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First published online January 11, 2008
doi: 10.1242/10.1242/dev.007047

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Zeb1 links epithelial-mesenchymal transition and cellular senescence

¹ James Graham Brown Cancer Center, Department of Ophthalmology and Visual Sciences, University of Louisville Health Sciences Center, Louisville, KY 40202, USA.
² Departments of Peiodontics, Endodontics and Dental Hygiene, Center for Oral Health and Systemic Disease, University of Louisville School of Dentistry, Louisville, KY 40292, USA.
³ Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.

View larger version (98K): [in this window] [in a new window]   Fig. 1. E-cadherin becomes ectopically expressed, whereas vimentin is diminished in palate and nasal mesenchyme in Zeb1 mutant mice. (A) Zeb1 immunostaining is shown in mesenchyme in the palate (P) and developing nasal cartilage (N). (B) E-cadherin immunostaining is present on the nasal (N), palatal (P) and tongue (T) epithelium. (C) Vimentin immunostaining is shown in palatal and nasal mesenchyme. (D) E-cadherin immunostaining is seen ectopically on nasal and palatal mesenchyme in Zeb1-null mice. Note the failure of palate closure. The arrow indicates mesenchyme in the nasal region. (E) Loss of vimentin immunostaining in the palatal and nasal mesenchyme of Zeb1-null mice. Sections of mice at E16.5 are shown. Scale bars: 100 µm.

View larger version (86K): [in this window] [in a new window]   Fig. 2. Mutation of Zeb1 leads to ectopic expression of E-cadherin, loss of vimentin, and defective proliferation in the perichondrial region of forming cartilage. (A) Immunostaining showing expression of Zeb1 in the perichondrium. (B) Immunostaining for vimentin in the perichondrium. (C) E-cadherin immunostaining is seen in the skin (arrow) but not in underlying mesenchymal cells. (D) Immunostaining for BrdU incorporation into the perichondrium. (E,F) Double immunolabeling for Zeb1 and BrdU in the perichondrium. (G) Overlay of E and F. (H) Diminished immunostaining for vimentin in the perichondrium of Zeb1-null mice. (I) Ectopic immunostaining of E-cadherin in the perichondrium of Zeb1-null mice. Arrow indicates skin. (J) Loss of BrdU immunostaining in the perichondrium of Zeb1-null mice. Sections of mice at E15.5 are shown. C, cartilage. Scale bars: 50 µm.

View larger version (105K): [in this window] [in a new window]   Fig. 3. Ectopic E-cadherin expression and diminished vimentin and GFAP expression in the embryonic eye of Zeb1-null mice. (A) Immunostaining for Zeb1. Retina (R); forming eye muscles (M); lens (L); cornea (C); eye lid (EL). (B) E-cadherin immunostaining is confined to the epithelium of the lens and cornea. (C) Vimentin immunostaining in the retina, lens, optic nerve (ON) and forming eyes muscles. (D) GFAP immunostaining in the retina, optic nerve and forming eye muscles. (E) Ectopic immunostaining for E-cadherin in the retina, optic nerve and forming eye muscle in Zeb1-null mice. (F) Double immunostaining for vimentin (red) and GFAP (green) shows loss of expression of both proteins in the retina, optic nerve and forming eye muscle in Zeb1-null mice. Sections of mice at E16.5 are shown. Scale bars: 100 µm.

View larger version (117K): [in this window] [in a new window]   Fig. 4. Mutation of Zeb1 leads to ectopic expression of E-cadherin, loss of vimentin and GFAP expression, and decreased proliferation in the ventricular zone of the brain. (A) Immunostaining for Zeb1 in the ventricular zone (arrow) of the lateral ventricle. (B,C) Immunostaining for vimentin and GFAP, respectively, in the ventricular zone. (D) Immunostaining for both vimentin (red) and GFAP (green) is lost in the ventricular zone of Zeb1 mutant mice. (E) Immunostaining for E-cadherin is not evident in the lateral ventricle in wild-type mice. (F,G) Ectopic immunostaining for E-cadherin in the ventricular zone of the lateral ventricle in Zeb1-null mice. (H) Immunostaining for E-cadherin in the third ventricle in Zeb1-null mice. (I) BrdU immunostaining in the ventricular zone of the lateral ventricle. (J,K) Double immunolabeling of the boxed region in I for BrdU and Zeb1, respectively. (L) Overlay of J and K. (M) Quantitation of BrdU incorporation into the ventricular zone of the left lateral ventricle (LLV), right lateral ventricle (RLV), hypothalamus (Hypo.) and, as a control, the tongue. For a representative view of areas counted, see Fig. S3 in the supplementary material. Sections at E15.5 are shown. Scale bars: 100 µm in E,F,I; 50 µm in A-D,G,H; 25 µm in J-L.

