Histone deacetylases 1 and 2 control the progression of neural precursors to neurons during brain development

Abstract

The molecular mechanism by which neural progenitor cells commit to a specified lineage of the central nervous system remains unknown. We show that HDAC1 and HDAC2 redundantly control neuronal development and are required for neuronal specification. Mice lacking HDAC1 or HDAC2 in neuronal precursors show no overt histoarchitectural phenotypes, whereas deletion of both HDAC1 and HDAC2 in developing neurons results in severe hippocampal abnormalities, absence of cerebellar foliation, disorganization of cortical neurons, and lethality by postnatal day 7. These abnormalities in brain formation can be attributed to a failure of neuronal precursors to differentiate into mature neurons and to excessive cell death. These results reveal redundant and essential roles for HDAC1 and HDAC2 in the progression of neuronal precursors to mature neurons in vivo.

Fig. 1.

HDAC1 and HDAC2 deletion causes severe hippocampal and cerebellar abnormalities. Gross brain, hippocampus, and cerebellum from wild-type, HDAC1^loxP/+; HDAC2^loxP/loxP; GFAP-Cre, and HDAC1^loxP/loxP; HDAC2^loxP/loxP; GFAP-Cre mice stained with hematoxylin and eosin showing loss of hippocampus and loss of foliation in cerebellum of double HDAC1 and HDAC2 mutants. One copy of a HDAC1 allele is sufficient for development and viability.

Fig. 2.

Neuronal defects in HDAC1^loxP/loxP; HDAC2^loxP/loxP; GFAP-Cre mice. (A) Immunohistochemical staining of MAP-2 on the neocortex of wild-type and mutant mice. Mutant mice show a lack of dendritic extensions compared with control. Scale bar = 40 μm. (B) Immunohistochemical staining for calbindin on wild-type and mutant cerebellum. Mutant cerebellum shows Purkinje cells have failed to migrate and remain among the deep cerebellar nuclei. Scale bar = 40 μm. (C) Immunohistochemistry of CSPGs (green) and Hoechst (blue) of wild-type and mutant cerebral cortex and hippocampus. Scale bar = 40 μm. Deletion of HDAC1 and HDAC2 results in disorganized molecular layers. Hippocampal structures are unidentifiable in mutant mice. MZ, marginal zone; CP, cortical plate; SP, subplate; IZ, intermediate zone; VZ, ventricular zone.

Fig. 3.

Neuronal birth date and proliferation analysis of HDAC1^loxP/loxP; HDAC2^loxP/loxP; GFAP-Cre mice. (A) Immunohistochemistry of BrdU (green) and Hoechst (blue) of wild-type and mutant cerebral cortex injected with BrdU at E12.5 or E14.5. Deletion of HDAC1 and HDAC2 results in normal neuronal migration but results in fewer differentiated neurons. (B) Quantification of BrdU⁺ cells from (A) showing a statistically significant reduction in neurons from double mutant (cko) mice. Scale bar = 40 μm. (C) Immunohistochemistry at E14.5 detecting Hoechst (blue), S-phase neuronal precursors labeled by BrdU (green), and Tuj1 (red) on wild-type and mutant cerebral cortex (Top) and developing hippocampus (Bottom). Scale bar = 40 microns. (D) Quantification of BrdU⁺ cells at E14.5 to assess proliferation. Deletion of HDAC1 and HDAC2 results in increased proliferation of neuronal precursors at the ventricular zone and reduced number of Tuj1-expressing neurons at E14.5. MZ, marginal zone; CP, cortical plate; SP, subplate; IZ, intermediate zone; VZ, ventricular zone; cKO, double conditional knockout.

Fig. 4.

E14.5 neuronal precursors lacking HDAC1 and HDAC2 cannot differentiate into neurons in vitro. Neuronal precursors expressing control, GFP (A, C, and E) or Cre-GFP (B, D, and F), in growth (FGF) (A and B), neuronal (Isoxazole-9) (C and D), and astrocyte (LIF+BMP) (E and F) conditions in 4-day cultures. Control GFP cells colabeled with Tuj1 (white arrows), whereas Cre-GFP cells are notably absent for Tuj1 (white arrowheads) in growth (A and B) and neuronal conditions (C and D). Both control GFP and Cre-GFP colabeled with GFAP in astrocyte conditions (E and F). (G) Quantification of Tuj1⁺ cells from total GFP or Cre-GFP cells to assess neuronal differentiation. (Scale bar: 15 μm.) Each experiment represents replicates from 2 independent experiments.

Fig. 5.

Deletion of HDAC1 and HDAC2 during neuronal differentiation induces cell death. (A) Live-staining of neuronal precursors after 24 h in neuronal differentiation media. Scale bar = 5 microns. Control-GFP are propidium iodide (PI)-negative, whereas Cre-GFP cells are PI-positive and show fragmented nuclei indicative of cell death. (B) TUNEL analysis of E15.5 wild-type and mutant mice shows significant lethality in the ventricular zone of mutant mice, whereas no cell death is observed in wild-type littermates. Scale bar: 100 microns.