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First published online 14 November 2007
doi: 10.1242/dev.008227

Development 134, 4369-4380 (2007)
Published by The Company of Biologists 2007
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Trk signaling regulates neural precursor cell proliferation and differentiation during cortical development

Katarzyna Bartkowska1,2,*, Annie Paquin1,3,*, Andrée S. Gauthier1,2,4, David R. Kaplan2,3,4 and Freda D. Miller1,3,4,5,{dagger}

1 Developmental and Stem Cell Biology, Hospital for Sick Children, University of Toronto, Toronto, Canada.
2 Cell Biology Programs, Hospital for Sick Children, University of Toronto, Toronto, Canada.
3 Institute of Medical Sciences, University of Toronto, Toronto, Canada.
4 Departments of Molecular and Medical Genetics, University of Toronto, Toronto, Canada.
5 Departments of Physiology, University of Toronto, Toronto, Canada.


Figure 1
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  		Fig. 1. TrkB and TrkC signaling are essential for the survival of cultured murine embryonic cortical precursor cells. (A) Fluorescence micrographs of cortical precursor cultures co-transfected with plasmids encoding EGFP and the empty vector (control), dnTrkB, dnTrkC or both (dnTrkB/C), and then analyzed at 2 days for EGFP (green, GFP), and cleaved caspase 3 (red, casp 3). Cells were counterstained with Hoechst 33258 (blue) to show all nuclei. Arrows denote double-labeled cells. (B,C) Quantification of the percentage of EGFP-positive cells (B) with condensed, fragmented nuclei, and (C) positive for cleaved caspase 3 in experiments similar to A. In each panel, two experiments of four are shown. Error bars denote the s.e.m. Scale bar: 100 µm. *P<0.05, **P<0.01, ***P<0.001 relative to control-transfected cultures.

 

Figure 2
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  		Fig. 2. TrkB and TrkC are essential for proliferation of cultured murine embryonic cortical precursor cells. (A) Immunostaining for EGFP (green, GFP) and Ki67 (red) for precursor cells transfected with EGFP and empty vector (control), dnTrkB, dnTrkC or both (dnTrkB/C) and cultured for 2 days. Cells were counterstained with Hoechst 33258 (blue). Arrows denote double-labeled cells. (B) Quantification of the percentage of transfected, Ki67-positive cells in experiments similar to A. Two experiments of four are shown. (C,D) Quantification of the percentage of apoptotic cells in experiments similar to A, except that cells were cultured with or without 100 µM ZVAD-FMK. Apoptosis was assessed by (C) analysis of condensed, fragmented nuclei and by (D) immunostaining for EGFP and cleaved caspase 3. In both panels, one experiment of two is shown. (E) Quantification of the percentage of transfected, Ki67-positive cells in experiments similar to A, except that cells were cultured with or without 100 µM ZVAD-FMK for 2 days. Error bars indicate s.e.m. Scale bar: 100 µm. *P<0.05, **P<0.01, ***P<0.001 relative to control-transfected cultures.

 

Figure 3
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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.

 

Figure 4
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  		Fig. 4. In mouse, Trk receptor signaling regulates embryonic cortical precursor cell proliferation but not survival in vivo. (A-E) Cortices were electroporated with EGFP and the empty vector (control), dnTrkB and/or dnTrkC and analyzed 1-3 days later. (A) Confocal micrographs of coronal sections through cortices immunostained for EGFP (GFP, green) and cleaved caspase 3 (casp 3, red) at 1 day. The right panels show the merges. Arrows indicate transfected, cleaved caspase-3-positive cells, and arrowheads cells that only express cleaved caspase 3. (B) Quantification of the total number of EGFP-positive cells in six sections through electroporated cortices at 3 days. n=at least nine animals per group. (C) Confocal micrographs of coronal VZ/SVZ sections immunostained for EGFP (GFP, green) and Ki67 (red) at 3 days. The right panels show the merges. Arrows indicate transfected, Ki67-positive cells. (D,E) Quantification of the percentage of transfected, Ki67-positive cells in sections like C, at 1 or 3 days post-electroporation. For D, n=at least three embryos. For E, n=at least ten embryos, four to five sections/embryo at each timepoint. (F,G) Quantification of the percentage of transfected, Ki67-positive (F) or phospho-histone-H3-positive (G) cells in the VZ/SVZ of cortices transfected with EGFP and control shRNA (control) or one of two TrkB shRNAs (shTrkB-1, shTrkB-2) at E13/14 and analyzed 3 days post-electroporation. n=at least four, five to six sections/embryo. Error bars indicate s.e.m. Scale bars: 100 µm. *P<0.05, **P<0.01, ***P<0.001 relative to control-transfected sections.

