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Figure 3


Fig. 3. In mouse, Trk signaling is necessary for development of neural precursors within the embryonic cortex. (A) Western blots for phosphotyrosine (pTyr) in full-length, panTrk immunoprecipitates of freshly isolated E14.5, P3 and adult cortical tissue stimulated for 5 minutes with BDNF or NT3. Blots were re-probed for TrkB (left panel) or TrkC (right panel). Arrowheads in upper panels indicate phosphotyrosine-positive full-length TrkB or TrkC, and in lower panels full-length TrkB and TrkC. Size markers are indicated to the left. (B) Fluorescence micrograph of a coronal section of a cortex co-electroporated with EGFP and myc-tagged dnTrkB and immunostained at 3 days for EGFP (green) and the myc-tag (red). Double-labeled cells are yellow in this merged image. (C) Fluorescence micrographs of coronal sections through cortices co-transfected with EGFP and the empty vector (control; top panels) or dnTrkB (bottom panels) and analyzed at 3 days. Sections were immunostained for HuD (red, left panels), and EGFP (GFP, green, middle panels; right panels are the merge). (D) Quantification of sections as in C for the percentage of total EGFP-positive transfected cells within the cortical mantle. n=at least ten animals each, five to six sections/embryo. (E) Quantification of the percentage of transfected precursors expressing high levels of TrkB 3 days after co-transfection with EGFP and control shRNA or one of two TrkB shRNAs (shTrkB-1, shTrkB-2). (F) Fluorescence micrographs of coronal sections through cortices electroporated with EGFP and control shRNA (control; top panels) or TrkB shRNA (shTrkB; bottom panels) and analyzed at 3 days. Sections were double-labeled for HuD (red, left panels) and EGFP (GFP, green, middle panels; right panels are the merge). (G) Quantification of sections as in F for the percentage of total EGFP-positive cells within the cortical mantle. n=six controls and four TrkB shRNA brains, five to six sections/embryo. *P<0.05, **P<0.01, ***P<0.001 relative to control-transfected sections. Error bars indicate s.e.m. Scale bars: 100 µm. CM, cortical mantle; V, ventricle; VZ/SVZ, ventricular zone/subventricular zone.