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First published online 8 April 2008
doi: 10.1242/jcs.020537

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E-catenin is not a significant regulator of β-catenin signaling in the developing mammalian brain

¹ Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
² Molecular and Cellular Biology Program, University of Washington, Seattle, WA 98109, USA
³ Department of Pathology and Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA 98195, USA

View larger version (87K): [in this window] [in a new window]   Fig. 1. Depletion of E-catenin has no major effect on interaction between β-catenin, N-cadherin and other β-catenin-binding proteins. (A,B) Total protein lysates from E14.5 wild-type (WT) and E-catenin–/– (knockout, KO) mouse brains were immunoprecipitated with control (IgG) or anti-β-catenin (β-cat) antibodies and the resulting protein complexes were separated by SDS-PAGE and stained with Colloidal Blue and Silver stain (A) or analyzed by western blot with anti-E-catenin, N-cadherin or β-catenin antibodies (B). Note that although E-catenin becomes depleted from β-catenin protein complexes, composition or relative abundance of other proteins does not change. Western blotting reveals no significant changes in the association between β-catenin and N-cadherin. (C-D") Despite disruption of apical junctional complexes and loss of cell polarity, β-catenin continues to colocalize with N-cadherin at the periphery of E-catenin–/– neural progenitor cells. Cortical sections from E13.5 wild-type (WT) and E-catenin–/– (KO) embryos were stained with anti-N-cadherin (N-cad) and anti-β-catenin (β-cat) antibodies. Scale bar: 15.9 µm.

View larger version (89K): [in this window] [in a new window]   Fig. 2. Depletion of E-catenin has no effect on overall level of β-catenin or its nuclear localization. (A,A') Total protein lysates from E12.5 and E13.5 wild-type (WT) and E-catenin–/– (KO) mouse brains were analyzed by western blotting with anti-E-catenin, anti-N-catenin, β-catenin and β-actin antibodies. Quantification of these results is shown in A'. Levels of β-catenin were normalized using β-actin and the results are shown as relative fold change. Data represent means ± s.d. (n=3). (B-C") Sagittal telencephalon sections from E13 embryos were immunostained for nuclear β-catenin. Sections were subjected to antigen retrieval, stained overnight with anti-β-catenin antibodies and processed using ABC MOM staining kit. Boxed areas in B and C are shown at higher magnification in B',B" and C',C", respectively. β-catenin was present in the nuclei of progenitor cells, which were localized around the ventricles. Prominent staining was also seen in the AJs at the ventricular surface (black arrows). There were no significant differences in the nuclear β-catenin between the wild-type (WT) and knockout (KO) mouse brains. HI, developing hippocampus. Scale bar in B: 0.12 mm for B,C and 0.012 mm for B'-C".

View larger version (82K): [in this window] [in a new window]   Fig. 3. Endogenous reporter for β-catenin transcriptional activity reveals no changes in E-catenin–/– mouse brains. (A) Schematic representation of TOPGAL reporter (DasGupta and Fuchs, 1999). The reporter contains three consensus Lef/Tcf-binding motifs (Lef) and a minimal c-fos promoter (P) to drive transcription of LacZ. (B-C') A similar pattern of β-catenin reporter expression in the telencephalon of wild-type (WT) and E-catenin–/– (KO) embryos. Sagittal sections of brains from E12.5 (B,B') and E13.5 (C,C') embryos positive for TOPGAL transgene were stained for β-galactosidase (blue) and counterstained with nuclear Fast Red. HI, developing hippocampus. Scale bar in B: 0.19 mm for B,B' and 0.26 mm for C,C'. (D) Similar levels of β-catenin reporter expression were observed in wild-type (WT) and E-catenin–/– (KO) mouse brains. Total protein lysates from E12.5 and E13.5 telencephalons fromTOPGAL-positive animals were analyzed by western blotting with anti-β-galactosidase (β-gal) and anti-β-actin antibodies. Con, control samples from TOPGAL-negative animals. The numbers indicate the relative amount of β-gal adjusted to the level of β-actin.

View larger version (22K): [in this window] [in a new window]   Fig. 4. Transition from E12.5 to E13.5 results in extensive hyperplasia in E-catenin–/– brains without significant changes in the expression of endogenous transcriptional targets of the β-catenin-signaling pathway. (A) Hyperplasia in E13.5 E-catenin–/– mouse brains. Total cells were isolated from E12.5 and E13.5 heterozygous and E-catenin–/– mouse brains and counted using a Coulter Counter. Data represent means ± s.d. n=3-5. *P<0.0001. (B) qPCR analysis of β-catenin pathway transcripts Myc, Ccnd1 (cyclin D1) and Axin2 in E12.5 and E13.5 heterozygous and E-catenin–/– mouse brains. The levels of expression are shown in arbitrary units with mean of levels in heterozygous embryos adjusted to 1. Data represent means ± s.d. n=4.

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