Loss of Apc in vivo immediately perturbs Wnt signaling, differentiation, and migration

Abstract

Although Apc is well characterized as a tumor-suppressor gene in the intestine, the precise mechanism of this suppression remains to be defined. Using a novel inducible Ahcre transgenic line in conjunction with a loxP-flanked Apc allele we, show that loss of Apc acutely activates Wnt signaling through the nuclear accumulation of beta-catenin. Coincidentally, it perturbs differentiation, migration, proliferation, and apoptosis, such that Apc-deficient cells maintain a "crypt progenitor-like" phenotype. Critically, for the first time we confirm a series of Wnt target molecules in an in vivo setting and also identify a series of new candidate targets within the same setting.

Figure 1.

Changes in intestinal crypt pathology 5 d after the first injection of the cre-inducing agent β-napthoflavone. (a,b). Hematoxylin-and-eosin-stained sections for control, induced Cre⁺Apc^+/+ (a), and induced Cre⁺Apc^fl/fl (b) mice, showing an enlarged crypt-like region in the induced Cre⁺Apc^fl/fl mice. (c,d) Apc immunofluorescence in induced Cre⁺Apc^+/+ (c) and in induced Cre⁺Apc^fl/fl (d) mice. Loss of Apc protein was observed in all morphologically atypical cells (below the arrow) in Cre⁺Apc^fl/fl mice. Apc staining was observed in unrecombined morphologically normal cells in the villus (above the arrow). (e,f) Wholemount preparations of intestines stained for lacZ activity to report cre-mediated recombination at the Rosa26R locus. Recombined control cells completely repopulate the crypt–villus axis (e) but fail to do so in the absence of Apc (f). (g) Sectioned material from f confirming that the pattern of recombination scored by LacZ activity directly overlays the pattern of histological change. Note that the staining for β-galactosidase exactly complements the Apc staining, with only the morphologically atypical cells (below the arrows) staining for β-galactosidase. For all panels, arrows indicate the point of demarcation between normal and atypical histology.

Figure 2.

Perturbation of differentiation in the absence of Apc. (a,b) Immunohistochemical staining for alkaline phosphatase in control, induced Cre⁺Apc^+/+ mice (a) and induced Cre⁺Apc^fl/fl intestines (b). Apc mutant cells do not express the villus marker alkaline phosphatase, with the arrow indicating demarcation between the residual wild-type and recombined Apc-deficient cells. (c,d) Staining for Goblet cells (blue cells stained with alcian blue) and Paneth cells (red cells stained with Pokeweed lectin) in the crypt–villus axis of control induced Cre⁺Apc^+/+ mice (c) and induced Cre⁺Apc^fl/fl intestines (d). Insets show detail of crypts. No Goblet or Paneth cell staining was observed in the induced Cre⁺Apc^fl/fl mice. (e,f) Staining for lysozyme in induced Cre⁺Apc^+/+ (e) and induced Cre⁺Apc^fl/fl (f) mice. Lyzozyme positivity is confined to the base of the crypt in the controls but is distributed throughout the crypt-like region in the induced Cre⁺Apc^fl/fl mice. (g,h) Mcm2 staining with positivity restricted to the crypt within the induced Cre⁺Apc^+/+ mice (g) but extending through the aberrant crypt structure in the induced Cre⁺Apc^fl/fl mice (h).

Figure 3.

