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Results: 5

Figure 4

Figure 4. From: Ets2 regulates colonic stem cells and sensitivity to tumorigenesis.

Increased proliferation at the bottom of crypts from Ets2 deficient mice. (A) DAB reaction (brown) indicates reaction of nuclei with antibody to PCNA. The height of individual crypts (vertical dotted line) was measured and used to divide the crypt into thirds (horizontal dotted lines). The arrow indicated PCNA positive cells in the bottom of crypts from Ets2flox/flox V-Cre (f/f V-Cre+) mice that are not as common in (B) Ets2flox/flox mice without V-Cre (f/f V-Cre−). (C) frequency of PCNA positive cells in the bottom third of colon crypt. Error bar indicates the standard deviation. (D) the number of PCNA positive cells in the middle third of colon crypts is not significantly different between the two genotypes.

Jorge Múnera, et al. Stem Cells. ;29(3):430-439.
Figure 1

Figure 1. From: Ets2 regulates colonic stem cells and sensitivity to tumorigenesis.

Increased representation of Ets2 deficient colons crypts. Representative whole mount images of distal colon areas stained for β-galactosidase. Examples are from areas 1 cm from the anus of 15 day female mice containing V-Cre, the R26R reporter gene and : (A) Ets2+/+; (B) Ets2flox/+; (C) Ets2flox/flox alleles. Note larger patches of blue stained crypts in Ets2flox/flox genotype. (D) Ets2 dependence of β-galactosidase activity of the colon. The colons of 18 day old mice were divided into four portions (proximal, mid-1, mid-2 and distal) homogenized and enzyme activity and protein content was determined. Two animals each of R26R, V-Cre mice with wt Ets2+/+, open bars, 1–2; Ets2flox/+, gray bars, 3–4; or Ets2 flox/flox, black bars, 5–6, were compared.

Jorge Múnera, et al. Stem Cells. ;29(3):430-439.
Figure 2

Figure 2. From: Ets2 regulates colonic stem cells and sensitivity to tumorigenesis.

Ets2 dependence of uniformly populated crypts. (A) representative sections of colon from Ets2 flox/flox, V-Cre, R26R; (B) Ets2flox/+, V-Cre, R26R mice stained for β-galactosidase (blue) and with neutral red. Black bars represent 100 microns. (C) agarose gel analyses of PCR products of approximately 10 pooled individual crypt sections recovered by laser capture microdissection of β-galactosidase positive and negative crypts from Ets2flox/flox, V-Cre, R26R mice. Mouse number from which the sections were obtained is indicated at the top. Ets2 genotype is indicated at right. Size marker are at left in bp. The “colon” sample represents whole tissue control. Ets2flox is recombined by Cre to generate the Ets2db2 allele in β-galactosidase positive crypts. (D) frequency of uniformly blue crypts was measured in sections of animals of different ages. The values represent the averages of at least 85 scored crypts from two sections of at least two mice of each genotype and age except for Ets2+/+ at 30 days and Ets2flox/+ at 90 days for which only one mouse was available. Two tailed Student t-test indicated the results for mice with the Ets2flox/flox genotype was significantly different from Ets2flox/+ or Ets2+/+.

Jorge Múnera, et al. Stem Cells. ;29(3):430-439.
Figure 3

Figure 3. From: Ets2 regulates colonic stem cells and sensitivity to tumorigenesis.

Crypt fission is increased in Ets2 deficient colons. Sections of V-Cre, R26R mice of varying Ets2 genotype and age stained for β-galactosidase were scored for bifurcating crypts. (A-C) examples of crypts in fission for each genotype at postnatal day 15 (P15). (D) frequency of dividing crypts as a function of age. For each measurement, at least 86 well oriented crypts were scored. Error bars represent the standard deviation. The numbers of mice analyzed are the same as for Figure 2. (E) increased fission of Ets2 deficient crypts. Only blue crypts were scored at 15 and 30 days. F, crypt patch size increases with age in Ets2flox/flox but not in Ets2flox/+ mice. Patches of two or more adjacent crypts were measured. In Ets2flox/flox mice, patch sizes is greater after 15 day. Patch size of Ets2flox/+. Patch size differences within each genotype were measured using a one-way ANOVA test. For Ets2flox/flox mice there was a significant difference in patch sizes within the group (p<0.0001). Patch size in Ets2flox/+ mice was not significantly different with age as revealed by a one-way ANOVA test (p=0.25). ns = not significant. Differences in patch size between the two genotypes was determined by Mann-Whitney test (p<0.0001).

Jorge Múnera, et al. Stem Cells. ;29(3):430-439.
Figure 5

Figure 5. From: Ets2 regulates colonic stem cells and sensitivity to tumorigenesis.

Adenoma formation in mice treated with azoxymethane and dextra sodium sulfate. Multiplicity but not size is increased in Ets2flox/flox, V-Cre mice compared to Ets2flox/+ V-Cre and Ets2flox/flox mice following the 9 week AOM/DSS regimen. (A) Representive hematoxylin and eosin stained section of colon tumors from an Ets2flox/flox V-Cre, scale bar = 500 microns. (B) Comparison of average number of tumors per mouse from AOM/DSS treated mice of the Ets2flox/flox, V-Cre, Ets2flox/+, V-Creand Ets2flox/flox genotypes. The solid black column labeled db2/db2 represents the Ets2flox/flox, V-Cre group adusted for the 30% of the crypts that do not express Cre at the age of AOM administration (Figure 2). N represents number of mice examined. Results of student T-test: * indicates P < 0.05, *** indicate P=0.001, ns, not significant. (C) increased sensitivity of Ets2A72/A72 mice to AOM/DSS induced tumors. Mice were examined 19 weeks after AOM/DSS administration. (D) tumor number of individual mice. Each of the mice is ranked by the number of tumors found. (E) Schematic diagram of possible relationships of Ets2 and Wnt signaling pathway components. Ets2 may be both a direct target of β-catenin/TCF4 and downstream of Ascl2. Activation of Ets2 by Ras occurs though phosphorylation of Thr72 by Erk. P16 has been suggested to be a target of Ets2 in primary cells. Cdx2 is a direct target of Ets2 in trophoblast cells and is decreased in Ets2 deficient mouse colon.

Jorge Múnera, et al. Stem Cells. ;29(3):430-439.

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