alphaE-catenin is not a significant regulator of beta-catenin signaling in the developing mammalian brain - PubMed (original) (raw)

. 2008 May 1;121(Pt 9):1357-62.

doi: 10.1242/jcs.020537. Epub 2008 Apr 8.

Affiliations

alphaE-catenin is not a significant regulator of beta-catenin signaling in the developing mammalian brain

Wen-Hui Lien et al. J Cell Sci. 2008.

Abstract

beta-Catenin is a crucial mediator of the canonical Wnt-signaling pathway. alpha-catenin is a major beta-catenin-binding protein, and overexpressed alpha-catenin can negatively regulate beta-catenin activity. Thus, alpha-catenin may be an important modulator of the Wnt pathway. We show here that endogenous alpha-catenin has little impact on the transcriptional activity of beta-catenin in developing mammalian organisms. We analyzed beta-catenin signaling in mice with conditional deletion of alphaE-catenin (Ctnna1) in the developing central nervous system. This mutation results in brain hyperplasia and we investigated whether activation of beta-catenin signaling may be at least partially responsible for this phenotype. To reveal potential quantitative or spatial changes in beta-catenin signaling, we used mice carrying a beta-catenin-signaling reporter transgene. In addition, we analyzed the expression of known endogenous targets of the beta-catenin pathway and the amount and localization of beta-catenin in mutant progenitor cells. We found that although loss of alphaE-catenin resulted in disruption of intercellular adhesion and hyperplasia in the developing brain, beta-catenin signaling was not altered. We conclude that endogenous alphaE-catenin has no significant impact on beta-catenin transcriptional activities in the developing mammalian brain.

PubMed Disclaimer

Figures

Fig. 1

Fig. 1

Depletion of αE-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 αE-catenin−/− (knockout, KO) brains were immunoprecipitated with control (IgG) or anti-β-catenin (β-cat) antibodies and resulting protein complexes were separated by SDS-PAGE and stained with Colloidal blue and Silver stain (A) or analyzed by Western blot with anti-αE-catenin, N-cadherin or β-catenin antibodies (B). Note that while αE-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 association between β-catenin and N-cadherin. (C-D″) Despite disruption of apical junctional complexes and loss of cell polarity, β-catenin continues to co-localize with N-cadherin at the periphery of αE-catenin−/− neural progenitor cells. Cortical sections from E13.5 wild-type (WT) and _αE-catenin_−/− (KO) embryos were stained with anti-N-cadherin (N-cad) and anti-β-catenin (β-cat) antibodies. Bar in C represents 15.9 μm.

Fig. 2

Fig. 2

Depletion of αE-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 _αE-catenin_−/− (KO) brains were analyzed by Western blotting with anti-αE-catenin, anti-αN-catenin, β-catenin and β-actin antibodies. Quantitation 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 ± SD (n=3). (B) 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. 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) brains. HI – developing hippocampus. Bar in B represents 0.12 mm for B, C and 0.012 mm for B′–C″.

Fig. 3

Fig. 3

Endogenous reporter for β-catenin transcriptional activity reveals no changes in _αE-catenin_−/− 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 the lacZ gene. (B-C′) Similar pattern of β-catenin reporter expression in the telencephalon of wild-type (WT) and _αE-catenin_−/− (KO) embryos. Sagital 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. Bar in B represents 0.19 mm in BB′ and 0.26 mm in C-C′. (D) Similar levels of β-catenin reporter expression between wild-type (WT) and αE-catenin−/− (KO) brains. Total protein lysates from E12.5 and E13.5 telencephalons of TOPGAL positive animals were analyzed by Western blotting with anti-β-galactosidase (β-gal) and anti-beta;-actin antibodies. Con - control samples from TOPGAL-negative animals. The numbers indicate the relative amounts of β-gal adjusted by the levels of β-actin.

Fig. 4

Fig. 4

Transition from E12.5 to E13.5 results in extensive hyperplasia without significant changes in expression of endogenous transcriptional targets of β-catenin signaling pathway. (A) Hyperplasia in E13.5 αE-catenin−/− brains. Total cells were isolated from E12.5 and E13.5 wild-type (WT) and _αE-catenin_−/− brains and counted using Coulter Counter. Data represent means ± SD. n=3 to 5. Asterisk indicates statistically significant difference with P<0.0001. (B) qPCR analysis of β-catenin pathway transcripts c-myc, Cyclin D1, Axin2 in E12.5 and E13.5 heterozygous and αE-catenin−/− brains. The levels of expression are shown in arbitrary units with mean of levels in heterozygous embryos adjusted to one. Data represent means ± SD. n=4.

References

    1. Behrens J, Jerchow BA, Wurtele M, Grimm J, Asbrand C, Wirtz R, Kuhl M, Wedlich D, Birchmeier W. Functional interaction of an axin homolog, conductin, with beta-catenin, APC, and GSK3beta. Science. 1998;280:596–9. - PubMed
    1. Behrens J, von Kries JP, Kuhl M, Bruhn L, Wedlich D, Grosschedl R, Birchmeier W. Functional interaction of beta-catenin with the transcription factor LEF-1. Nature. 1996;382:638–42. - PubMed
    1. Brembeck FH, Schwarz-Romond T, Bakkers J, Wilhelm S, Hammerschmidt M, Birchmeier W. Essential role of BCL9-2 in the switch between beta-catenin’s adhesive and transcriptional functions. Genes Dev. 2004;18:2225–30. - PMC - PubMed
    1. Chenn A, Walsh CA. Regulation of cerebral cortical size by control of cell cycle exit in neural precursors. Science. 2002;297:365–9. - PubMed
    1. Clevers H. Wnt/beta-catenin signaling in development and disease. Cell. 2006;127:469–80. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources