Article Figures & Data
Figures
- Figure 1.
Expression of GFRα1 in the developing mouse OE. A, Overview of GFRα1 expression in the mouse OE at E12.5, E14.5, P0, and 2 weeks as assessed by immunohistochemistry. Scale bars: (E12.5), 100 μm (E14.5), 50 μm (P0), and 50 μm (2 week). B, Expression of Gfra1 mRNA in the developing mouse OE analyzed by in situ hybridization at P0 and 2 weeks. Scale bars: 200 μm (P0), 50 μm (2 week low power), and 50 μm (2 week high power). Box illustrates region in D. C, Expression of GFRα1 in GAP43-positive immature axons and OSNs in the newborn mouse OE. Scale bar, 50 μm. D, Expression of Gfra1 mRNA in basal cells and GAP-43-positive immature OSNs in the newborn OE. Scale bar, 50 μm. E, Expression of GFRα1 in LN-expressing OECs of the newborn OE. Scale bar, 50 μm. F, At P0 and 2 weeks, GFRα1 did not colocalize with OMP-positive mature OSNs and was restricted to the immature and basal cell layers of the OE. Scale bars: 50 μm (P0 and 2 week).
- Figure 2.
Expression of GFRα1 in the developing mouse OB. A, Overview of GFRα1 expression in the mouse OB at E12.5, E14.5, P0, and 2 weeks assessed by immunohistochemistry. Boxes indicate high-magnification images in P0 and 2 week right panels. Scale bars: 200 μm (E12.5), 100 μm (E14.5), 200 μm (P0 low power), 50 μm (P0 high power), 500 μm (2 week low power), 100 μm (2 week high power). B, Expression of Gfra1 mRNA in the developing mouse OB analyzed by in situ hybridization at P0 and 2 weeks. Scale bars: 200 μm (P0) and 100 μm (2 week). C, Double labeling for GFRα1 and GAP-43 (immature OSN axons), OMP (mature OSN axons), NCAM (all OSNs axons), and LN (OECs) in the ONL of the newborn OB. OMP was detected in OMP-GFP transgenic mice and visualized by GFP fluorescence. The LN panel shows two ONLs from adjacent OBs at the midline. Scale bar, 50 μm. D, Double immunofluorescence for GFRα1 and Tbr1 or Reelin in the newborn OB. Scale bar, 50 μm. E, Lack of colocalization between GFRα1 and GABAergic markers in the newborn OB. GFRα1 (red) was detected by immunohistochemistry in OB from GAD65-GFP and GAD67-GFP transgenic mice as indicated. GAD65 and GAD67 were visualized by GFP fluorescence. Left, Shows whole OB coronal sections. Middle, Shows high-magnification of solid-line boxes. Right, Shows high magnification of dash-line boxes. Size bars; 100 μm (left), 50 μm (middle), 20 μm (right). F, Double immunofluorescence for GFRα1 (red) and interneuron markers TH, CB, and CR (green) in the newborn OB. No overlap was observed with TH. However, a few CB- and CR-positive interneurons colocalized with GFRα1, as depicted by arrows. Scale bar, 100 μm.
- Figure 3.
Gdnf mRNA expression in the developing olfactory system. A, Expression of Gdnf mRNA in the developing mouse OE analyzed by in situ hybridization at P0 and 2 weeks. Arrow denotes an OEC expressing Gdnf mRNA below the BL. iOSNs, immature OSNs; mOSNs, mature OSNs. Scale bars: 100 μm (P0), 100 μm (2 week). B, Expression of Gdnf mRNA in the developing mouse OB analyzed by in situ hybridization at P0 and 2 weeks. Arrow denotes OECs expressing Gdnf mRNA in the ONL. Scale bars: 100 μm (P0), 200 μm (2 week low magnification), and 100 μm (2 week box, high magnification).
- Figure 4.
Loss and disorganization of OSNs in the OE of mice lacking GFRα1. A, Mature (OMP, green) and immature (GAP43, red) OSNs and their axons in wild-type and Gfra1−/− mutant OE at birth. Arrows denote axon bundles (AB). Asterisks indicate immature ABs. Lamina propria (LP), containing OECs, is marked by dotted lines. Scale bars: 100 μm (top row), 50 μm (bottom row-OMP and GAP-43). iOSNs, immature OSNs; mOSNs, mature OSNs. B, OECs (LN, red), all OSNs (NCAM, green) visualized by immunohistochemistry in newborn wild-type and Gfra1−/− mutant OE in relation to all cells (DAPI, blue, top row) or mature OSNs (OMP, blue, bottom row). Arrows depict normal size ABs of mature and immature axons (coexpression of OMP and NCAM). Asterisk denotes enlarged AB in mutant of only immature axons (no coexpression of NCAM and OMP). Scale bar, 50 μm. C, Activated cleaved caspase-3 (arrows) and BrdU incorporation in newborn wild-type and Gfra1−/− mutant OE. Counterstaining with DAPI (blue). Arrows indicate the cell types with the greatest increase. Scale bars: 100 μm. D, OMP-positive mature OSN (mOSN) (left) cell density is significantly decreased (*p < 0.001, t = 28.6) in newborn Gfra1−/− mutants (529.7 ± 31.3, n = 10) compared with wild-type (722.3 ± 20.6, n = 10) per OE. GAP-43-positive immature OSN (iOSN) (right) cell density per OE is dramatically reduced (*p < 0.0001, t = 42.0) in P0 Gfra1−/− mutant (561.6 ± 34.0, n = 10) compared with wild-type (840.6 ± 35.0, n = 10). Values are mean ± SD. E, Total number of activated caspase-3 cells per OE (right) reveal a significant increase (*p < 0.0001, t = 28.0) in Gfra1−/− knock-outs (457.6 ± 51.3, n = 10) compared with control (219.3 ± 26.0, n = 10). BrdU incorporating cells (left) are remarkably increased (*p < 0.0001, t = 61.6) in P0 Gfra1−/− mutants (804.9 ± 34.3, n = 10) compared with wild-type (439.8 ± 20.9, n = 10). Values are mean ± SD. F, Colocalization of activated caspase-3 with NCAM (asterisks in top), OMP (arrows in top) and integrin-α6, an alternative marker of OECs (asterisks in bottom) in the OE of newborn wild-type and Gfra1−/− mutants. Scale bar, 50 μm. G, Colocalization of BrdU incorporation with GAP43 (arrows in top), OMP, and LN (arrows in bottom) in the OE of newborn wild-type and Gfra1−/− mutants. Scale bar, 50 μm.
- Figure 5.
Reduced olfactory nerve layer and loss of OECs in the OB of Gfra1−/− mutant mice. A, Immunohistochemistry for OMP, GAP43, NCAM, and LN in the ONL of newborn wild-type and Gfra1−/− mutant OBs. ONL thickness is denoted with broken lines and is clearly reduced in mature (OMP), immature (GAP-43), and all OSN axons (NCAM). LN-expressing OECs are also significantly reduced in Gfra1−/− mutants, thickness denoted by broken lines of both OBs. Scale bar, 100 μm. B, Colocalization of activated caspase-3 with NCAM, OMP, and integrin-α6 (an alternative marker of OECs) in the ONL of newborn wild-type and Gfra1−/− mutant OBs. Scale bar, 100 μm.
- Figure 6.
Glomerular deficits in newborn homozygous and adult heterozygous Gfra1 mutants. A, Glomeruli in wild-type and Gfra1−/− newborn mice were visualized by immunohistochemistry for VGlut2 (red). A superposition with OMP (green) and DAPI (blue) in the bottom was used to define GL boundaries. Scale bar, 100 μm. Histograms show significantly larger (*p < 0.0003, t = 5.752) average glomerular area in Gfra1−/− knock-outs (2640 ± 333.7, n = 570) compared with controls (1427 ± 128.6, n = 866). Values are shown as mean ± SEM. Total number of glomeruli was also reduced (*p < 0.0001, t = 37.5) per OB in Gfra1−/− mutants at birth (210.9 ± 22.2, n = 10) compared with wild type (317.6 ± 24.7, n = 10). Values are shown as mean ± SD). B, Glomeruli of 7-week-old wild-type and Gfra1+/− heterozygous mice. Scale bar, 100 μm. Histograms show significantly smaller (*p < 0.0001, t = 6.933) average glomerular area in Gfra1+/− heterozygous mice (5947 ± 332.9, n = 711) compared with controls (6916 ± 226.0, n = 933). Values are shown as mean ± SEM. Total number of glomeruli were also drastically fewer (*p < 0.0001, t = 65.39) per OB in Gfra1+/− heterozygous mice (665.6 ± 35.32, n = 10) compared with wild type (937.1 ± 44.82, n = 10). Values are shown as mean ± SD.
- Figure 7.
Abnormal distribution and loss of projection neurons in OB of newborn homozygous and adult heterozygous Gfra1 mutants. A, Immunohistochemistry for Reelin (red) and Tbr1 (green) in OB of newborn wild-type and Gfra1−/− mice. ONL, GL, EPL, and MCL are indicated. Scale bar, 50 μm. Histograms show a significant reduction in both mitral cell density (Reelin-positive cells per OB left, *p < 0.0001, t = 52.8) and glutamatergic cell density (Tbr1-positive cells per OB right, *p < 0.0001, t = 16.2) at P0 in Gfrα1−/− mutants (Reelin: 4237 ± 230, n = 10;Tbr1: 3674 ± 260, n = 10) compared with wild type (Reelin: 5750 ± 194, n = 10; Tbr1: 5074 ± 192, n = 10). Values are mean ± SD. B, Staining for activated caspase-3 in the newborn OB of Gfra1 mutant mice does not overlap with Reelin in the mitral cell layer. Scale bar, 50 μm. C, Immunohistochemistry for Reelin in 7-week-old OB of wild-type and Gfra1+/− heterozygous mice. Scale bar, 100 μm. Histograms show significant losses in Gfra1+/− heterozygous mitral cell density (Reelin-positive cells in the MCL, left, *p < 0.0001, t = 37.42; 450.9 ± 33.26, n = 10) and tufted cell density (Reelin-positive tufted cells in the EPL, right, *p < 0.0001, t = 34.25; 864.0 ± 54.50, n = 10) compared with wild type (mitral cell density: 554.4 ± 37.76, n = 10; tufted cell density: 1181.0 ± 76.25, n = 10). Values are mean ± SD.
