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Figure 5


Fig. 5. Inhibition of Trk, but not Akt, signaling regulates murine cortical precursors in vivo. (A,B) Akt is not essential for survival or proliferation of cortical precursors in vivo. (A) Western blot of HEK 293 cells transfected with HA-tagged dnAkt or GFP and probed for the HA-tag or GFP. The upper arrow denotes an appropriately sized HA-positive band. (B) Quantification of the percentage of transfected, Ki67-positive cells in the VZ/SVZ of cortices electroporated with EGFP and empty vector (control) or dnAkt and analyzed at 2 days. n=six each, five to six sections/embryo. (C-F) Trk receptor signaling is necessary for appropriate embryonic neurogenesis. Cortices were electroporated with EGFP and the empty vector (control), dnTrkB, dnTrkC or both (dnTrkB/C) and analyzed at 3 days. (C) Confocal micrographs of coronal sections immunostained at 3 days for HuD (red) and EGFP (GFP, green). Upper panels are higher magnification images through the cortical plate, and the lower panels show both the VZ/SVZ and part of the cortical mantle (CM). Arrows indicate double-labeled cells. (D,E) Quantification of micrographs similar to C analyzing the percentage of cells co-expressing EGFP and HuD (D) within the cortical mantle or (E) the entire cortex. n=at least ten mice, four to five sections/embryo. (F) Quantification of the percentage of transfected, HuD-positive cells in cortices electroporated with EGFP and control shRNA (control) or one of two TrkB shRNAs (shTrkB-1, shTrkB-2) at 3 days. n=six controls and four TrkB shRNA brains, five to six sections/embryo. (G-I) Trk receptors collaborate to regulate precursor proliferation and neurogenesis. Quantification of the percentage of (G) EGFP-positive cells in the cortical mantle, (H) EGFP, Ki67-positive cells in the VZ/SVZ and (I) EGFP, HuD-positive neurons in cortices electroporated with EGFP and 1.5 µg of dnTrkB or dnTrkC alone (1/2 dnTrkB, 1/2 dnTrkC), 1.5 µg dnTrkB plus dnTrkC (dnTrkB/C) or 3 µg dnTrkB plus dnTrkC (double B/C) at 3 days. n=six controls, four dnTrkB, five dnTrkC, ten dnTrkB/C and ten double B/C, four sections/embryo. Error bars indicate s.e.m. Scale bar: 100 µm. *P<0.05; **P<0.01; ***P<0.001; NS, nonsignificant.