Gastroenterology

Gastroenterology

Volume 140, Issue 2, February 2011, Pages 517-528.e8
Gastroenterology

Basic—Alimentary Tract
Cdx2 Levels Modulate Intestinal Epithelium Maturity and Paneth Cell Development

https://doi.org/10.1053/j.gastro.2010.11.033Get rights and content

Background & Aims

Caudal-related homeobox protein 2 (Cdx2) is an intestine-specific transcription factor that is important for intestinal development and intestine-specific gene expression. Cdx2 regulates intestinal cell-cell adhesion, proliferation, and the transcriptional activities of Wnt and β-catenin in cell culture systems. We generated transgenic mice that overexpress Cdx2 in the small intestinal and colonic epithelium to investigate the role of Cdx2 in differentiation and function of the intestinal epithelium.

Methods

We established 4 different lines of villin−Cdx2 transgenic mice. Intestines were collected from infant, 3-month old, and wild-type mice. Genes of interest and cell lineage markers were examined by polymerase chain reaction and immunohistochemistry.

Results

Villin−Cdx2 transgenic mice had complex phenotypes that were associated with transgene expression levels. The 2 lines that had the greatest levels of transgene expression had significant, preweaning failure to grow and death; these were the result of early epithelial maturation and alterations in nutrient digestion and absorption. Fat malabsorption was a prominent feature. Other effects associated with the transgene expression included loss of Paneth cell markers, increases in goblet cells, and migration of proliferating, EphB2-expressing cells to the crypt base. Loss of Paneth cell markers was associated with reduced nuclear localization of β-catenin but not homeotic posteriorization of the epithelium by Cdx2.

Conclusions

Overexpression of Cdx2 in the small intestine is associated with reduced post-natal growth, early epithelial maturation, alterations in crypt base organization, and changes in Paneth and goblet cell lineages. Cdx2 is a critical regulator not only of intestine-specific genes, but also processes that determine epithelial maturity and function.

Section snippets

Generation of Villin-Cdx2 Transgenic Mice

Mouse Cdx2 complementary DNA (cDNA) with an N-terminal FLAG-tag19 was subcloned into a pCMV-Tag3c (Stratagene, La Jolla, CA) plasmid. The 12.4-kb mouse Villin promoter was then subcloned before the cDNA to generate the final Villin-(FLAG)-Cdx2 construct. The Villin-Cdx2 DNA was linearized and injected into the male pronuclei of fertilized eggs and implanted into pseudopregnant females by the Transgenic and Chimeric Mouse Core Facility at the University of Pennsylvania. Founder animals were

Development of Transgenic Mouse Lines Overexpressing Cdx2 in the Mouse Intestine

Transgenic mice were generated using the 12.4-kb mouse villin regulatory sequences (kindly provided by Dr. Deborah Gumucio, University of Michigan) to drive expression of a mouse Cdx2 complementary DNA with an N-terminal FLAG tag that differentiates transgenic from endogenous Cdx2 (Figure 1A).22 Four distinct founders expressing the transgene were obtained from 2 injections. All 4 lines expressed the transgene messenger RNA (mRNA) as detected by PCR, and levels of total intestinal Cdx2 protein

Discussion

It has been established that the homeodomain transcription factor Cdx2 plays a critical role in regulating intestinal epithelial differentiation and function.12 Our transgenic mice unexpectedly display a complex phenotype, suggesting Cdx2 plays important roles in other intestinal cell processes.

Acknowledgments

We thank William Hockheimer and Abena Kwaa for their technical support.

At the time of the study, Rong-Jun Guo and Shinsuke Funakoshi were at the Division of Gastroenterology, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.

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    Conflicts of interest The authors disclose no conflicts.

    Funding This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases grants DK068366 and DK085551 (JL), an National Cancer Institute Program Project P01 CA098101, and Core Facilities of the Center for Molecular Studies in Digestive and Liver Disease at the University of Pennsylvania (P30-DK50306).

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