Article Figures & Data
Figures
- Figure 1.
OTX2 knockdown in the choroid plexus reduces newborn neurons in the olfactory bulb. A–C, Conditional Otx2 knockdown with Otx2lox/lox mice. A, Staining for OTX2 in lateral ventricle choroid plexus 35 d after intracerebroventricular injection of vehicle or Cre-Tat. B, Comparison of Otx2 expression levels between fourth and lateral ventricles from the same brain (linked data points represent individual mouse) after vehicle or Cre-Tat intracerebroventricular injection. Dotted line represents 30% decrease in Otx2 expression. C, Quantitative PCR analysis of Otx2 expression in fourth ventricle (****p < 0.0001, Mann–Whitney test, 5 experiments). D, Schematic of adult (3 months old) neurogenesis study paradigm. E, Staining for BrdU in a coronal section of olfactory bulb 3 weeks after BrdU injections. F, Quantification of BrdU-positive cells in GCL and GL of the olfactory bulb (**p < 0.01, ns = p > 0.05, t test, 3 experiments). G, Staining for caspase and DCX in a sagittal section of the RMS. Arrows indicate caspase-labeled neuroblasts throughout the entire RMS. H, Quantification of caspase/DCX colabeled cells in RMS (ns = p > 0.05, t test, 3 experiments). I, Schematic of adult V-SVZ proliferation study paradigm. J, Staining for BrdU in a coronal section of the V-SVZ 2 h after BrdU injection. K, Quantification of BrdU-positive cells in V-SVZ (ns = p > 0.05, t test, 2 experiments). L, Schematic of adult migration study paradigm. M, Staining for BrdU and DCX in a coronal section of the RMS. N, Quantification of BrdU/DCX colabeled cells in RMS (*p < 0.05, t test, 2 experiments). IP, Intraperitoneal; icv, intracerebroventricular; inj, injection; veh, vehicle. Error bars represent SD.
- Figure 2.
OTX2 in RMS and V-SVZ is non-cell-autonomous. A–C, Absence of Otx2 expression in V-SVZ (A, C) and RMS (B), as shown by GFP staining in coronal sections of Otx2+/GFP mice (A, B) and by in situ hybridization for Otx2 mRNA in coronal sections of wild-type mice (C). Note the expression of GFP in choroid plexus (A) and of Otx2 in septum (C). D, Representative sagittal section of RMS microdissection. E, Western blot (WB) analysis of OTX2 immunoprecipitation from lysates of various brain regions (ctrl, IgG control). Bands include OTX2 (<40 kDa), IgG (25 and 55 kDa), and unknown (∼45 kDa). SC, Superior colliculus; Str, striatum; M.W., molecular weight.
- Figure 3.
OTX2 transfers into supporting cells of neurogenic niches. A, In dorsal V-SVZ, OTX2 staining is detected in some GFAP-labeled astrocytes but not in DCX-labeled neuroblasts. Arrowheads highlight astrocytes containing OTX2. B, Along the lateral ventricular wall, OTX2 staining is detected in vimentin (VIM)-labeled ependymal cells. C, In the RMS, OTX2 staining is detected in GFAP-labeled astrocytes but not in DCX-labeled neuroblasts. Arrowheads highlight astrocytes containing OTX2. D, Orthogonal projection shows OTX2 in the nucleus of astrocytes within the RMS.
- Figure 4.
Non-cell-autonomous OTX2 is sufficient to regulate adult neurogenesis. A, Efficient recombination in choroid plexus of scFv-Otx2 mice after intracerebroventricular injection of Cre-Tat assessed by GFP reporter. B, Change in OTX2 staining within V-SVZ 35 d after Cre-Tat intracerebroventricular injection. C, Quantification of OTX2 mean intensity per cell in V-SVZ (ns = p > 0.05, Mann–Whitney test). D, Schematic of adult (3 months old) neurogenesis study paradigm with scFv-Otx2 mice. E, Quantification of BrdU-positive cells in olfactory bulb GCL and GL (ns = p > 0.05, **p < 0.01, t test, 3 experiments). F, TUNEL analysis of GCL and GL to assess cell death in olfactory bulb of scFv-Otx2 mice (ns = p > 0.05, Mann–Whitney test, 2 experiments). IP, Intraperitoneal; icv, intracerebroventricular; inj, injection; veh, vehicle. Error bars represent the SD.
- Figure 5.
OTX2 regulates expression of ECM and signaling proteins. A, Quantitative PCR analysis of V-SVZ gene expression in scFv-Otx2 after intracerebroventricular injection of Cre-Tat or vehicle (ns = p > 0.05, *p < 0.05, **p < 0.01, Mann–Whitney test). B, Quantitative PCR analysis of OTX2-treated astrocyte cultures (**p < 0.01, ***p < 0.001, Mann–Whitney test). Error bars represent the SD.
Tables
t df p Mean of vehicle Mean of Cre-TAT Welch’s two-sample t tests Fig. 1F, GCL 3.590 24.96 0.0014** 214.70 146.90 Female 2.510 9.97 0.0310* 197.40 137.80 Male 2.684 12.95 0.0188* 229.5 153.7 Fig. 1F, GL 1.541 24.95 0.1358 63.900 50.090 GCL+GL area 1.100 18.63 0.2851 2.756 2.932 Fig. 1H 0.768 12.57 0.4567 32.390 29.200 Female 0.3007 6.670 0.7728 32.39 30.58 Male 0.7001 4.389 0.5192 32.39 27.82 Fig. 1K 0.028 11.79 0.9782 49.890 49.760 Fig. 1N 2.300 10.03 0.0442* 1706.0 1182.0 Fig. 4E, GCL 3.761 15.72 0.0018** 180.70 120.40 Female 1.593 4.88 0.1734 183.1 130.5 Male 2.593 9.188 0.0286* 178.7 124.9 Fig. 4E, GL 1.688 11.96 0.1172 49.570 39.320 n of Veh n of Cre-Tat p Mean of vehicle Mean of Cre-TAT Mann–Whitney tests Fig. 1C 21 25 <0.0001**** 102.6 40.40 Fig. 4C 84 103 0.3283 44.77 41.25 Fig. 4F, GCL 5 5 0.3095 1.524 2.388 Fig. 4F, GL 5 5 >0.9999 0.933 1.187 Fig. 5A Efna1 5 7 0.0177* 101.50 153.00 Efna2 5 7 0.2677 100.20 117.40 Epha4 5 7 0.8763 100.30 103.10 Robo2 5 7 0.2020 100.90 90.33 Thbs1 5 7 0.0480* 108.30 164.70 Thbs4 7 7 0.0012** 102.10 179.90 Tnc 7 7 0.0239* 99.02 139.80 n of Ctrl n of OTX2 p Mean of Control Mean of OTX2 Fig. 5B Efna1 11 8 0.0003*** 102.500 58.680 Efna2 11 8 0.0001*** 103.300 52.690 Epha4 11 8 0.0025** 102.600 72.510 Robo2 11 8 0.17740 103.700 81.590 Thbs1 11 8 0.0001*** 104.600 50.430 Tnc 12 12 0.0015** 105.800 73.870 *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
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