View larger version (54K): [in this window] [in a new window]   Fig. 5. E-cadherin is ectopically expressed on embryo fibroblasts from Zeb1 mutant mice, and the cells display an abnormal epithelial-like morphology. Left-hand and middle panels show light micrographs of cells at different time points after isolation from wild-type (+/+) and Zeb1 mutant (-/-) mice. Note the increase in cells with epithelial-like morphology; such clusters of epithelial-like cells were not evident in the wild-type population. Right-hand panels show immunostaining for E-cadherin. Scale bars: 25 µm.

View larger version (13K): [in this window] [in a new window]   Fig. 6. Effect of Zeb1 mutation on expression of Zeb2 and Snail1/2 mRNAs, and TGF-β-dependent induction of vimentin and repression of E-cadherin mRNAs. (A) Real-time PCR analysis shows that TGF-β is unable to repress E-cadherin mRNA levels in Zeb1-null MEFs. Concentrations of TGF-β are shown. No E-cadherin mRNA was detected in wild-type cells (see also Fig. 8B below). (B) Real-time PCR was used to assess the effect of Zeb1 mutation on expression of other E-box-binding repressor mRNAs (Zeb2, Snail1 and Snail2) in MEFs. Results were normalized to β-actin and Gapdh mRNA, with similar results. (C) Real-time PCR analysis shows that basal vimentin mRNA expression is unaffected by Zeb1 mutation, but TGF-β induction is lost in Zeb1-null MEFs. (D) Real-time PCR shows that the basal plasminogen activator inhibitor 1 (PAI-1) mRNA level is unaffected by Zeb1 mutation, and it remains inducible by TGF-β in Zeb1-null MEFs.

View larger version (16K): [in this window] [in a new window]   Fig. 7. MEFs from Zeb1 mutant mice undergo gene dosage-dependent premature replicative senescence. (A) Effect of Zeb1 mutation on MEF proliferation. (B) Micrographs of cells at early versus later passage numbers. Note the appearance of large, flat senescent-like cells associated with growth arrest. (C) Arrested Zeb1 mutant cells express senescent β-galactosidase (SA-β-Gal; blue X-Gal staining).

View larger version (21K): [in this window] [in a new window]   Fig. 8. Mutation of Zeb1 does not trigger the classic Ink4A replicative senescence pathway in MEFs; instead, it is associated with induction of p15Ink4b and p21Cdkn1a. (A) Real-time PCR was used to compare mRNA expression from the Ink4a locus (p16Ink4a and Arf), and genes known to regulate the Ink4a locus (Ets1, Bmi1, Tbx2, Tbx3), in proliferating wild-type MEFs (P3) and senescent Zeb1 heterozygous (P5) and null (P2) cells. (B) p15Ink4b, p21Cdkn1a and E-cadherin mRNAs are induced in a gene dosage-dependent fashion in Zeb1 mutant MEFs. Real-time PCR results using the same samples as in A are shown. (C) ChIP assays. Input, starting chromatin used for the immunoprecipitations; IgG, preimmune serum. (D) Control ChIP assay showing that Zeb1 does not bind to the Gapdh promoter. Histone H3 and H4 are positive controls for binding to the Gapdh promoter. (E) Real-time PCR quantification of the results in C and D. The same input DNA was used for each ChIP assay, and the relative input value is set at 100. Primers for the promoters are shown in Table 2.

View larger version (73K): [in this window] [in a new window]   Fig. 9. p15Ink4b is ectopically expressed in the perichondrium, on forming cartilage, and in the ventricular zone of the lateral ventricle in Zeb1-null mice. (A,C) Immunostaining for p15Ink4b on forming cartilage. (B,D) Overlay of the immunostaining in A and C with a Nomarski image. (E,F) Immunostaining for p15Ink4b in the ventricular zone of the lateral ventricle. (G,H) Immunostaining for BrdU and p15Ink4b, respectively, in the ventricular zone of the lateral ventricle. (I) Overlay of G and H. Immunostaining of E15.5 embryos is shown. Scale bars: 50 µm.

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