Journal: The Journal of Cell Biology

Article Title: Effects of Forced Expression of an NH 2 -terminal Truncated β-Catenin on Mouse Intestinal Epithelial Homeostasis

doi:

Figure Lengend Snippet: Forced expression of ΔN89β-catenin has no discernible effects on epithelial cell differentiation. Frozen sections were prepared from PLP-fixed jejunums of 6-mo-old chimeric-transgenic animals. ( A ) A polyclonal villus stained with biotin-conjugated Dolichos biflorus agglutinin ( DBA ), Cy3-conjugated avidin, rabbit anti–β-gal, and FITC-conjugated donkey anti–rabbit Ig. Glycoconjugates containing GalNAcα3GalNAc and GalNAcα3Gal recognized by DBA appear yellow-orange; β-gal appears green. The polarity and differentiation of enterocytes appears to be unaffected, as judged by the distribution of these glycoconjugates in apical membranes and supranuclear Golgi apparatus ( closed arrow ). Similarly, based on their reaction with DBA, the number and differentiation of goblet cells ( open arrows ) is equivalent in the 129/Sv and B6-ROSA26 components of the polyclonal villus. ( B ) The base of a polyclonal villus with its crypt-villus junction indicated by closed arrows . Three crypts are seen ( open arrows at their base): the one on the left is supplying cells to another villus. The section was incubated with rat anti-β 4 integrin subunit, Cy3 donkey anti–rat Ig, rabbit anti–β-gal, FITC donkey anti–rabbit Ig, and bis-benzimide. Nuclei ( blue ); the β 4 integrin subunit ( orange ); β-gal ( green-brown ). The location of β 4 integrin at the base of epithelial cells and its distribution along the crypt-villus unit are unaffected by ΔN89β-catenin. ( C ) Villi sectioned perpendicular to their crypt-villus axis. The tight junction protein ZO-1 ( orange ) was detected with rat anti-ZO-1 and Cy3 donkey anti–rat Ig. β-Gal ( green ) was visualized with the same reagents used in the preceding sections. The levels and location of ZO-1 in the 129/Sv(ΔN89β-catenin) and B6-ROSA26 components of polyclonal villi are similar (e.g., open arrows ). ( D ) Villi sectioned perpendicular to their crypt-villus axis as in C . The section was incubated with rat anti-β 7 integrin, Cy3-donkey anti–rat Ig, rabbit anti-laminin, rabbit anti–β-gal, FITC donkey anti–rabbit Ig, and bis-benzimide. B6-ROSA26 cells exhibit diffuse staining of their cytoplasm due to the presence of β-gal ( green ). β 7 integrin is confined to intraepithelial lymphocytes ( orange ). Comparable numbers of these cells are seen in the B6-ROSA26 and 129/ Sv(ΔN89β-catenin) components of polyclonal villi and in wholly 129/Sv(ΔN89β-catenin) villi. Laminin appears as linear green immunoreactivity underlying 129/Sv and B6-ROSA26 epithelium (e.g., closed arrows ). The intensity of staining is similar under cells of both genotypes. Bars, 25 μm.

Article Snippet: PLP-fixed frozen sections of jejunum were stained with a 19-member panel of antibodies: ( a ) affinity-purified rabbit anti– Escherichia coli β-galactosidase (β-gal) (1:500; 5′→ 3′ Inc., Boulder, CO); ( b ) rabbit anti– β-catenin sera (see above, final dilution in PBS/blocking buffer = 1:500); ( c ) affinity-purified rabbit anti–c-myc (see above, 1:100); ( d ) affinity-purified rabbit antibodies raised against amino acids 1034–2130 of human APC (APC2, a gift of P. Polakis; ; ); ( e ) affinity-purified rabbit anti–α-catenin (1:500; gift of J. Nelson); ( f ) a monoclonal rat antibody to E-cadherin (1:1,000; Sigma Chemical Co. ; Hermiston et al., 1995 a ); ( g ) rat anti–ZO-1 (polyclonal antibodies, 1:50; Chemicon International, Inc., Temecula, CA); ( h ) rabbit anti-laminin (1: 1,000; Chemicon International Inc.); ( i ) rabbit anti-mouse fibronectin (1: 1,000; Chemicon International Inc.); ( j ) rabbit anti-mouse collagen type IV (1:1,000; Chemicon International Inc.); ( k ) rat anti-mouse β 1 integrin (1:500; PharMingen , San Diego, CA); ( l ) rat anti-mouse β 7 integrin (1:500; PharMingen ); ( m ) rat anti-mouse β 4 integrin (1:500; PharMingen ); ( n ) rat anti-mouse α 6 integrin (1:500; PharMingen ); ( o ) goat anti-BrdU (1:1,000; ); ( p ) rabbit anti-serotonin (1:1,000, a marker of the predominant enteroendocrine subpopulation in the adult mouse intestine; Incstar, Stillwater, MN); ( q ) rabbit anti-chromagranin A (1:1,000, a general marker of enteroendocrine cells; Incstar); and ( r ) rabbit anti-liver fatty acid binding protein (1:1,000, an enterocyte lineage marker; ).

Techniques: Expressing, Cell Differentiation, Transgenic Assay, Staining, Avidin-Biotin Assay, Incubation