A dual function of Bcl11b/Ctip2 in hippocampal neurogenesis

Abstract

The development of the dentate gyrus is characterized by distinct phases establishing a durable stem-cell pool required for postnatal and adult neurogenesis. Here, we report that Bcl11b/Ctip2, a zinc finger transcription factor expressed in postmitotic neurons, plays a critical role during postnatal development of the dentate gyrus. Forebrain-specific ablation of Bcl11b uncovers dual phase-specific functions of Bcl11b demonstrated by feedback control of the progenitor cell compartment as well as regulation of granule cell differentiation, leading to impaired spatial learning and memory in mutants. Surprisingly, we identified Desmoplakin as a direct transcriptional target of Bcl11b. Similarly to Bcl11b, postnatal neurogenesis and granule cell differentiation are impaired in Desmoplakin mutants. Re-expression of Desmoplakin in Bcl11b mutants rescues impaired neurogenesis, suggesting Desmoplakin to be an essential downstream effector of Bcl11b in hippocampal development. Together, our data define an important novel regulatory pathway in hippocampal development, by linking transcriptional functions of Bcl11b to Desmoplakin, a molecule known to act on cell adhesion.

Figure 1

Bcl11b expression is restricted to postmitotic neurons during prenatal- and postnatal development of the hippocampus. (A–L) Immunohistological analysis of co-expression of Bcl11b (green) and NeuroD (red) at embryonic stage E18 (A, D) and postnatal stages P7 (B, E) and P30 (C, F); Doublecortin (Dcx; red) at P30 (G, J); NeuN (red) at P7 (H) and P30 (K); Calbindin (red) at P7 (I) and P30 (L). (M, N) Co-expression analysis of Bcl11b (red) and Sox2 (M, green) as well as Bcl11b (red) and Tbr2 (N, green) at P14. (O) Statistical analysis of M and N; no co-expression of Bcl11b with Sox2 and Tbr2 was detected (error bar, s.d.; n=3). Confocal images taken at × 20 (A–C, G) and × 63 (D–F, H–N) magnification. Scale bar: 100 μm (A), 20 μm (D) and 25 μm (M). Rectangular box indicates area of higher magnification. Arrow indicates early migrating NeuroD expressing cells. DGA, dentate gyrus anlage; GCL, granule cell layer; SGZ, subgranular zone.

Figure 2

Ablation of Bcl11b expression causes morphological changes of the dentate gyrus. Representative coronal sections of control (A, C) and Bcl11b^flox/flox;Emx-1Cre (B, D) animals at P30 stained with cresyl violet. Earlier developmental stages are presented in Supplementary Figure S3. Loss of Bcl11b expression causes reduced numbers of granule cells resulting in a smaller dentate gyrus. Statistical analysis of dentate gyrus area (E) and granule cell number (F) (t-test, *P<0.05; **P<0.005; error bars, s.e.m.; n=3). Brightfield images taken at × 5 (A, B) and × 63 (C, D) magnification. Hi, Hilus; SG, Stratum granulosum; SM, Stratum moleculare.

Figure 3

Ablation of Bcl11b results in reduced proliferation of progenitor cells, depletion of neural stem cells as well as increased apoptosis. Confocal images of coronal sections of control (A, D, F, G, I) and Bcl11b^flox/flox;Emx1-Cre (B, E, J) at P14. Earlier developmental stages are presented in Supplementary Figure S4. Co-staining of TUNEL (green) and NeuroD (red) (A, B); BrdU (green) and NeuroD (red) (D, E); BrdU (green) and Bcl11b (red) (F, G); Sox2 (green), BrdU (red) and NeuN (blue) (I, J); Statistical analysis of TUNEL assays (C), BrdU-positive cells (K, L) as well as co-staining of Bcl11b/BrdU (H). (K–O) Statistical analysis of Sox2- and Tbr2-positive cells of control and Bcl11b mutant dentate gyrus. Bcl11b mutants exhibit a reduced progenitor cell pool and Sox2 cells are aberrantly located in the granule cell layer in Emx1-Cre (K, M, O) as well as NexCre (L, N, O) mutants. t-test, *P<0.05 error bars, s.e.m. (C, K–O), s.d. (H); n=3 (C, H, O (Tbr2/BrdU and Tbr2)); n=5, (K, M, O (Sox2/BrdU); n=6 (L, N, O (NexCre)). Images taken at × 10 (A, B, D–F), × 40 (G, I, J) magnification. Scale bar: 100 μm (A); 20 μm (H).

Figure 4

Loss of Bcl11b expression disrupts dentate granule cell differentiation. (A–H) Immunohistological analysis at P14 of Dcx (green) and NeuN (red) (A–D) as well as NeuroD (green) (E–H) expression in the dentate gyrus of control (A, C, E, G) and Bcl11b^flox/flox;Emx1-Cre (B, D, F, H) animals at P30. (I–N) Immunohistological analysis of Calbindin (red) and NeuN (green) expression of control (I, K, M) and Bcl11b^flox/flox; Emx1-Cre (J, L, N) granular cells at P7 (I, J) and P30 (K–N). (O–S) Analysis of mosaic deletions of Bcl11b by ex-utero electroporation of pCIG2 (O, Q) and pCIG2-Cre (P, R) into Bcl11b^flox/flox brains at E15.5. (O–R) Immunohistological analysis after 18 DIV of DAPI (O, P) as well as GFP (green) amd NeuroD (red) (Q, R). Inserts in (Q) and (R) demonstrating Bcl11b and GFP expression. (S) Statistical analysis of GFP, NeuroD and GFP/NeuroD-positive cells. t-test, *P<0.005; error bars, s.d.; n=5. Scale bar: 100 μm (E, I, P); 50 μm (A, G); 20 μm (C).

