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The conditional inactivation of the beta-catenin gene in endothelial cells causes a defective vascular pattern and increased vascular fragility.

Cattelino A, Liebner S, Gallini R, Zanetti A, Balconi G, Corsi A, Bianco P, Wolburg H, Moore R, Oreda B, Kemler R, Dejana E - J. Cell Biol. (2003)

Bottom Line: We found that early phases of vasculogenesis and angiogenesis were not affected in mutant embryos; however, vascular patterning in the head, vitelline, umbilical vessels, and the placenta was altered.These changes paralleled a decrease in cell-cell adhesion strength and an increase in paracellular permeability.We conclude that in vivo, the absence of beta-catenin significantly reduces the capacity of endothelial cells to maintain intercellular contacts.

View Article: PubMed Central - PubMed

Affiliation: FIRC Institute of Molecular Oncology, 16-20139, Milan, Italy.

ABSTRACT
Using the Cre/loxP system we conditionally inactivated beta-catenin in endothelial cells. We found that early phases of vasculogenesis and angiogenesis were not affected in mutant embryos; however, vascular patterning in the head, vitelline, umbilical vessels, and the placenta was altered. In addition, in many regions, the vascular lumen was irregular with the formation of lacunae at bifurcations, vessels were frequently hemorrhagic, and fluid extravasation in the pericardial cavity was observed. Cultured beta-catenin -/- endothelial cells showed a different organization of intercellular junctions with a decrease in alpha-catenin in favor of desmoplakin and marked changes in actin cytoskeleton. These changes paralleled a decrease in cell-cell adhesion strength and an increase in paracellular permeability. We conclude that in vivo, the absence of beta-catenin significantly reduces the capacity of endothelial cells to maintain intercellular contacts. This may become more marked when the vessels are exposed to high or turbulent flow, such as at bifurcations or in the beating heart, leading to fluid leakage or hemorrhages.

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Cre recombinase efficiently recombines the β-catenin flox allele in vivo. (A) Genomic PCR on E10.5 embryos from Tie2-Cre x β-catenin flox/flox crossings for the β-catenin gene and for Tie2-Cre gene. The four lanes represent the four different genotypes obtained: β-catenin flox del/flox, Cre− (+/−, lane 1), β-catenin flox del/flox, Cre+ (−/−, lane 2), β-catenin flox/wt, Cre− (+/+, lane 3), and β-catenin flox/wt, Cre+ (+/−, lane 4). In this last lane, note the presence of the recombined β-catenin flox del band when the flox allele is in the presence of Tie2-Cre recombinase. (B) Western blot of β-catenin protein in endothelial cell lines established from wt (control) and β-catenin flox/flox del, Tie2-Cre+ embryos (−/−). (C-J) Immunofluorescence for anti-PECAM and anti-β-catenin antibodies on serial cryosections from wild-type (+/+, control) and β-catenin flox/flox del, Tie2-Cre+ (−/−, mutant) E10.5 embryos. Heart sections show positive staining of the endocardium (e) with PECAM antibodies in both control (C) and β-catenin −/− (E) animals. β-Catenin staining is undetectable in mutant embryos (F). β-Catenin is also absent in the dorsal aorta (G and H, Ao) and in the yolk sac vessels (I and J, arrow) of the mutant embryos. Bar in H refers to C–H, and bar in J refers to I and J.
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fig1: Cre recombinase efficiently recombines the β-catenin flox allele in vivo. (A) Genomic PCR on E10.5 embryos from Tie2-Cre x β-catenin flox/flox crossings for the β-catenin gene and for Tie2-Cre gene. The four lanes represent the four different genotypes obtained: β-catenin flox del/flox, Cre− (+/−, lane 1), β-catenin flox del/flox, Cre+ (−/−, lane 2), β-catenin flox/wt, Cre− (+/+, lane 3), and β-catenin flox/wt, Cre+ (+/−, lane 4). In this last lane, note the presence of the recombined β-catenin flox del band when the flox allele is in the presence of Tie2-Cre recombinase. (B) Western blot of β-catenin protein in endothelial cell lines established from wt (control) and β-catenin flox/flox del, Tie2-Cre+ embryos (−/−). (C-J) Immunofluorescence for anti-PECAM and anti-β-catenin antibodies on serial cryosections from wild-type (+/+, control) and β-catenin flox/flox del, Tie2-Cre+ (−/−, mutant) E10.5 embryos. Heart sections show positive staining of the endocardium (e) with PECAM antibodies in both control (C) and β-catenin −/− (E) animals. β-Catenin staining is undetectable in mutant embryos (F). β-Catenin is also absent in the dorsal aorta (G and H, Ao) and in the yolk sac vessels (I and J, arrow) of the mutant embryos. Bar in H refers to C–H, and bar in J refers to I and J.