Altered proliferation, migration, and apoptosis in the absence of Apc. (a–f) BrdU staining in control and Apc-deficient intestinal epithelium. (a) BrdU-positivite cells in wild-type-induced Cre⁺Apc^+/+ crypts 2 h following BrdU injection are confined to a proliferative zone within the crypt. (b) The position of BrdU-positive cells in crypt–villus axis of control, induced Cre⁺Apc^+/+ mice 24 h after BrdU injection, indicating migration of labeled cells onto the villus. (c) BrdU staining in induced Cre⁺Apc^fl/fl epithelium 2 h after BrdU injection show that positively stained cells are present throughout the entire aberrant crypt-like structure. (d) BrdU staining 24 h after injection of BrdU in induced Cre⁺Apc^fl/fl intestinal epithelium. (e) Position of BrdU-positive cells within the control crypt–villus axis at 2 h (black bars) and 24 h (open bars). Position 0 represents the base of the crypt. Note the increase in position at 24 h, reflecting migration. (f) Position of BrdU cells within the crypt-like structure in induced Cre⁺Apc^fl/fl mice at 2 h (black bars) and 24 h (open bars). Unlike in the controls, no migration of labeled cells is evident. (g,h) Increased apoptosis in Apc mutant crypts. Immunohistochemical staining of active caspase 3 in induced Cre⁺Apc^+/+ mice (g) and induced Cre⁺Apc^fl/fl mutant crypts (h). This staining confirmed apoptosis counts from H&E-stained sections that showed significantly increased apoptosis in Apc deficient crypts (11.1% ± 1.2%) compared with control crypts (3.5% ± 0.45%; p = 0.01; Mann Whitney, n = 5).

Figure 4.

(a) Immunoblasts show the levels of Apc, β-catenin, and dephosphorylated β-catenin. Equal amounts of protein from induced control Cre⁺Apc^+/+, uninduced Cre⁺Apc^fl/fl, and induced Cre⁺Apc^fl/fl mice were separated on a 4%–12% gradient gel and probed with antibodies against Apc, β-catenin, actin, and dephospho-β-catenin as shown. Probing with dephospho-β-catenin was performed first and the blot stripped before it was reprobed with the antibody that detects all β-catenin so that these two images reflect identical samples. No image processing was performed on the films, and they are shown in their original appearance. Although the total amount of β-catenin is not grossly different between the different samples, the amount of dephosphorylated β-catenin is increased after inactivation of Apc in the tissue obtained from induced Cre⁺Apc^fl/fl mice, whereas detectable Apc levels drop significantly. (Lanes 1,2) Induced Cre⁺Apc^+/+. (Lane 3) Noninduced Cre⁺Apc^fl/fl. (Lanes 4–6) Induced Cre⁺Apc^fl/fl. The migration of proteins with molecular weights of 220, 160, 120, 100, 90, 80, 60, 50, 40, 30, and 20 kDa (top) and 120, 100, 90 80, and 50 kDa (bottom) are indicated to the left of the blots. (b,c) Increased nuclear β-catenin staining in crypts. (b) β-catenin staining in control crypts 5 d after the first of four inductions with β-napthoflavone, showing nuclear staining confined to the cells at base of the crypts. (c) Nuclear β-catenin staining observed throughout the whole of the aberrant structure in induced Cre⁺Apc^fl/fl mice 5 d after induction. (d,e) Immunohistochemical confirmation of c-Myc up-regulation. (d) Staining for c-Myc in control Cre⁺Apc^+/+ intestine at day 5. The inset shows crypt detail; note the absence of nuclear staining. (d) Nuclear up-regulation of c-Myc in the induced Cre⁺Apc^fl/fl mice, with inset showing crypt detail. (f,g) Immunohistochemical confirmation of CD44 up-regulation. (f) No detectable CD44 staining in control Cre⁺Apc^+/+ intestines at day 5. (g) Up-regulation of CD44 in the induced Cre⁺Apc^fl/fl mice. The inset shows detail.

Figure 5.

Perturbation of the EphB/ephrinB system. (a) The pattern of EphB2 staining in crypt enterocytes, predominantly confined to the lower half of control crypts. (b) EphB2 staining throughout the aberrant crypt structure in the induced Cre⁺Apc^fl/fl mice. Arrow denotes leading edge of aberrance, where unrecombined villus cells with wild-type morphology do not stain for EphB2. (c) EphB3 staining confined to the base of control crypts. (d) EphB3 staining throughout the aberrant crypt structure in the induced Cre⁺Apc^fl/fl mice. (e,f) Ephrin B1 and B2 staining respectively of villus cells within control intestine. (g) Lack of staining for Ephrin B2 within the aberrant cells in the induced Cre⁺Apc^fl/fl mice. Arrow denotes leading edge of aberrance, where unrecombined villus cells stain for Ephrin B2.