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. 2013 Apr 25:8:7.
doi: 10.1186/1749-8104-8-7.

AKT activation by N-cadherin regulates beta-catenin signaling and neuronal differentiation during cortical development

Jianing Zhang  1 , Julie R Shemezis, Erin R McQuinn, Jing Wang, Maria Sverdlov, Anjen Chenn
Affiliations

AKT activation by N-cadherin regulates beta-catenin signaling and neuronal differentiation during cortical development

Jianing Zhang et al. Neural Dev. .

Abstract

Background: During cerebral cortical development, neural precursor-precursor interactions in the ventricular zone neurogenic niche coordinate signaling pathways that regulate proliferation and differentiation. Previous studies with shRNA knockdown approaches indicated that N-cadherin adhesion between cortical precursors regulates β-catenin signaling, but the underlying mechanisms remained poorly understood.

Results: Here, with conditional knockout approaches, we find further supporting evidence that N-cadherin maintains β-catenin signaling during cortical development. Using shRNA to N-cadherin and dominant negative N-cadherin overexpression in cell culture, we find that N-cadherin regulates Wnt-stimulated β-catenin signaling in a cell-autonomous fashion. Knockdown or inhibition of N-cadherin with function-blocking antibodies leads to reduced activation of the Wnt co-receptor LRP6. We also find that N-cadherin regulates β-catenin via AKT, as reduction of N-cadherin causes decreased AKT activation and reduced phosphorylation of AKT targets GSK3β and β-catenin. Inhibition of AKT signaling in neural precursors in vivo leads to reduced β-catenin-dependent transcriptional activation, increased migration from the ventricular zone, premature neuronal differentiation, and increased apoptotic cell death.

Conclusions: These results show that N-cadherin regulates β-catenin signaling through both Wnt and AKT, and suggest a previously unrecognized role for AKT in neuronal differentiation and cell survival during cortical development.