Mentions: To specifically inactivate the β-catenin gene in endothelial cells, mice containing a floxed β-catenin allele were intercrossed with transgenic mice expressing Cre under the control of Tie2 endothelial-specific promoter (Schlaeger et al., 1997; Brault et al., 2001; Kisanuki et al., 2001). The β-catenin flox mice have been used successfully to inactivate β-catenin in vivo (Brault et al., 2001; Hari et al., 2002; Lickert et al., 2002). The four genotypes obtained from the crossing are shown in Fig. 1 A (see Materials and methods for the mating strategy). The ability of Tie2-Cre to recombine the flox allele specifically in the endothelium was tested by genomic PCR from embryonic yolk sacs. As reported in Fig. 1 A, the flox del allele was detected only in E10.5 animals positive for Tie2-Cre (Fig. 1 A, compare lane 3 and lane 4), suggesting that Cre expression in the endothelial cells of the yolk sac was capable of recombining the loxP sites within β-catenin gene. The presence of the unrecombined flox allele in the same lane is attributable to the nonendothelial cells abounding in the yolk sac used for the genomic PCR. The flox del allele was detected as early as E8.5 (unpublished data). Examination of the offspring indicated that heterozygous (Fig. 1 A, lane 1 and lane 4) were viable and fertile and appeared to be normal. In contrast, no mouse with endothelial β-catenin flox del/flox del (Fig. 1 A, lane 2) was born (Table I). Analyzing the embryos at different times after conception, we found that up to 9.5 d, β-catenin mutants were morphologically indistinguishable from control embryos (either wild types or littermates that had not inherited the complete set of alleles). However, ∼50 and 100% of embryos died within E11.5 and E13.5, respectively (Table I).


The conditional inactivation of the beta-catenin gene in endothelial cells causes a defective vascular pattern and increased vascular fragility.

Cattelino A, Liebner S, Gallini R, Zanetti A, Balconi G, Corsi A, Bianco P, Wolburg H, Moore R, Oreda B, Kemler R, Dejana E - J. Cell Biol. (2003)

Cre recombinase efficiently recombines the β-catenin flox allele in vivo. (A) Genomic PCR on E10.5 embryos from Tie2-Cre x β-catenin flox/flox crossings for the β-catenin gene and for Tie2-Cre gene. The four lanes represent the four different genotypes obtained: β-catenin flox del/flox, Cre− (+/−, lane 1), β-catenin flox del/flox, Cre+ (−/−, lane 2), β-catenin flox/wt, Cre− (+/+, lane 3), and β-catenin flox/wt, Cre+ (+/−, lane 4). In this last lane, note the presence of the recombined β-catenin flox del band when the flox allele is in the presence of Tie2-Cre recombinase. (B) Western blot of β-catenin protein in endothelial cell lines established from wt (control) and β-catenin flox/flox del, Tie2-Cre+ embryos (−/−). (C-J) Immunofluorescence for anti-PECAM and anti-β-catenin antibodies on serial cryosections from wild-type (+/+, control) and β-catenin flox/flox del, Tie2-Cre+ (−/−, mutant) E10.5 embryos. Heart sections show positive staining of the endocardium (e) with PECAM antibodies in both control (C) and β-catenin −/− (E) animals. β-Catenin staining is undetectable in mutant embryos (F). β-Catenin is also absent in the dorsal aorta (G and H, Ao) and in the yolk sac vessels (I and J, arrow) of the mutant embryos. Bar in H refers to C–H, and bar in J refers to I and J.
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Related In: Results  -  Collection

