Structure-function integrity of the adult hippocampus depends on the transcription factor Bcl11b/Ctip2

Abstract

The dentate gyrus is one of the only two brain regions where adult neurogenesis occurs. Throughout life, cells of the neuronal stem cell niche undergo proliferation, differentiation and integration into the hippocampal neural circuitry. Ongoing adult neurogenesis is a prerequisite for the maintenance of adult hippocampal functionality. Bcl11b, a zinc finger transcription factor, is expressed by postmitotic granule cells in the developing as well as adult dentate gyrus. We previously showed a critical role of Bcl11b for hippocampal development. Whether Bcl11b is also required for adult hippocampal functions has not been investigated. Using a tetracycline-dependent inducible mouse model under the control of the forebrain-specific CaMKIIα promoter, we show here that the adult expression of Bcl11b is essential for survival, differentiation and functional integration of adult-born granule cell neurons. In addition, Bcl11b is required for survival of pre-existing mature neurons. Consequently, loss of Bcl11b expression selectively in the adult hippocampus results in impaired spatial working memory. Together, our data uncover for the first time a specific role of Bcl11b in adult hippocampal neurogenesis and function.

Keywords: Adult neurogenesis; Bcl11b; dentate gyrus; hippocampus; spatial memory.

Figure 1

Conditional Bcl11b mutants exhibit a progressive phenotype at 6 months of age. (a,b) Immunofluorescence staining of 6‐month‐old hippocampal control (a) and conditional Bcl11b mutant (b) sections. (c–h) Cresyl violet staining of plastic sections of control (c,e,g) and conditional Bcl11b mutant (d,f,h) hippocampi. (i,j) Quantitative analysis of the dentate gyrus area (i) and granule cell number (j). (k,l) Quantitative analysis and distribution of BrdU at 4 (4d) and 28 (28d) days after the initial BrdU injection (k) and TUNEL (l), positive cells of control and conditional Bcl11b mutant hippocampi sections. Supra, suprapyramidal blade; Infra, infrapyramidal blade; scale bar, 25 µm (h), 75 µm (b) and 100 µm (d); t‐test, numbers indicate P‐values; error bars, SEM; n = 3 (k; 4 days mutant; 28 days control; l), n = 4 (i–j; k: 4 days control; 28 days mutant).

Figure 2

Bcl11b expression is required for the differentiation of adult‐born neurons. Quantitative analysis and distribution of genes involved in adult neurogenesis, Sox2 (a), Tbr2 (b) and Doublecortin (c). t‐test, numbers indicate P‐values; error bars, SEM; n = 6 (a), n = 5 (b); n = 3 (c).

Figure 3

Differentiation analysis of adult‐born granule cells. (a,c) Immunofluorescence staining of BrdU (red), Tbr2 (green) and NeuroD (blue) (a) as well as BrdU (red), CB (green) and NeuN (blue) (c) positive cells. Scale bar, 7.5 µm. (b,d) Schematic representation of the quantitative analysis of co‐localization of BrdU, Tbr2 and NeuroD (b) as well as BrdU, Calbindin and NeuN (d). n = 3; NN, NeuN; ND, NeuroD; CB, Calbindin. (e) Alignment of marker genes used to their respective differentiation stages.

Figure 4

Generation and examination of the adult‐induced Bcl11b mutation. (a) Schematic representation of the tet‐off system and time course of induction of the Bcl11b mutation. (b) Quantitative analysis of Bcl11b and Dsp mRNA expression levels of control and adult‐induced Bcl11b mutants at 3 months of age administered doxycycline at all times (+Dox) or at 4 weeks after doxycycline removal (−Dox). t‐test, numbers indicate P‐values; error bars, SD; n = 3. (c) Immunofluorescence staining employing a Bcl11b specific antibody on hippocampal sections of control and Bcl11b mutants administered doxycycline at all times (Bcl11b + Dox) and at 4 weeks after doxycycline removal (Bcl11b − Dox). Scale bar, 100 µm.

Figure 5

Analysis of adult‐induced Bcl11b mutant mice at 2 and 4 months after doxycycline removal. (a–h) Cresyl‐violet staining of control (a,c,e,g) and Bcl11b mutant (b,d,f,h) hippocampal sections at 2 months after doxycycline removal. Scale bar, 20 µm (c); 100 µm (a). (i‐o) Quantitative analysis of the dentate gyrus area (i), granule cell number (j), CA1 cell number (k), BrdU incorporation at 4 (4d) and 28 (28d) days after the initial BrdU injection (l) as well as TUNEL (m), NeuroD (n) and Doublecortin (o) positive cells at 2 (2m‐Dox) and 4 (4m‐Dox) months after doxycycline removal. Infra, infrapyramidal blade; Supra, suprapyramidal blade; t‐test, numbers indicate P‐values; error bars, SEM; n = 3 [4m‐Dox in i,j,l,n (4d)]; n = 4 [4m‐Dox in l (28d), m; 2m‐Dox in n; mutant in o]; n = 5 (2m‐Dox in i,j); n = 8 (2m‐Dox in l; control in o).

Figure 6

Analysis of hippocampal mossy fiber terminals of adult‐induced Bcl11b mutants. (a,b) Timm staining of mossy fibers of control (a) and adult‐induced Bcl11b mutant (b) hippocampi. (c) Quantitative analysis of apical thorny excrescences by Golgi impregnation of control and adult‐induced Bcl11b mutant hippocampal sections at 2 (2m‐Dox) and 4 (4m‐Dox) months after doxycycline removal. t‐test, numbers indicate P‐values; error bars, SEM; n = 3.

Figure 7

Adult‐induced ablation of Bcl11b impairs spatial learning and working memory capacities at 4 months after doxycycline removal. (a–d) Open field test to determine locomotor activity (a), leaning (b) and rearing (c) events as well as distance traveled (d). (e–h) Radial maze test analysis of spatial learning behavior by determining new entries into radial arms (e) and working memory (f) at day 3–5 as well as number of errors at five successive days at 2 (2m‐Dox) (g) and 4 (4m‐Dox) (h) months after doxycycline removal. (i,j) Elevated plus maze test analyzing anxiety behavior. t‐test, numbers indicate P‐values; error bars, SEM; n = 6 (2m‐Dox); n = 7 (4m‐Dox).