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Figures

Figure 1
Figure 1
Conditional knockout of N-cadherin reduces β-catenin signaling in developing cortical precursors. (A) Immunostaining for d2EGFP (green) in E12.0 littermate control (Axin2-d2EGFP; Nes11Cre; NcadFlox/+) and Ncad cKO brain (Axin2-d2EGFP; Nes11Cre; NcadFlox/Flox) embryonic cortex reveals that conditional tissue-wide knockout of N-cadherin leads to reduced EGFP expression in the developing ventricular zone (VZ) (DNA stained with DAPI, pseudocolored blue; bar = 100 µm). (B) Focal elimination of N-cadherin reduces β-catenin transcriptional activity. β-catenin mediated transcriptional activation was examined through expression of destabilized GFP controlled by the TOP promoter. E13.5 embryos were electroporated with pTOP-dGFP, pCAG-mCherry, and pCAGLacZ in the control treatment, and with pTOP-dGFP, pCAG-mCherry, and pCAG-Cre in the experimental condition, and analyzed at E14.5. The dot image below shows the positions of the individual electroporated cells (yellow dots represent double-labeled mCherry/dGFP + cells and red dots represent dGFP- cells (expressing mCherry only)). Only electroporated cells in the VZ were included in the analysis, and the proportion of cells expressing dGFP was compared to the total number of electroporated cells in the VZ. *P = 0.0242 by unpaired Student’s t-test, n = 3 brains for each. Error bars represent 1 SEM. Bar = 50 µm. EP’d, electroporated.
Figure 2
Figure 2
N-cadherin functions in Wnt-induced β-catenin signaling in a cell-autonomous manner. (A) Separate populations of 293 T cells were transfected with Wnt3a expression and pSuper8TOPFlash reporter constructs and plated either on the opposite of a polycarbonate transwell filter (“membrane bound”) or on the bottom of the transwell assay (“separated”). In a TOPFlash reporter assay, Wnt3a-expressing signaler cells induced β-catenin signaling in reporter cells adherent on the opposite face of the transwell membrane, but not in reporter cells plated separately at the bottom of the chamber. P = 0.004 by repeated measures analysis of variance (ANOVA), ***P < 0.001, Neuman Keuls post-hoc test, n = 3. (B) Reduction of N-cadherin by shRNA (Ncad-shRNA) or by overexpression of C-terminal truncated N-cadherin (NcadΔC) in Wnt-responsive cell results in reduced Wnt-activated β-catenin transcriptional activation. Wnt3a transfected signaler cells were co-cultured with pTOPflash-transfected reporter cells co-transfected with either shRNA to N-cadherin (P = 0.0012 by repeated measures ANOVA, **P < 0.01, *P < 0.05 by Neuman Keuls post-hoc test; n = 4) or NcadΔC (P = 0.0006 by repeated measures ANOVA, ***P < 0.001, **P < 0.01 by Neuman Keuls post-hoc test; n = 3), and luciferase activity was measured 24 hours after co-culture. (C) Inhibition of N-cadherin in the Wnt-producing signaling cell does not affect Wnt-mediated β-catenin signaling. Wnt3a transfected signaler cells were co-transfected with either Ncad-shRNA (P = 0.0024 by repeated measures ANOVA, **P < 0.01 by Neuman Keuls post hoc test; n = 4) or NcadΔC (P = 0.0002 by repeated measures ANOVA, ***P < 0.001 by Neuman Keuls post-hoc test; n = 3), co-cultured with pTOPflash-transfected reporter cells, and luciferase activity was measured 24 hours after co-culture.
Figure 3
Figure 3
Reduction of N-cadherin leads to reduction of AKT and LRP6 phosphorylation. (A) Transfection of N-cadherin-shRNA (Ncad-shRNA) reduced the amount of AKT phosphorylated at Serine 473 (P-AKT [S473P]) and AKT targets β-catenin phospho-Serine 552, β-catenin [S552-P]) and phosphorylated GSK3β (P-GSK3β [Ser 9]). N-cadherin, P = 0.0151 by paired t-test, n = 7; P-AKT [S473], P = 0.0182 by paired t-test, n = 6; P-β-catenin [S552], P = 0.0083 by paired t-test, n = 3; P-GSK3β [Ser 9], P = 0.0085 by paired t-test, n = 3. Consistent with the stabilizing role of phosphorylated Ser 552, there was a trend for reduction in total β-catenin in N-cad-shRNA transfected cells (P = 0.1383 by paired t-test, n = 2). (B) Cells stably expressing N-cad-shRNA knockdown reveal reduced levels of AKT phosphorylation (P-AKT [S473], phospho-GSK3β (P-GSK3β [S9]), and phospho-β-catenin (P-β-catenin [S552]). N-cadherin knockdown also reduced baseline phosphorylation of LRP-6 (P-LRP6 [S1490]) as well as Wnt3a-induced phosphorylation of LRP6. (C) Primary mouse cortical progenitors were nucleofected with two different shRNA to N-cadherin or EGFP and cell extracts were blotted for phospho-β-catenin Serine 552 (P-β-catenin [S552]). In both cases, N-cadherin knockdown resulted in reduced phosphorylation of β-catenin at Ser 552. shRNA1: P = 0.0119 by paired t-test (n = 3); shRNA2: P = 0.0055 by paired t-test (n = 3). (D) Primary mouse neural stem cells derived from E14.5 mouse cortices were treated for 24 hours with either control IgG or N-cadherin function blocking antibody 20 µg/ml, and cell extracts were blotted for phospho-LRP6 [S1490]. Both biological replicates are shown.
Figure 4
Figure 4
