E-cadherin loss of function in the murine intestine
<p>E-cadherin (<em>Cdh1</em>), is a major component of epithelial adherens junctions, binds the Wnt pathway effector β-catenin and is lost at the invasive edge of colon cancers. Crypt stem cells give rise to 4 cell lineages that, with the exception of Paneth cells, travel along the...
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Format: | Thesis |
Language: | English |
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2012
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author | Matheson, JAH |
author2 | Hassan, AB |
author_facet | Hassan, AB Matheson, JAH |
author_sort | Matheson, JAH |
collection | OXFORD |
description | <p>E-cadherin (<em>Cdh1</em>), is a major component of epithelial adherens junctions, binds the Wnt pathway effector β-catenin and is lost at the invasive edge of colon cancers. Crypt stem cells give rise to 4 cell lineages that, with the exception of Paneth cells, travel along the crypt-villus axis over 3 days and are shed by anoikis. Gain of function of the Wnt pathway by a mutation in Adenomatous Polyposis Coli (<em>Apc</em>) disrupts enterocyte turnover to result in adenoma formation. Homozygote null <em>Cdh1</em> is embryonic lethal, and heterozygote <em>Cdh1</em> can promote <em>Apc<sup>1638N</sup></em> induced adenoma formation, but we lack models that assess additional functions of Cdh1 in the adenoma to carcinoma transition. The aim of this thesis was to evaluate the effect of <em>Cdh1</em> loss of function in the murine intestine, including in the setting of Wnt pathway activation. To do this, germline and intestinal specific conditional <em>Cdh1</em> loss of function models were generated.</p> <p>Conditional homozygous deletion of <em>Cdh1</em> resulted in embryonic lethality using <em>Villin-Cre</em>. In adults, homozygous <em>Cdh1</em> loss using the tamoxifen inducible <em>Villin-CreER<sup>T2</sup></em> led to intestinal inflammation, bacteraemia and disrupted crypt-villus architecture. Combined conditional homozygous deletion of <em>Cdh1</em> and <em>Apc</em> resulted in Wnt pathway upregulation assessed by β-catenin immunolabelling. Strain dependent effects of <em>Cdh1</em> heterozygosity were apparent on the <em>Apc</em> heterozygote background: <em>Apc<sup>Min/&plus;</sup></em> <em>Cdh1<sup>&plus;/-</sup></em> (C57BL/6J) had no effect on survival or adenoma phenotype compared to littermate <em>Apc<sup>Min/&plus;</sup></em>; <em>Cdh1<sup>+/fl</sup></em> increased adenoma burden in <em>Apc<sup>&plus;/fl</sup> Vil-Cre</em> animals (B6D2/C57BL/6J).</p> <p>Low frequency recombination of <em>Apc<sup>fl/fl</sup></em> using <em>Lgr5-EGFP-IRES-CreER<sup>T2</sup></em> bypasses the loss of heterozygosity event relied on in heterozygous <em>Apc</em> tumour models achieving a large adenoma burden within 4 weeks. <em>Cdh1</em> loss did not alter survival or adenoma phenotype in this model, including the development of large caecal tumours. Immunolabelling of tumours from <em>Apc<sup>fl/fl</sup></em> <em>Cdh1<sup>fl/fl</sup></em> animals showed persistent E-cadherin protein expression, suggesting incomplete recombination or that double homozygote enterocytes failed to survive. <em>In vitro</em> adenoma culture was used to test whether E-cadherin loss was incompatible with enterocyte survival in the setting of Wnt activation. <em>Apc<sup>fl/fl</sup> Cdh1<sup>&plus;/&plus;</sup></em> and <em>Apc<sup>fl/fl</sup> Cdh1<sup>fl/fl</sup></em> adenoma were cultured in matrigel and treated with an adenovirus expressing Cre recombinase under a CMV promoter (Ad-Cre). Ad-Cre had no effect on <em>Apc<sup>fl/fl</sup> Cdh1<sup>+/+</sup></em> adenoma growth. Ad-Cre treated <em>Apc<sup>fl/fl</sup> Cdh1<sup>fl/fl</sup></em> adenoma organoids showed cells where E‑cadherin loss resulted in Wnt pathway upregulation as assessed by nuclear β-catenin and Axin2 expression, and epithelial mesenchymal transition shown by upregulation of fibronectin, twist and vimentin. This work supports a role for E-cadherin in modulation of the Wnt pathway. Further investigation is required to define the cell adhesion versus Wnt regulatory functions of E-cadherin.</p> |
first_indexed | 2024-03-07T04:04:22Z |
format | Thesis |
id | oxford-uuid:c5a61fcd-e0a2-4af7-bb53-c78c3a47874f |
institution | University of Oxford |
language | English |
last_indexed | 2024-03-07T04:04:22Z |
publishDate | 2012 |
record_format | dspace |
