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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.
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