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fig1: Cre recombinase efficiently recombines the β-catenin flox allele in vivo. (A) Genomic PCR on E10.5 embryos from Tie2-Cre x β-catenin flox/flox crossings for the β-catenin gene and for Tie2-Cre gene. The four lanes represent the four different genotypes obtained: β-catenin flox del/flox, Cre− (+/−, lane 1), β-catenin flox del/flox, Cre+ (−/−, lane 2), β-catenin flox/wt, Cre− (+/+, lane 3), and β-catenin flox/wt, Cre+ (+/−, lane 4). In this last lane, note the presence of the recombined β-catenin flox del band when the flox allele is in the presence of Tie2-Cre recombinase. (B) Western blot of β-catenin protein in endothelial cell lines established from wt (control) and β-catenin flox/flox del, Tie2-Cre+ embryos (−/−). (C-J) Immunofluorescence for anti-PECAM and anti-β-catenin antibodies on serial cryosections from wild-type (+/+, control) and β-catenin flox/flox del, Tie2-Cre+ (−/−, mutant) E10.5 embryos. Heart sections show positive staining of the endocardium (e) with PECAM antibodies in both control (C) and β-catenin −/− (E) animals. β-Catenin staining is undetectable in mutant embryos (F). β-Catenin is also absent in the dorsal aorta (G and H, Ao) and in the yolk sac vessels (I and J, arrow) of the mutant embryos. Bar in H refers to C–H, and bar in J refers to I and J.
Mentions: To specifically inactivate the β-catenin gene in endothelial cells, mice containing a floxed β-catenin allele were intercrossed with transgenic mice expressing Cre under the control of Tie2 endothelial-specific promoter (Schlaeger et al., 1997; Brault et al., 2001; Kisanuki et al., 2001). The β-catenin flox mice have been used successfully to inactivate β-catenin in vivo (Brault et al., 2001; Hari et al., 2002; Lickert et al., 2002). The four genotypes obtained from the crossing are shown in Fig. 1 A (see Materials and methods for the mating strategy). The ability of Tie2-Cre to recombine the flox allele specifically in the endothelium was tested by genomic PCR from embryonic yolk sacs. As reported in Fig. 1 A, the flox del allele was detected only in E10.5 animals positive for Tie2-Cre (Fig. 1 A, compare lane 3 and lane 4), suggesting that Cre expression in the endothelial cells of the yolk sac was capable of recombining the loxP sites within β-catenin gene. The presence of the unrecombined flox allele in the same lane is attributable to the nonendothelial cells abounding in the yolk sac used for the genomic PCR. The flox del allele was detected as early as E8.5 (unpublished data). Examination of the offspring indicated that heterozygous (Fig. 1 A, lane 1 and lane 4) were viable and fertile and appeared to be normal. In contrast, no mouse with endothelial β-catenin flox del/flox del (Fig. 1 A, lane 2) was born (Table I). Analyzing the embryos at different times after conception, we found that up to 9.5 d, β-catenin mutants were morphologically indistinguishable from control embryos (either wild types or littermates that had not inherited the complete set of alleles). However, ∼50 and 100% of embryos died within E11.5 and E13.5, respectively (Table I).

Bottom Line: We found that early phases of vasculogenesis and angiogenesis were not affected in mutant embryos; however, vascular patterning in the head, vitelline, umbilical vessels, and the placenta was altered.These changes paralleled a decrease in cell-cell adhesion strength and an increase in paracellular permeability.We conclude that in vivo, the absence of beta-catenin significantly reduces the capacity of endothelial cells to maintain intercellular contacts.

View Article: PubMed Central - PubMed

Affiliation: FIRC Institute of Molecular Oncology, 16-20139, Milan, Italy.

ABSTRACT
Using the Cre/loxP system we conditionally inactivated beta-catenin in endothelial cells. We found that early phases of vasculogenesis and angiogenesis were not affected in mutant embryos; however, vascular patterning in the head, vitelline, umbilical vessels, and the placenta was altered. In addition, in many regions, the vascular lumen was irregular with the formation of lacunae at bifurcations, vessels were frequently hemorrhagic, and fluid extravasation in the pericardial cavity was observed. Cultured beta-catenin -/- endothelial cells showed a different organization of intercellular junctions with a decrease in alpha-catenin in favor of desmoplakin and marked changes in actin cytoskeleton. These changes paralleled a decrease in cell-cell adhesion strength and an increase in paracellular permeability. We conclude that in vivo, the absence of beta-catenin significantly reduces the capacity of endothelial cells to maintain intercellular contacts. This may become more marked when the vessels are exposed to high or turbulent flow, such as at bifurcations or in the beating heart, leading to fluid leakage or hemorrhages.

Show MeSH
Related in: MedlinePlus