Animals and Ethics Statement
All animal experiments were conducted in accordance with the European Communities Council Directive (86/609/EEC) and received ethical approval by the committee for Animal Research of the Bavarian State authorities. The generation of the knockout allele has been described previously (13). All animals - except for the animals in the enriched environment experiments - were housed in standard cages (size: 37 x 21 x 15 cm) with 3-4 mice per cage under a 12 h light/dark cycle with unlimited access to water and standard rodent food. Mice were housed in the animal facilities of the Helmholtz Center Munich and the Friedrich-Alexander-Universität Erlangen. Animal care was in accordance with institutional guidelines.
Genotyping of the mice was done using PCR and the following primers:
Tcf4Het fwd Mut TCG TGG TAT CGT TAT GCG CC
fwd WT CCG ATG ACA GTG ATG ATG GT
rev AAG TTA AGC TGA AGT AAA TAC CCA CA
lacZ fwd ATC ACG ACG CGC TGT ATC
lacZ rev ACA TCG GGC AAA TAA TAT CG
BrdU injections and Enriched environment
At the age of eight weeks intraperitoneal 5-bromo-2'-deoxyuridine (BrdU) injections were performed twice a day on three consecutive days (0.1mg BrdU/g body weight, per dose). Animals were sacrificed either three hours (6 control and 6 haploinsufficient mice) after last injection or after additional four weeks (9 control and 5 haploinsufficient mice).
An additional cohort of eight week old Tcf4 haploinsufficient mice (5 animals) was placed in an enriched environment (EE cage size: 60 x 26 x 33 cm, containing running wheels, toys, tunnels and nest materials). Mice were injected twice intraperitoneal with BrdU on three consecutive days (0.1mg BrdU/g body weight, per dose). Animals were sacrificed four weeks after the last injection.
Tissue preparation
Mice were killed using CO2 and perfused transcardially with PBS for 5 min (20 ml/min), followed by fixation with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB), pH 7.4, for 5 min. Brains were postfixed overnight in 4% PFA at 4°C, followed by dehydration in 30% sucrose in 0.1 M PBS at 4°C. Brains were cut coronally at 40 µm, using a sliding microtome. Sections were stored at −20°C.
Immunohistochemistry
Free‐floating sections were rinsed six times for 10 min in Tris‐buffered saline (TBS: 1 M Tris–HCL, pH 7.5/0.9% NaCl), and incubated for 72 h at 4 °C with primary antibodies (Table 1) in blocking solution, containing 0.25% Triton X-100 and 3% donkey serum in TBS. Sections were rinsed in TBS six times for 10 min, incubated overnight at 4°C with fluorochrome‐labelled secondary antibodies (Table 2) diluted in blocking solution, and rinsed in TBS three times for 10 min. Nuclei were counterstained with DAPI (1:10,000 in 1xTBS) for 10 min, followed by three rinses in TBS for 10 min. Sections were mounted on slides and covered with Aqua-Poly/Mount (Polysciences).
For BrdU stainings, slices were first stained for all antigens of interest except for BrdU. Slices were then postfixed in 4% PFA for 10 min at room temperature. Sections were rinsed three times in TBS, incubated in 2N HCl for 10 min at 37°C. After two rinses in 0.1 M borate buffer, sections were washed three times with PBS. Detection of BrdU immunoreactivity was conducted as described above.
Imaging and quantification
For volume, BrdU, and MCM2 quantification, fluorescence signal was detected with an AF6000 Modular Systems Leica fluorescent microscope and documented with a SPOT-CCD camera and the Leica software LAS AF (Version 2.6.0.7266; Leica Microsystems, Wetzlar Germany). For co-localization analyses, fluorescence signal was detected using a Zeiss LSM 780 confocal microscope with four lasers (405, 488, 550, and 633 nm) and × 25 and × 40 objective lens. Images were processed using ImageJ.
For each animal, a series of every 12th section of the dentate gyrus was selected. Volume measurements were performed with ImageJ by tracing the granular zone of dentate gyrus. For BrdU and MCM2 quantification, cells in the granule cell layer and contiguous subgranular zone were counted (52). For co-localization analyses, all BrdU+ cells within the granule cell layer and contiguous subgranular zone in at least one section were analysed for expression of DCX or PROX1. A minimum 50 cells per animal were analysed per marker and animal. Statistical analysis was performed with GraphPad Prism (Graphpad Software Inc.), using unpaired two tailed t-test. The data are expressed as mean values ± SEM. Significant differences were assumed at a level of p < 0.05.
Antibodies
Table 1: Primary antibodies
Antigen
|
Host
|
Manufacturer
|
Dilution
|
Catalog number
|
RRID
|
BrdU
|
Rat
|
Serotec
|
1:500
|
OBT0030CX
|
AB_609566
|
DCX
|
Goat
|
Santa Cruz
Biotechnology
|
1:500
|
sc-8066
|
AB_2088494
|
CALBINDIN
|
Mouse
|
Swant
|
1:300
|
C9638
|
AB_2314070
|
MCM2
|
Mouse
|
BD Bioscience
|
1:500
|
610700
|
AB_2141952
|
MCM2
|
Rabbit
|
Cell Signaling Technology
|
1:500
|
4007S
|
AB_2142134
|
NESTIN
|
Mouse
|
Millipore
|
1:500
|
MAB353
|
AB_94911
|
PROX1
|
Rabbit
|
Chemicon International
|
1:500
|
AB5475
|
AB_177485
|
TCF4
|
Rabbit
|
Abcam
|
1:500
|
AB130014
|
-
|
Table 2: Secondary antibodies
Fluorophore
|
Epitope
|
Manufacturer
|
Dilution
|
Catalog number
|
RRID
|
Alexa488
|
Anti-Goat
|
Invitrogen
|
1:500
|
A11055
|
AB_2534102
|
Alexa488
|
Anti-Rabbit
|
Invitrogen
|
1:500
|
A21206
|
AB_2535792
|
Cy3
|
Anti-Rat
|
Jackson
|
1:500
|
712-165-153
|
AB_2340667
|
Cy3
|
Anti-Goat
|
Jackson
|
1:500
|
705-165-147
|
AB_2307351
|
Cy5
|
Anti-Mouse
|
Jackson
|
1:500
|
715-175-151
|
AB_2340820
|
Cy5
|
Anti-Rabbit
|
Jackson
|
1:500
|
711-495-152
|
AB_2315775
|