Figure 5

Analysis of granule cell layer processes of Bcl11b^flox/flox; Emx1-Cre mice. (A, B) Timm-stained mossy fibres of adult hippocampi of control (A) and Bcl11b mutant (B) animals. In control animals, the largest part of the mossy fibres of the hilus form synapses on apical dendrites of CA3 neurons (suprapyramidal, arrow) and only a small part runs infrapyramidally. In contrast, Bcl11b mutant mossy fibres run mostly infrapyramidally (arrowhead) exhibiting a scattered distribution. (C) Statistical analysis of thorny excrescences on apical dendrites of CA3 pyramidal neurons of Golgi impregnated control and Bcl11b mutant hippocampi (t-test, *P<0.05; error bar, s.d.; n=3). (D, E) Golgi stained dendritic spines of control (D) and Bcl11b mutant (E) dentate granule cells. Note dendrites of mutants are shaped and distributed more irregularly. (F) Statistical analysis of dendritic spine numbers of control and Bcl11b mutants. Dendrites were subdivided into 50 μm sections starting at the cell soma (t-test, *P<0.05; error bar, s.d.; n=3). Scale bar: 10 μm.

Figure 6

Desmoplakin is a direct transcriptional target of Bcl11b. (A–D) Desmoplakin RNA in-situ hybridization of hippocampal cryostat sections of control (A, C) and Bcl11b^flox/flox;Emx1-Cre (B, D) animals at P10 (A, B) and P30 (C, D). (E–H) Co-expression analysis of Dsp (green) and Bcl11b (red) (E, G) as well as Sox2 (red) (F, H) of wild-type dentate gyrus at P14. Images taken at × 20 (E, F) and × 63 (G, H) magnification. Scale bars: 100 μm (E); 20 μm (G). (I) Determination of hippocampal relative mRNA expression levels of Desmoplakin and Calbindin by quantitative RT–PCR at P14 (2^−δδC_T method; t-test, *P<0.05; **P<0.005; error bar, s.d.; n=4). (J) Determination of direct interaction of Bcl11b and Desmoplakin by Chromatin immunoprecipitation (ChIP). ChIP assays were performed on hippocampus tissue of P14 animals employing a Bcl11b-specific antibody (Bcl11bAB), IgG as a negative control and an RNA polymerase II (RNA Pol II AB)-specific antibody as positive control. Direct interactions were determined by qPCR using specific primer pairs for Desmoplakin regulatory regions as well as a specific primer pair for the Gapdh promoter region (2^−δδC_T method; t-test, ***P<0.001; ****P<0.0001; error bar, s.d.; hippocampi of 10 animals were used per assay).

Figure 7

Analysis of the hippocampus-specific Dsp^flox/flox;Emx1-Cre phenotype at P14. (A–F) Morphological analysis by cresyl-violet staining of control (A, D) and Dsp^flox/flox;Emx1-Cre (B, E) coronal forebrain sections as well as statistical analysis of the dentate gyrus area (C) and cell number (F). (G–I) Immunohistochemical analysis of progenitor proliferation by BrdU incorporation of control (G) and Dsp^flox/flox;Emx1-Cre (H) animals. BrdU, red; NeuN, blue. (J–L) Analysis of cell death of control (J) and Dsp^flox/flox;Emx1-Cre (K) by TUNEL assay. TUNEL, green; DAPI, blue; arrows indicate TUNEL-positive cells. (M–Q) Immunohistochemical analysis of NeuroD (green)-positive cells in control (M, P) and Dsp^flox/flox;Emx1-Cre (N, Q) animals (t-test, *P<0.05; **P<0.01; ***P<0.005; error bar, s.d.; n=4 (C, F); n=3 (I, L, O)). Scale bar: 250 μm (B), 100 μm (H), 50 μm (E, Q).

Figure 8

Re-introduction of Desmoplakin expression rescues Bcl11b phenotype. (A–F) Immunohistological analysis of control (A, D) and Bcl11b^flox/flox;Emx1-Cre (B, C, E, F) hippocampal slice cultures after 11 DIV electroporated with control pRC-CMV (A, B, D, E) and pRC-CMV-Dsp (C, F) expression vectors as well as vector pCAGGS expressing GFP using DAPI (blue) as morphological marker, GFP (green) as marker for electroporation efficiency and BrdU (red) as marker for cell proliferation. Inserts in (E) and (F) showing Dsp and GFP expression. Images taken at × 20 magnification. (G) Statistical analysis of BrdU-positive cells in control and mutant hippocampal slice cultures. (H) Statistical analysis of co-expression of GFP/NeuroD- and GFP/BrdU-positive cells (t-test, *P<0.01; error bar, s.e.m.; n=4).

Figure 9

Ablation of Bcl11b causes impaired learning and memory behaviour. (A–C) Open field test analysing locomotor activity (A), leaning (B) and rearing (C) behaviour (t-test, *P=0.01; **P=0.0005; ***P=0.00001; error bar, s.e.m.; n=8). (D, E) Radial maze test analysing spatial learning behaviour by determining the number of errors at 5 successive days (t-test, *P<0.05; **P<0.005; ***P=0.0005; ****P=0.0001; error bar, s.e.m.; n=9) (D) and number of entries into new radial arms of days 3–5 (t-test, *****P<0.00001; error bar, s.e.m.; n=9) (E). (F) Elevated plus maze test analysing anxiety behaviour by determining number of arm entries in relation to locomotor activity. No significant differences between control and mutant animals were observed (n=9).