Growth of intestinal epithelium in organ culture is dependent on EGF signalling

Abstract

Differentiation of endoderm into intestinal epithelium is initiated at E13.5 of mouse development when there are significant changes in morphology resulting in the conversion of undifferentiated stratified epithelium into a mature epithelial monolayer. Here we demonstrate that monolayer formation is associated with the selective apoptosis of superficial cells lining the lumen while cell proliferation is progressively restricted to cells adjacent to the basement membrane. We describe an innovative embryonic gut culture system that maintains the three-dimensional architecture of gut and in which these processes are recapitulated in vitro. Explants taken from specific regions of the gut and placed into organ culture develop and express molecular markers (Cdx1, Cdx2 and A33 antigen) in the same spatial and temporal pattern observed in vivo indicating that regional specification is maintained. Inhibition of the epidermal growth factor receptor (EGFR) tyrosine kinase using the specific inhibitor AG1478 significantly reduced the proliferation and survival of cells within the epithelial cell layer of cultured gut explants. This demonstrates an essential role for the EGF signalling pathway during the early stages of intestinal development.

Introduction

Adult intestine consists of a lumen surrounded by a layer of epithelium that is extensively folded into crypts and villi and enclosed by supporting mesenchymal tissue. The development and maintenance of this tissue architecture is essential for the function of intestinal epithelial cells [1], [2]. From E13.5 of rodent development, the endoderm cell layer destined to become intestinal epithelium begins remodelling, with the conversion of a stratified cell layer into a differentiated polarised epithelial monolayer [3].

The morphology and homeostasis of the intestinal epithelium result from a highly regulated equilibrium between cell proliferation, movement, differentiation and apoptosis [4], [5], [6], [7], [8], [9], [10]. Hyperproliferation associated with the loss of cell polarity and epithelial monolayer morphology is a hallmark of adenomatous polyps that precede metastatic colon cancer [11], [12], [13], [14]. An understanding of the establishment and maintenance of epithelial monolayers is therefore likely to provide insight into tumour initiation and progression.

The study of intestinal epithelial cell growth and differentiation has been limited by the inability to culture either normal adult or embryonic intestinal epithelial cells in a manner that allows manipulation and observation. Despite many attempts, reliable culture systems that mimic normal intestinal epithelial cell differentiation have yet to be established [15]. As soon as intestinal epithelial cells are removed from the basement membrane and underlying stroma, apoptosis is initiated in a few hours [10], [16]. This has limited the study of gene function to some studies in transgenic animals [17] or colonic carcinoma-derived cell lines that often contain substantial chromosomal aberrations and mutations in many genes of interest [18]. These cell lines are unable to reproduce the complex changes in gene expression observed during intestinal development in vivo. These difficulties, along with evidence that there is significant crosstalk between the epithelium and adjacent cell layers [19], [20], [21], [22], [23], suggest that the only efficient way to study normal intestinal epithelial differentiation in vitro may be an organ culture system where the epithelial cells remain in contact with the underlying mesenchyme.

A novel method of culturing embryonic gut to study the development of the enteric nervous system has been described [24]. Explants of embryonic gut were cultured in suspension by attachment to pieces of filter paper (catenary culture), thereby maintaining the embryonic gut as a tube with both the mesoderm and endoderm cell layers intact. The preservation of overall structure of the gut may allow signalling between adjacent cell layers to be conserved and therefore more faithfully recapitulate development in vivo. This system has been used successfully to study the sequence of appearance of migratory enteric neuron precursors and the role of GDNF as a chemoattractant [25]. We sought to further develop this technique for the study of endoderm/epithelial cell differentiation and intestinal epithelial monolayer formation during mouse development.

Many studies have implicated the epidermal growth factor (EGF) signalling pathway in the regulation of intestinal epithelial cell growth and differentiation [26], [27], [28], [29], [30], [31]. Overexpression or mutation of the EGF receptor (EGFR) has been associated with many different carcinomas including colonic carcinoma [32]. EGFR null animals that die early in postnatal life exhibit severe defects in intestinal cell proliferation and organisation along with many other abnormalities [28]. These observations indicate that the EGFR signalling pathway plays an important role in regulating intestinal epithelial cell production and is required for normal gut development. The majority of reports of EGF action have concentrated on postnatal stages of epithelial cell maturation. The catenary culture system allows the function of this pathway to be examined in earlier embryonic stages.

We analysed the patterns of cell proliferation and programmed cell death in normal tissue and accurately compared this to growth in catenary culture over several days of development. We also assessed the capacity of cultured gut endoderm to differentiate into mature cell lineages using a combination of histology and electron microscopy. The specification of mid- and hindgut segments was examined using a panel of markers that exhibit specific spatial and temporal expression patterns during the very early stages of intestinal epithelial development [29], [30], [31]. We then used the catenary culture model to examine the effects of exogenous EGF and AG1478, which specifically inhibits EGFR tyrosine kinase, on the patterns of cell growth and apoptosis in gut explants. We expected these observations to provide further insight into the function of EGF signalling in early intestinal development and interpretation of the phenotypes observed in EGFR^−/− mice.