spelling | oxford-uuid:c5a61fcd-e0a2-4af7-bb53-c78c3a47874f2022-03-27T06:32:31ZE-cadherin loss of function in the murine intestineThesishttp://purl.org/coar/resource_type/c_db06uuid:c5a61fcd-e0a2-4af7-bb53-c78c3a47874fTransgenicsMolecular geneticsGenetics (life sciences)EnglishOxford University Research Archive - Valet2012Matheson, JAHHassan, AB<p>E-cadherin (<em>Cdh1</em>), is a major component of epithelial adherens junctions, binds the Wnt pathway effector β-catenin and is lost at the invasive edge of colon cancers. Crypt stem cells give rise to 4 cell lineages that, with the exception of Paneth cells, travel along the crypt-villus axis over 3 days and are shed by anoikis. Gain of function of the Wnt pathway by a mutation in Adenomatous Polyposis Coli (<em>Apc</em>) disrupts enterocyte turnover to result in adenoma formation. Homozygote null <em>Cdh1</em> is embryonic lethal, and heterozygote <em>Cdh1</em> can promote <em>Apc<sup>1638N</sup></em> induced adenoma formation, but we lack models that assess additional functions of Cdh1 in the adenoma to carcinoma transition. The aim of this thesis was to evaluate the effect of <em>Cdh1</em> loss of function in the murine intestine, including in the setting of Wnt pathway activation. To do this, germline and intestinal specific conditional <em>Cdh1</em> loss of function models were generated.</p> <p>Conditional homozygous deletion of <em>Cdh1</em> resulted in embryonic lethality using <em>Villin-Cre</em>. In adults, homozygous <em>Cdh1</em> loss using the tamoxifen inducible <em>Villin-CreER<sup>T2</sup></em> led to intestinal inflammation, bacteraemia and disrupted crypt-villus architecture. Combined conditional homozygous deletion of <em>Cdh1</em> and <em>Apc</em> resulted in Wnt pathway upregulation assessed by β-catenin immunolabelling. Strain dependent effects of <em>Cdh1</em> heterozygosity were apparent on the <em>Apc</em> heterozygote background: <em>Apc<sup>Min/&plus;</sup></em> <em>Cdh1<sup>&plus;/-</sup></em> (C57BL/6J) had no effect on survival or adenoma phenotype compared to littermate <em>Apc<sup>Min/&plus;</sup></em>; <em>Cdh1<sup>+/fl</sup></em> increased adenoma burden in <em>Apc<sup>&plus;/fl</sup> Vil-Cre</em> animals (B6D2/C57BL/6J).</p> <p>Low frequency recombination of <em>Apc<sup>fl/fl</sup></em> using <em>Lgr5-EGFP-IRES-CreER<sup>T2</sup></em> bypasses the loss of heterozygosity event relied on in heterozygous <em>Apc</em> tumour models achieving a large adenoma burden within 4 weeks. <em>Cdh1</em> loss did not alter survival or adenoma phenotype in this model, including the development of large caecal tumours. Immunolabelling of tumours from <em>Apc<sup>fl/fl</sup></em> <em>Cdh1<sup>fl/fl</sup></em> animals showed persistent E-cadherin protein expression, suggesting incomplete recombination or that double homozygote enterocytes failed to survive. <em>In vitro</em> adenoma culture was used to test whether E-cadherin loss was incompatible with enterocyte survival in the setting of Wnt activation. <em>Apc<sup>fl/fl</sup> Cdh1<sup>&plus;/&plus;</sup></em> and <em>Apc<sup>fl/fl</sup> Cdh1<sup>fl/fl</sup></em> adenoma were cultured in matrigel and treated with an adenovirus expressing Cre recombinase under a CMV promoter (Ad-Cre). Ad-Cre had no effect on <em>Apc<sup>fl/fl</sup> Cdh1<sup>+/+</sup></em> adenoma growth. Ad-Cre treated <em>Apc<sup>fl/fl</sup> Cdh1<sup>fl/fl</sup></em> adenoma organoids showed cells where E‑cadherin loss resulted in Wnt pathway upregulation as assessed by nuclear β-catenin and Axin2 expression, and epithelial mesenchymal transition shown by upregulation of fibronectin, twist and vimentin. This work supports a role for E-cadherin in modulation of the Wnt pathway. Further investigation is required to define the cell adhesion versus Wnt regulatory functions of E-cadherin.</p> |
spellingShingle | Transgenics Molecular genetics Genetics (life sciences) Matheson, JAH E-cadherin loss of function in the murine intestine |
title | E-cadherin loss of function in the murine intestine |
title_full | E-cadherin loss of function in the murine intestine |
title_fullStr | E-cadherin loss of function in the murine intestine |
title_full_unstemmed | E-cadherin loss of function in the murine intestine |
title_short | E-cadherin loss of function in the murine intestine |
title_sort | e cadherin loss of function in the murine intestine |
topic | Transgenics Molecular genetics Genetics (life sciences) |
work_keys_str_mv | AT mathesonjah ecadherinlossoffunctioninthemurineintestine |