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Brain-specific deletion of histone variant H2A.z results in cortical neurogenesis defects and neurodevelopmental disorder

Abstract Defects in neurogenesis alter brain circuit formations and may lead to neurodevelopmental disorders such as autism and schizophrenia. Histone H2A.z, a variant of histone H2A, plays critical roles in chromatin structure and epigenetic regulation, but its function and mechanism in brain development remain largely unknown. Here, we find that the deletion of H2A.z results in enhanced proliferation of neural progenitors but reduced neuronal differentiation. In addition, neurons in H2A.z knockout mice exhibit abnormal dendrites during brain development. Furthermore, H2A.zcKO mice exhibit serial behavioral deficits, such as decreased exploratory activity and impaired learning and memory. Mechanistically, H2A.z regulates embryonic neurogenesis by targeting Nkx2–4 through interaction with Setd2, thereby promoting H3K36me3 modification to activate the transcription of Nkx2–4. Furthermore, enforced expression of Nkx2–4 can rescue the defective neurogenesis in the H2A.z-knockdown embryonic brain. Together, our findings implicate the epigenetic regulation by H2A.z in embryonic neurogenesis and provide a framework for understanding how disruption in the H2A.z gene may contribute to neurological disorders.

H2A.z regulates the proliferation of neural progenitor cells (A) Mouse embryos were electroporated in utero with control, H2A.z-sh1, H2A.z-sh2 at E13 and the harvested brain sections were immunostained with mitotic marker pH3 at E16.
(C) BrdU was injected intraperitoneally for 2 hours of pulse labeling and the harvested brain sections were immunostained with anti-BrdU.
(E) Immunostaining shows that the expression of H2A.z is depleted in E13 H2A.z cKO brains.
(F) The apoptosis levels between H2A.z fl/fl and H2A.z cKO brains show no significant difference. E16 brain sections of H2A.z fl/fl and H2A.z cKO mice were subjected to TUNEL assay.

(G)
Graph shows the numbers of apoptotic cells per field. n = 6 for all samples.
(H) Ablation of H2A.z does not affect the proliferation of RGs. H2A.z fl/fl mice and H2A.z cKO mice were administered BrdU (100 mg/kg) for 2 hours of pulse labeling and were euthanized at E16. The harvested brain sections were immunostained with anti-Pax6 and anti-BrdU.
Representative images from at least three independent experiments. Error bars represent means ± s.e.m.; H2A.z fl/fl , WT; H2A.z cKO , cKO. Two-tailed unpaired ttest. p < 0.05 (*), p < 0.01(**). n.s., not significant. Scale bar represents 50 μm (A and C); 20 μm (E; F and H). (A) Western blot shows that the expression of Tbr2 is increased, while the expression of Pax6 is not significantly altered when H2A.z is deleted in NPCs. E12 primary neural progenitor cells (NPCs) were isolated from H2A.z cKO and H2A.z fl/fl mice, and cultured in proliferated medium (suspension culture) for 3 days, and then collected the neurospheres for further analysis (A-C).
(C) Relative Tbr2 mRNA levels in neurospheres derived from H2A.z fl/fl and H2A.z cKO mice. n = 7 for all samples.
(E) Quantification of the percentage of GFP-expressing cells in different regions of the developing cortex. n = 4 for all samples.
(F) Coronal brain sections were immunostained with anti-BrdU.
(D) Graphs of the percentage of GFP-positive cells in the VZ/SVZ, IZ and CP.
(F) The surface of VZ/SVZ at E16 cortices labeled for Tuj1, Pax6 and DAPI. n = 4 for all samples.
(D) Neurons that incorporated BrdU at E13 were revealed by immunostaining for BrdUand Ctip2 (green; marker of layer V).

(E) Statistics of Ctip2 + cells in deep layer.
(F) Statistics of BrdU + and Ctip2 + cells in Ctip2 + cells.

(G)
Neurons that incorporated BrdU at E13 were revealed by immunostaining for BrdU and Tbr1 (green; marker of layer VI).
(H) Statistics of Tbr1 + cells in deep layer.

Figure S11 The expression and localization of Nkx2-4 in the developing cortex (A)
Representative images of brain sections at E12, E15, and E18 stained for Nkx2-4 and Tuj1. Note that Nkx2-4 is expressed in a subpopulation of Tuj1 + neurons.
(C) Images of coronal sections of the developing cortex labeled for Nkx2-4 and Sox2.
(D) Western blot analysis shows that Nkx2-4 is efficiently reduced when Nkx2-4 is depressed. Primary progenitors from E12 brain cultured in proliferation conditions were infected with lentivirus expressing the control or Nkx2-4-shRNA for analysis. Protein lysates of infected cells were probed with the antibodies indicated.
(E) Nkx2-4-shRNA or control plasmids were electroporated into the brains of E13 embryos. At E16, the brains were harvested to investigate the cell distribution and were immunostained with the mitotic marker pH3.  (C) Graph shows the percentage of pH3 + GFP + cells. n = 4 for all samples.
(D) Representative images of E16 coronal brain sections immunostained for Tbr2.
(E) Graph shows the percentage of GFP + Tbr2 + cells. n = 4 for all samples. (B) The levels of H3K36me3 are reduced when the expression of Setd2 is downregulated. Primary neural progenitors were infected with control or Setd2-shRNA lentivirus. Three days later, the cell lysates were detected by western blotting with the antibodies indicated.
(C) Interaction between H2A.z and Setd2 is verified by co-immunoprecipitation experiments. HA-Setd2-and Flag-H2A.z-expressing plasmids were cotransfected into N2A cells. Three days later, the transfected cells were subjected to immunoprecipitation using HA beads. Bound proteins were detected by western blotting for the antibodies indicated.
(D) Images of E16 mouse cortices electroporated at E13 with plasmid indicated.

Figure S14
The model for H2A.z-mediated neurogenesis.
H2A.z regulates neural progenitor cell differentiation through Nkx2-4. The loss of function of H2A.z leads to a reduction of Nkx2-4 expression, neural progenitor differentiation defects and abnormal behaviors.