 

Figure 5
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  		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.

 

Figure 6
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  		Fig. 6. Increased BDNF expression in the cortical neuroepithelium promotes precursor proliferation and enhances neurogenesis in mouse. Cortices were electroporated with EGFP and BDNF or the empty vector, and analyzed at 3 days post-electroporation. (A-C) Quantification of the percentage of (A) EGFP, Ki67-positive cells in the VZ/SVZ, (B) EGFP-positive cells within the cortical mantle and (C) EGFP, HuD-positive cells. n=eight each, five to six sections/embryo. (D) Confocal micrographs of coronal sections immunostained for HuD (red) and EGFP (green). The left panels of each pair are HuD staining, and the right panels are merges of HuD and EGFP staining. Arrows indicate a cluster of EGFP-negative, HuD-positive cells. Error bars indicate s.e.m. Scale bar: 100 µm. *P<0.05; ***P<0.001. V, ventricle.

 

Figure 7
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  		Fig. 7. In mouse, Trk signaling in embryonic precursors is necessary for maintenance of normal numbers of precursors in the postnatal SVZ, but not for astrocyte formation. Cortices were electroporated with EGFP and the empty vector (control), dnTrkB, dnTrkC or both (dnTrkB/C), and analyzed at P3. (A) Confocal micrographs of coronal sections through the electroporated VZ/SVZ immunostained for EGFP (GFP, green) and GFAP (red). The right panels show the merges. Arrows indicate double-labeled cells. (B,C) Quantification of micrographs similar to those in A for the percentage of (B) EGFP-positive cells and (C) EGFP, GFAP-positive cells in the VZ/SVZ. n=five, five sections/cortex. (D) Quantification of confocal micrographs similar to A where cortices were co-electroporated with EGFP and control shRNA (control) or TrkB shRNA (shTrkB) to determine the percentage of EGFP, GFAP-positive cells within the VZ/SVZ. n=four control and three TrkB shRNA brains. Error bars indicate s.e.m. Scale bar: 100 µm. **P<0.01; ***P<0.001 relative to control-transfected sections.

 

Figure 8
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  		Fig. 8. Inhibition of Trk signaling in embryonic cortical precursors leads to postnatal perturbations in cortical neurons in mouse. Cortices were electroporated with EGFP and the empty vector (control), dnTrkB, dnTrkC or both, and analyzed at P3. (A) Confocal micrographs of coronal sections through cortices immunostained for EGFP (GFP, green) and NeuN (red). The right panels show the merges. Arrows indicate double-labeled cells. (B,C) Quantification of micrographs similar to A for percentage of (B) EGFP, NeuN or (C) EGFP, HuD-positive cells within the cortical layers. n=five, five sections/brain. (D) Photomicrographs of coronal sections immunostained for EGFP (green) and counterstained with Hoechst (blue). Cortical layers are denoted on the left, and regions defined as the upper and lower parts of layers II/III are demarcated by the white lines. (E) Quantification of the percentage of EGFP-positive cells in the upper (white columns) and in the lower (black column) parts of layers II/III in sections similar to (D). n=five, five sections/brain. (F) Photomicrographs of coronal sections immunostained for EGFP (green) and GAD67 (red) and counterstained for Hoechst (blue). Shown are EGFP and Hoechst (left) or EGFP and GAD67 (middle). The right panel is a higher-magnification view showing a rare, double-labeled cell (arrow), and several EGFP-positive, GAD67-negative cells (arrowhead). Error bars indicate s.e.m. Scale bars: 100 µm in A; 200 µm in D; 50 µm in F. ***P<0.001 relative to control-transfected sections.

 

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© The Company of Biologists Ltd 2007