One week after birth, newly generated neurons end their migration, and the MIF requirement is reduced. and mRNA improved from P0, reached a maximum at P7, and stably indicated until P30 before declining dramatically at 3 months. MIF was localized in materials of GFAP- and BLBP-positive radial glial precursor cells in dentate gyrus (DG). DCX-expressing newly generated neurons were MIF-negative. Inhibition of MIF by MIF antagonist S, R-3-(4-hydroxyphenyl)-4, 5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1) reduced BrdU-positive cells. Interestingly, MIF was indicated by NeuN-positive GABAergic interneurons including parvalbumin-and Reelin-expressing cells in the DG. Neither NeuN-positive granule cells nor NeuN-positive pyramidal neurons indicated MIF. In transgenic mice, POMC-EGFPCpositive immature dentate granule cells and Thy1-EGFPCpositive mature granule cells were MIF-negative. Treatment of neuronal cultures with ISO-1 inhibited neurite outgrowth. Consequently, we conclude that MIF might be important for feature maintenance of neural stem cells and neurite outgrowth during hippocampal development. hybridization shows a strong labeling of TC-DAPK6 mRNA in cytoplasm of granule cells and pyramidal neurons of hippocampus (Bacher et al., 1998). Until 2011, Conboy and his colleagues shown that MIF is definitely indicated by NeuroD-positive immature granule neurons in hippocampus, but not by NeuN-positive adult neurons (Conboy et al., 2011). However, it is still elusive on MIF manifestation and characteristics, as well as potential functions, especially in the stage of hippocampal development. To address this issue, we systematically examined the spatial and temporal manifestation pattern of MIF protein and mRNA by European blotting and real-time polymerase chain reaction (PCR) and characterized MIF-positive cells in hippocampus of mice aged from postnatal day time 0 (P0) to 3 months by immunostaining. Using antagonist of MIF, we clogged MIF function and observed impairment of cell proliferation and neurite outgrowth in hippocampus. Our results suggest that MIF might maintain the feature of neural stem cells and promote outgrowing of neuronal processes in developing hippocampus. Materials and Methods Animals P7 POMC-EGFP and 3-month Thy1-EGFP transgenic mice were from the animal facility of Hamburg University or college, UKE, Germany. Animals were housed under standard laboratory conditions in the animal facility at the Center for Molecular Neurobiology Hamburg. All experiments were performed in accordance TC-DAPK6 with the institutional guidebook for animal care (license quantity ORG 850). Genotyping was performed by PCR analysis of genomic DNA, as explained previously (Overstreet et al., 2004; Vuksic et al., 2008). P0, P3, P7, P14, P30, and 3-month Kunming mice were from the Experimental Animal Center of Xian Jiaotong University or college. All procedures were performed in accordance with the protocol authorized by the Institutional Animal Care of Northwest A&F University or college and Xian Medical University or college. Antibodies and Inhibitor The following primary antibodies were utilized for immunofluorescence studies and Western blot analyses: rabbit polyclonal anti-MIF (1:500, ABclonal, United States), mouse monoclonal anti-Reelin G10 (dilution 1:1,000, Millipore, United States), mouse monoclonal anti-Parvalbumin (1:500, Abcam, United States), mouse polyclonal anti-GFAP (1:500, DAKO, United States), mouse monoclonal anti-NeuN (1:500, Millipore, United States), mouse monoclonal antiCneurofilament 200 (1:1,000, Millipore, United States), mouse monoclonal antiC-tubulin (1:500; Sigma-Aldrich, United States), goat polyclonal ZNF538 anti-DCX (1:1,000; Santa Cruz, United States), mouse monoclonal anti-BLBP (1:300, Raybiotech, Inc., United States), and rat polyclonal anti-BrdU (1:500, Bio-Rad Laboratories Inc., United States). The secondary antibodies utilized for Western blotting were horseradish peroxidase (HRP)Cconjugated goat antiCrabbit IgG and goat antiCmouse IgG TC-DAPK6 (1:2,000; Cell Signaling Technology, United States). The secondary antibodies for immunostaining were as follows: Alexa Fluor 488-conjugated goat antiCmouse, Alexa Fluor 568Cconjugated goat antiCmouse,.