- Figure 8.
Loss of GABAergic interneurons in OB of newborn homozygous and adult heterozygous Gfra1 mutants. A, Immunohistochemistry for TH (upper row), CB (middle row), and CR (bottom row) in OB of newborn wild-type and Gfra1−/− mice. ONL, GL, EPL, and MCL are indicated. Scale bar, 100 μm. Histograms show significant losses of TH (*p < 0.0001, t = 36.79), CB (*p < 0.0001, t = 33.19), and CR (*p < 0.0001, t = 14.56) in Gfra1−/− mutants (TH: 628.7 ± 52.29, CB: 1141 ± 36.45, and CR: 1721 ± 172.2, n = 10 for all groups) compared with controls (TH: 1010 ± 70.43, CB: 1550 ± 43.02, and CR: 2056 ± 207.8, n = 10 for all groups). Images are high-power representation of results. Values are mean ± SD. B, TH-, CB-, and CR-expressing interneurons in 7-week-old OB of wild-type and Gfra1+/− heterozygous mice. Scale bar, 100 μm. Histograms show quantification of TH, CB, and CR cell density. Gfra1+/− heterozygous mice have significantly fewer TH (*p < 0.0001, t = 21.05; 709.6 ± 58.58), CB (*p < 0.0001, t = 38.81; 1207 ± 80.44), and CR (*p < 0.0001, t = 11.39; 2081 ± 354.1) interneurons compared with wild type (TH: 1052 ± 70.19, CB: 1647 ± 79.15, and CR: 2498 ± 446.8, n = 10 for all groups). Values are shown as mean ± SD. Scale bar, 100 μm.
- Figure 9.
Deficits in the olfactory system of newborn mice lacking GDNF. A, Mature (OMP, green) and immature (GAP43, red) OSNs and their axons in wild-type and Gdnf−/− mutant OE at birth. Arrows denote mature axon bundles. Asterisks indicate immature axon bundles. Lamina propria is marked by dotted lines. Scale bar, 50 μm. B, Immunohistochemistry for OMP (green, top) in the ONL of newborn wild-type and Gdnf−/− mutant OBs. ONL/GL thickness is denoted with broken lines and is clearly reduced in Gdnf−/− mutants. Asterisks denote irregularly shaped glomeruli in the mutants. DAPI staining (blue, bottom) shows the reduction in OB layers of newborn Gdnf−/− mutant mice. Arrow indicates round discrete glomeruli in wild type. Scale bar, 100 μm. C, Immunohistochemistry for TH (upper row), CB (middle row), and CR (bottom row) in OB of newborn wild-type and Gdnf−/− mice. ONL, GL, EPL, and MCL are indicated. Histograms show quantification of TH, CB, and CR cell density. Newborn Gdnf−/− mutant mice have significantly fewer TH (*p < 0.0001, t = 13.75; 940.0 ± 97.96), CB (*p < 0.0001, t = 9.548; 1317 ± 127.5), and CR (*p < 0.0001, t = 20.89; 2021 ± 122.2) interneurons compared with wild type (TH: 1532 ± 228.3, CB: 1844 ± 226.7, and CR: 2443 ± 117.5, n = 10 for all groups). Values are shown as mean ± SD. Scale bar, 100 μm.
- Figure 10.
Impaired olfactory function in heterozygous Gfra1 mutants. A, Buried food assay. Adult male heterozygous Gfra1 mutant mice displayed a significantly (*p < 0.0001, t = 6.286) longer latency measured in seconds (56.10 ± 8.51, n = 20) to retrieve a buried food pellet compared with wild-type controls (169.8 ± 15.95, n = 20). Values are shown as mean latency in seconds ± SEM. B, Innate olfactory preference and sensitivity test. Histogram depicts the duration in which wild-type and Gfrα1 heterozygous mice investigated a scented filter paper with various odorants measured over a 3 min test period. All odorants tested at concentrations given in Material and Methods (1:1); female mouse urine and TMT were also tested at 1:5 and 1:10 dilutions. Dotted line denotes the neutral level of response (water). *p < 0.0001 versus wild type for each odor; #p < 0.0001 versus wild-type water, n = 14 for each genotype and odorant. Values are shown as mean investigation time in seconds ± SEM.
