Rapid loss of intestinal crypts upon conditional deletion of the Wnt/Tcf-4 target gene c-Myc

Abstract

Inhibition of the mutationally activated Wnt cascade in colorectal cancer cell lines induces a rapid G1 arrest and subsequent differentiation. This arrest can be overcome by maintaining expression of a single Tcf4 target gene, the proto-oncogene c-Myc. Since colorectal cancer cells share many molecular characteristics with proliferative crypt progenitors, we have assessed the physiological role of c-Myc in adult crypts by conditional gene deletion. c-Myc-deficient crypts are lost within weeks and replaced by c-Myc-proficient crypts through a fission process of crypts that have escaped gene deletion. Although c-Myc(-/-) crypt cells remain in the cell cycle, they are on average much smaller than wild-type cells, cycle slower, and divide at a smaller cell size. c-Myc appears essential for crypt progenitor cells to provide the necessary biosynthetic capacity to successfully progress through the cell cycle.

FIG. 1.

Assessment of c-Myc deletion in β-napthoflavone-induced Cre⁺ Myc^fl/fl intestines over a 5-day time course. (A) β-Galactosidase staining on intestinal whole mounts 3 days p.i. [dpi], indicating near-100% recombination at the Rosa26 reporter allele. Left, Cre⁺ Myc^+/+; right, Cre⁺ Myc^fl/fl. (B and C) c-Myc staining at days 1 and 2 p.i., showing reduced levels of c-Myc protein but still significant numbers of c-Myc-positive cells decorating the crypts. (D to F) Large areas of c-Myc-deficient epithelium at later time points (white arrows). Black arrows in panels E and F depict occasional c-Myc-proficient cells. Note that at later time points c-Myc-proficient cells clustered. (G) Representative section of Cre⁺ Myc^+/+ control mice, where c-Myc protein was uniformly expressed at the lower half of the crypt region.

FIG. 2.

c-Myc-proficient cells in Cre⁺ Myc^fl/fl mice are dominant over c-Myc-deficient cells and form enlarged crypts. (A and E) Representative sections from Cre⁺ Myc^fl/fl mice at 7 and 14 days p.i. [dpi], respectively, stained with anti c-Myc antibody. Enlarged crypts are composed exclusively of c-Myc-proficient cells (black arrows), while the significantly smaller crypts contain cells expressing no c-Myc protein (white arrows). (B and F) Ki-67 staining showed cell proliferation in both c-Myc-proficient and -deficient crypts in Cre⁺ Myc^fl/fl mice (black arrows versus white arrows). Note that c-Myc staining and Ki-67 staining were performed on consecutive sections. (C) Consecutive paraffin section of Cre⁺ Myc^fl/fl mice at 7 days p.i. stained with antibody to β-catenin. The dashed lines depict the position until nuclear β-catenin translocation could be observed. A much higher cell position with nuclear β-catenin from a c-Myc-proficient crypt is also presented as a magnification of the squared area (left lower panel). In contrast, similar cell positions in a Myc-deficient crypt have no nuclear β-catenin (right lower panel). (D) Serial paraffin sections of Cre⁺ Myc^fl/fl mice at 7 days postdeletion stained with antibody to CD44. The CD44 staining pattern follows that of nuclear β-catenin (dashed lines). Expanded CD44 expression in c-Myc-proficient crypts is marked with black arrows. Cells at equivalent positions in c-Myc-deficient crypts have no CD44 expression (white arrows).

FIG. 3.

Myc^fl/fl crypts are lost in Cre⁺ Myc^fl/fl mice after 4 weeks of induction and are replaced by Myc^+/+ crypts through crypt fission. (A to C) LacZ-stained whole-mount preparations from Cre⁺ Myc^fl/fl and control mice at day 5 (A), day 7 (B), and day 28 (C) p.i., indicating preferential repopulation with unrecombined cells in the induced Cre⁺ Myc^fl/fl mice (right intestinal strip in each panel) which was almost complete at 28 days p.i. (D, E, and F) c-Myc staining at 7, 14, and 28 days p.i., respectively, in sections from Cre⁺ Myc^fl/fl mice, indicating repopulation from c-Myc-proficient, hyperplastic crypts through crypt fission (black arrows). (G) Cre⁺ Myc^+/+ control mice. (H) Graph showing reduced cell numbers per crypt in c-Myc-deficient crypts (myc-) from Cre⁺ Myc^fl/fl mice compared to c-Myc-expressing crypts (myc+) of control Cre⁺ Myc^+/+ mice at 7 days p.i. (Cre⁺ Myc^fl/fl, 25 ± 6 [n = 2]; Cre⁺ Myc^+/+, 40 ± 7 [n = 3]; *, P = 0.025 by Mann-Whitney U test). Data are means ± standard deviations; n is the number of mice analyzed. The first bar in the graph represents numbers of cells in c-Myc-proficient crypts (MycP) in Cre⁺ Myc^fl/fl mice (103 ± 24; n = 2). A total of 100 crypts was analyzed per mouse.

