Draxin regulates hippocampal neurogenesis in the postnatal dentate gyrus by inhibiting DCC-induced apoptosis

Hippocampal neurogenesis in the dentate gyrus (DG) is controlled by diffusible molecules that modulate neurogenic processes, including cell proliferation, differentiation and survival. To elucidate the mechanisms underlying hippocampal neurogenesis, we investigated the function of draxin, originally identified as a neural chemorepellent, in the regulation of neuronal survival in the DG. Draxin was expressed in Tbr2 (+) late progenitors and NeuroD1 (+) neuroblasts in the dentate granule cell lineage, whereas expression of its receptor DCC (deleted in colorectal cancer) was mainly detectable in neuroblasts. Our phenotypic analysis revealed that draxin deficiency led to enhanced apoptosis of DCC-expressing neuroblasts in the neurogenic areas. Furthermore, in vitro assays using a hippocampal neural stem/progenitor cell (HNSPC) line indicated that draxin inhibited apoptosis in differentiating HNSPCs, which express DCC. Taken together, we postulate that draxin plays a pivotal role in postnatal DG neurogenesis as a dependence receptor ligand for DCC to maintain and promote survival of neuroblasts.


EXPERIMENTAL PROCEDURES
Measuring the area size of the dentate gyrus. Brains, fixed with 4 % paraformaldehyde (PFA; Wako Pure Chemical) in PBS, were dissected from various developmental stages of mice, and then cut into 18 µm-thick coronal sections using a cryostat (Leica). To compare the area size of the dentate gyrus (DG) at the similar histological location between draxin wild-type (WT) and knockout (KO) mice, sections containing the habenular nucleus were collected, and then immersed in Mayer's hematoxylin (Sigma-Aldrich) and 1 % eosin solutions (Wako Pure Chemical) to visualize the DG structure.
After taking photos of the DG, its area size was measured using ImageJ software provided by the National Institutes of Health (NIH), and shown as a percentage of the area size in the WT DG.
Immunochemical analyses. Immunohistochemistry on hippocampal sections was performed as described previously (Tawarayama et al., 2010). Briefly, dissected whole brains were fixed with 4 % paraformaldehyde (PFA; Wako Pure Chemical) in PBS overnight at 4 °C, and then cut into 18-or 80 µm-thick sections using a cryostat (Leica) or vibratome (Leica), respectively. Sections were treated with 5 % normal donkey serum (Merck-Millipore) in PBS containing 0.1 % Triton-X100 to block non-specific binding, then reacted with primary antibodies overnight at 4 °C. After removing the primary antibodies, sections were incubated with fluorophore-or horseradish peroxidase (HRP)conjugated secondary antibodies for 1 hour at room temperature. Color development was done using the Vector VIP Peroxidase Substrate Kit (Vector laboratories).
Immunocytochemistry was performed following the immunohistochemical procedure described above with a modified fixation time (30 min). The details for antibodies and fluorophores used in immunochemical analyses are shown in the Supplementary Table.

Expression analysis of neogenin in the postnatal subgranular zone (SGZ).
To examine neogenin expression in the DG, we used a mutant mouse line, in which a ß-gal genetrapping vector was integrated into one of the neogenin alleles Mitchell et al., 2001). In this mutant line, truncated neogenin-ß-gal fusion proteins are generated and trapped in the endoplasmic reticulum of the expressing cells, where they appear as punctate and perinuclear ß-gal immunoreactivity. Thus, the cells immunoreactive to ß-gal were regarded as neogenin-expressing cells. To determine the cell type of SGZ cells expressing neogenin, hippocampal slices from P30 neogenin heterozygous mice were coimmunostained for ß-gal and expression markers, specific to various differentiation stages in the granule cell lineage, including GFAP, nestin, Tbr2, NeuroD1 and Prox1.

Evaluation of efficiency of siRNA transfection and protein knockdown.
To evaluate transfection efficiency of siRNA duplexes, the siGLO-Red transfection indicator (GE Healthcare) was introduced into rat HNSPCs using Lipofectamine RNAiMAX Reagent (Invitrogen) at a final concentration of 20 nM. HNSPCs were fixed with 4 % PFA in PBS one day after transfection, followed by counter staining with DAPI. Number of cells incorporating siGLO-Red was counted and shown as a percentage of that of total cells.
To evaluate protein knockdown efficiency of siRNA duplexes specific to DCC and neogenin, these siRNA duplexes (20 nM) were introduced into HNSPCs, pretreated with differentiation inducers for one day to induce expression of DCC and neogenin, using Lipofectamine RNAiMAX Reagent. Next day, whole cell lysates were extracted from the transfectants, and then expression level of each target protein was analyzed by the Western blot analysis.

Western blot analyses.
To perform the Western blot analysis, rat hippocampal neural