Targets of AKT activity are expressed in dividing radial glia during cortical development. Immunohistofluorescence staining of cortical sections at embryonic day 12.5 reveals AKT activity as assessed by targets of AKT phospho-β-catenin [S552] and phosphor-GSK3β [S9] in the ventricular zone (VZ). (A) Phospho-beta-catenin-Ser 552 (pseudocolored red) is expressed in dividing radial glial cortical precursors identified by the expression of phosphorylated vimentin 4A4 (P-vim, pseudocolored green) located at the apical (lumenal) surface of the VZ. (B) Phospho-GSK3beta-Ser 9 (pseudocolored red) is expressed in dividing radial glial cortical precursors expressing phosphorylated vimentin 4A4 (P-vim, pseudocolored green) at the apical ventricular surface. Scale bar = 20 µm. 74.3±5.9% (1 SEM) of P-Vim expressing cells were also expressing P-β-catenin (n = 3 brains), and 82.5±3.6% (1 SEM) of P-Vim-expressing cells were also P-GSK3β positive (n = 3 brains).
Figure 5
Figure 5
Inhibition of AKT signaling reduces β-catenin transcriptional activityin vivo. β-catenin mediated transcriptional activation was examined through expression of destabilized GFP controlled by the TOP promoter. E13.5 embryos were electroporated with pTOP-dGFP, pCAG-mCherry, and pcDNA in the control treatment, and with pTOP-dGFP, pCAG-mCherry, and dominant negative AKT (DN-AKT) in the experimental condition and analyzed 30 hours after electroporation. The dot image below shows the positions of the electroporated cells (green dots represent electroporated cells co-expressing dGFP and mCherry, and red dots represent dGFP- cells (expressing mCherry only)). Only electroporated cells in the ventricular zone (VZ) were included in the analysis, and the proportion of cells expressing dGFP was compared to the total number of electroporated cells (red, labeled by mCherry) in the VZ. Compared to pcDNA control, DN-AKT expression reduced the fraction of TOPdGFP-expressing cells from 73 to 53% (n = 3 brains each), **P = 0.0043 by unpaired t-test. Error bars represent 1 SEM. Scale bar = 100 µm. EP’d, electroporated.
Figure 6
Figure 6
AKT prevents premature differentiation of neural progenitorsin vivo. (A) E13.5 forebrains were electroporated in utero with pCAG-GFP and 4-fold (by mass) excess of pcDNA control or K197M dominant negative AKT (DN-AKT) and analyzed at E14.5 (n = 4 brains each). Electroporated cells were identified using antibody staining against GFP and sections counterstained with DAPI. The pial surface is indicated by the white line. Scale bar = 100 µm. (B) To quantify changes in cortical positioning of electroporated cells, ten equal sized bins were drawn over each image covering the cortical plate. Each dot corresponds with the soma of an electroporated cell. The fraction of the total GFP + cells was then graphed. The x-axis denotes the fraction of the total number of electroporated cells in each bin. Brackets indicate 1 SEM. (C-F) Sections of electroporated brains were stained for Pax6 (C), Tbr2 (D), and Tbr1 (E). Electroporated cells are green, and the respective antigens are red. Bar = 100 µm. The dot plots show the electroporated cells co-expressing the marker as yellow and electroporated cells not expressing the marker as green. (F) Cell histograms show the fraction of electroporated cells that express each marker after electroporation (yellow/yellow + green dots). DN-AKT causes premature neuronal differentiation as defined by the alterations in Tbr1, Tbr2, and Pax6 expression. For Pax 6, DN-AKT versus control (n = 4 brains for each, **P = 0.0071), Tbr2 (n = 2 for DN-AKT, n = 4 for pcDNA control, *P = 0.0406), and Tbr1 (n = 4 for each, *P = 0.0277), unpaired t-test. Error bars represents SEM. Scale bar = 100 µm. (G) DN-AKT increases the fraction of cells that lose contact with ventricular surface. n = 4 brains, *P = 0.0368, paired t-test. Error bar represents SEM. EP’d, electroporated.
Figure 7
Figure 7
Dominant negative AKT increases apoptotic cell deathin vivo. E13.5 forebrains were electroporated in utero with pCAG-GFP and 4-fold (by mass) excess of pcDNA control or K197M dominant negative AKT (DN-AKT) and analyzed at E14.5. Sections of electroporated brains were stained for GFP, cleaved caspase 3, and DNA stained with DAPI. Arrows point to electroporated cells that are expressing cleaved caspase 3. Bright red round spots are non-specific staining not associated with cells. Bar = 50 µm. Compared with control, electroporation of DN-AKT causes increased cell death, P = 0.0067 by paired students t-test. n = 3 brains for each condition. EP’d, electroporated.
Figure 8
Figure 8
Model for the function of N-cadherin in β-catenin activation and cortical development. (A) N-cadherin functions in the activation of β-catenin signaling via AKT-mediated phosphorylation of β-catenin and Wnt-mediated activation of the Wnt co-receptor, LRP6. In Wnt-mediated signaling, N-cadherin is required in a cell-autonomous manner in the responding cell and is involved in signaling via phosphorylation of the Wnt co-receptor LRP6. N-cadherin also functions to stimulate β-catenin signaling via AKT activation and subsequent phosphorylation of β-catenin at Ser 552. (B) In cortical radial glial neural progenitors, N-cadherin regulates epithelial integrity, and enhances Wnt-mediated and AKT-mediated β-catenin activation. Downstream of N-cadherin, AKT signaling functions to promote self-renewal via activation of β-catenin signaling as well as inhibiting apoptosis.
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