FIG. 4.

Size and biosynthetic capacity are reduced in c-Myc-deficient cells. (A) Graph summarizing cell size reduction in c-Myc-deficient cells (myc-). Data were analyzed at two time points after induction, and similar results were obtained (Cre⁺Myc^fl/fl, 459 ± 46 μm², 4 days p.i. [dpi]; Cre⁺ Myc^+/+, 662 ± 61 μm², 4 days p.i.; Cre⁺ Myc^fl/fl, 437 ± 43 μm², 5 days p.i.; Cre⁺ Myc^+/+, 672 ± 53 μm², 5 days p.i., n = 3). The last bar represents the cell size of c-Myc-proficient cells (MycP) of Cre⁺ Myc^fl/fl mice at 7 days p.i. (611 ± 70 μm²; n = 2). Data are means ± standard deviations, and n is the number of mice analyzed per time point. (B) Underrepresentation of AgNor dots, regions of ribosomal gene synthesis, in c-Myc-deficient cells and crypts of Cre⁺ Myc^fl/fl mice at 4 days p.i. (C) Corresponding control Cre⁺ Myc^+/+ mice with multiple large AgNor dots per nucleus. Insets are magnifications of representative squared areas. (D) Sections from Cre⁺Myc^fl/fl mice at 7 days p.i. and incubated with AgNor. Note that at this stage, smaller c-Myc-deficient crypts (white arrows, right-hand magnification inset) clearly have fewer and smaller AgNors compared in the same section with c-Myc-proficient crypts (black arrows, left-hand magnification inset). (E) Consecutive paraffin section stained with c-Myc Ab, showing smaller crypts with fewer AgNors being c-Myc negative (white arrows).

FIG. 5.

Nucleophosmin (B23) protein, involved in ribosomal gene synthesis and a component of AgNor dots, is downregulated in Cre⁺ Myc^fl/fl mice. (A) Northern blot showing downregulation of B23 RNA in Cre⁺ Myc^fl/fl mice (two left lanes, upper panel) compared with Cre⁺ Myc^+/+ mice (two right lanes, upper panel). Lower panel: ethidium bromide-stained 1% denatured agarose gel. (B and C) IHC for B23 protein in mouse small intestine samples. Crypt cells from Cre⁺ Myc^fl/fl mice express almost undetectable levels of B23 protein (white arrows in panel B), and expression in the villus is reduced (black arrows in panel B). Cre⁺ Myc^+/+ control intestine samples show uniform B23 expression throughout crypt villus units (white arrows in panel C). (D) B23 IHC at 14 days p.i. of intestine from Cre⁺ Myc^fl/fl mice, clearly underscoring the difference in B23 levels between c-Myc-deficient (black arrows, left inset) and -proficient crypts (white arrows, right inset). (E) IHC with c-Myc Ab on a consecutive paraffin section, confirming that B23-negative crypts are also c-Myc negative. dpi, days p.i.

FIG. 6.

c-Myc-deficient crypt cells from Cre⁺ Myc^fl/fl mice cycle profoundly slower than c-myc-expressing cells from Cre⁺ Myc^+/+ control mice. (A and B) BrdU time course. Mice at 4 days p.i. (dpi) were injected with BrdU and harvested 2, 12, 24, 36, and 48 h later. BrdU-positive cells were scored as a percentage where the number of positive cells was normalized with the total number of cells per crypt (blue lines) or villus (red lines). Villus BrdU⁺ cells reached maximum numbers at 36 h, while the maximum number for control Cre⁺Myc^+/+ mice was already reached at 24 h. (C and D) IHC to BrdU antibody 24 h postinjection. Significant numbers of BrdU⁺ cells did not exit the crypt in Cre⁺ Myc^fl/fl mice (C, black arrows), while only a few cells decorated the crypts in Cre⁺ Myc^+/+ mice (D, black arrows), with the majority of cells present in the villus (D, white arrows). (E) Graph summarizing the percentage of BrdU-labeled cells in the crypt and villus in Cre⁺ Myc^fl/fl and Cre⁺ Myc^+/+ mice induced for 6 days and pulsed for 24 h. Data are means ± standard deviations, and a minimum of three mice were analyzed per genotype per BrdU time point.