Cre^ERT2; Kdm6b^F/F;Ai14 mice at 10dpi. D, Quantification of (B), two-tailed paired t test (* = p < 0.05). E, IHC for tdTomato (red), SOX2 (green), and DAPI (blue) in coronal sections of DG in control and Nestin-Cre^ERT2; Kdm6b^F/F;Ai14 mice at 30dpi. F, Quantification of (D), two-tailed paired t test (* = p < 0.05).

Expression of Kdm6b during DG development.
A, Schematic illustration of DG in development. B, ISH for Kdm6b in coronal DG sections of control mice at E16.5. C, ISH for Kdm6b in coronal DG sections of control mice at P1. D, ISH for Kdm6b in coronal DG sections of control mice at P7. E, ISH for Kdm6b in coronal DG sections of control mice at P21. F, ISH for Kdm6b in coronal DG sections of hGFAP-Cre;Kdm6b^F/Fmice at E16.5. G, ISH for Kdm6b in coronal DG sections of hGFAP-Cre;Kdm6b^F/Fmice at P1.

Kdm6b-deleted mice do not display locomotive defects
A, Quantification of total movement over 10 minutes (measured in units) in the open field paradigm. Two-tailed unpaired t test (* = p < 0.05). B, Quantification of locomotion by minute (measured in units) in the open field paradigm. Repeated measures ANOVA with Bonferroni post-hoc test (* = p < 0.05).

Reduction of NSCs in the DG of hGFAP-Cre;Kdm6b^F/F mice at P15.
A, IHC for PAX6 (white), SOX2 (green), GFAP (red), and DAPI (blue) in coronal sections of DG in control and hGFAP-Cre;Kdm6b^F/F mice at P15. B, Quantification of DG neural precursor/NSCs in control and hGFAP-Cre;Kdm6b^F/Fmice at P15 (n = 3 each), two-tailed unpaired t test (* = p < 0.05).

Increased cell death is not observed in hGFAP-Cre;Kdm6b^F/F mice
A-B, IHC for cleaved caspase 3 (CC3) (red) and DAPI (blue) in coronal DG sections of P0 control (A) and hGFAP-Cre;Kdm6b^F/F mice (B). C-D, IHC for CC3 (red) and DAPI (blue) in coronal DG sections of P7 control (C) and hGFAP-Cre;Kdm6b^F/F mice (D). E, Quantification of (A-D) two-tailed unpaired t test (NS = not significant).

Similar number of SGZ NSC precursor cells at E15.5 between Kdm6b-deleted and control brain
A-D, IHC for SOX2 (green), EdU (white) and DAPI (blue) in coronal DG sections of E15.5 control (A, B) and hGFAP-Cre;Kdm6b^F/F mice (C, D). C, Quantification of (A, C) two-tailed unpaired t test (NS = not significant).

Single-cell RNA sequencing resolves the tissue heterogeneity in the developing DG
A, unlabeled t-SNE plot of DG cells from hGFAP-Cre;Kdm6b^F/F and control mice. B-C, t-SNE plot of well-known marker expression for each cell type. D, t-SNE plot of DG cells based on the genotype of mice. E, Gene ontology terms identified for statistically significant down-regulated genes in Kdm6b-deleted NSCs.

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