Apoptosis in neural crest cells by functional loss of APC tumor suppressor gene

Abstract

Apc is a gene associated with familial adenomatous polyposis coli (FAP) and its inactivation is a critical step in colorectal tumor formation. The protein product, adenomatous polyposis coli (APC), acts to down-regulate intracellular levels of beta-catenin, a key signal transducer in the Wnt signaling. Conditional targeting of Apc in the neural crest of mice caused massive apoptosis of cephalic and cardiac neural crest cells at about 11.5 days post coitum, resulting in craniofacial and cardiac anomalies at birth. Notably, the apoptotic cells localized in the regions where beta-catenin had accumulated. In contrast to its role in colorectal epithelial cells, inactivation of APC leads to dysregulation of beta-catenin/Wnt signaling with resultant apoptosis in certain tissues including neural crest cells.

Figure 1

Craniofacial and cardiac defects in mutant mice (P0-Cre/Apc^580S/580S). (A and B) Lateral (A) and frontal (B) views of a wild-type littermate (Left) and a mutant (Right) at birth. (C) Lateral views of faces of a wild-type littermate (Left) and a mutant (Right). (D) Lateral views of skeletal preparations of a P0-Cre/Apc^580S/+ mouse (Left) and a mutant (Right) at 18.5 dpc. Part of the frontal bone is abnormal in mutants (arrow). fr, frontal; pr, parietal; eo, exoccipital; md, mandibullar bone. (E) Ventral views of skeletal preparations of a P0-Cre/Apc^580S/+ mouse (Left) and a mutant (Right) at 18.5 dpc. n, nasal; mx, maxillary; j, jugal; ps, presphenoid; bs, basisphenoid; as, alisphenoid; tr, tympanic ring; bo, basioccipital bone. (F) Coronal sections of the trunk of P0-Cre/Apc^580S/+ mice and mutants at 15.5 dpc. A ventricular septal defect (VSD) is shown in the heart of a mutant (arrow in Upper Right). The aorta and pulmonary artery are still adjoined and the pulmonary artery comes directly from the truncus arteriosus (arrow in Lower Right) in another mutant. RV, right ventricle; LV, left ventricle; Ao, aorta; PA, pulmonary artery. [Scale bars, 1 mm (A–E) and 0.5 mm (F).]

Figure 2

Embryonal development of mutant and P0-Cre/Apc^580S/+mice. Appearance of P0-Cre/Apc^580S/+ embryos (A–C) and mutants (D–F) at various developmental stages indicated below the panels. Embryos at 9.5 and 11.5 dpc were stained with X-gal. In 11.5 dpc P0-Cre/Apc^580S/+ embryos, frontonasal mass, trigeminal ganglia, branchial arches, and DRG were positive for LacZ. Fnm, frontonasal mass; Tg, trigeminal ganglia; Ba, branchial arch; Ot, otocyst; DRG, dorsal root ganglia. (Scale bars, 0.5 mm.)

Figure 3

Apoptosis and β-catenin accumulation in neural crest-derived tissues of mutant mice. Cre expression confirmed by LacZ staining (A–F), β-catenin immunoreactivity (G–L), and TUNEL stains (M–R) of the sections of ventral craniofacial mesenchyme (VCM), branchial arch (BA), and cardiac outflow tract (COT) from mutant and P0-Cre/Apc^580S/+ embryos at 11.5 dpc are shown. Apoptotic cells are clustered in the branchial arch and cardiac outflow tract in one mutant (arrows in P and R). The number of apoptotic cells in VCM, BA, and COT of P0-Cre/Apc^580S/+ mice was 32.4 ± 5.9, 6.2 ± 3.0, and 30.6 ± 19.1, respectively, and in mutants was 107.6 ± 15.8, >200, and 65.4 ± 21.4, respectively (mean ± SD, n = 3). β-catenin accumulated in TUNEL-positive cell-clustered regions (arrows in J and L). (Scale bars, 0.1 mm.)

Figure 4

Collagen type 2a1-positive cells are lost and enter into apoptosis in the first branchial arch in the mutant mice. In situ hybridization for β-catenin, collagen type 2a1 (Col2a1), and MyoD on sections of the first branchial arch of wild-type (Left) and mutant mice (Right). (Scale bars, 0.1 mm.)

Figure 5

Apoptosis and morphological appearance of Schwann cells and DRG in mutant mice. (A) Peripheral nerves in the legs of wild-type (Left) and mutant mice (Right) at 15.5 dpc. The number of Schwann cells in peripheral nerves at 18.5 dpc is 34.0 ± 11.5 in wild-type mice and 37.5 ± 1.9 in the mutants (mean ± SD, n = 3). (B) LacZ expression (Cre expression) and TUNEL in 11.5 dpc DRG. The number of LacZ-positive cells per DRG is 176.5 ± 23.9 in heterozygotes, and 50.1 ± 27.7 in the mutants (mean ± SD, n = 3). The number of apoptotic cells per DRG is 8.2 ± 5.6 in heterozygotes, and 19.9 ± 9.9 in mutants (mean ± SD, n = 3). (C) Whole views of DRG from 15.5 dpc wild-type (Left) and mutant mice (Right). The number of DRG neurons at 18.5 dpc is 116.7 ± 10.8 in wild-type and 105.8 ± 14.7 in the mutant mice (mean ± SD, n = 3). [Scale bars, 25 μm (A) and 50 μm (B and C).]

Figure 6

Effects of p53 deficiency on phenotypes of conditional mutants. (A) Frontal (Left) and lateral (Right) views of a P0-Cre/Apc^580S/580S/p53^+/+ (Upper) and a P0-Cre/Apc^580S/580S/p53^−/− (Lower) at birth. (B and C) β-catenin accumulation (Left) and TUNEL (Right) in branchial arch (B) and cardiac outflow tract (C) of mutant mice on p53 heterozygous (Upper) or homozygous (Lower) background. Yellow arrows indicate accumulation of β-catenin. (Insets) Magnified views of apoptotic lesions (yellow box). The number of TUNEL-positive cells within the region where β-catenin was accumulated in branchial arch is 48.4 ± 1.9 in P0-Cre/Apc^580S/580S/p53^+/− and 50.7 ± 4.7 in P0-Cre/Apc^580S/580S/p53^−/− (mean ± SD, n = 2, P < 0.05). In cardiac outflow tract, the number is 96.7 ± 1.9 in P0-Cre/Apc^580S/580S/p53^+/− and 76.2 ± 7.3 in P0-Cre/Apc^580S/580S/p53^−/− (mean ± SD, n = 2, P < 0.05). [Scale bars, 1 mm (A) and 0.25 mm (B and C).]