The mammalian esophagus exhibits a remarkable change in epithelial organization during the transition from embryonic to adult tissue. Initially, the esophagus is lined by a simple columnar epithelial layer, which is gradually replaced by a stratified squamous tissue comprised a clearly distinct basal layer surrounded by the spinous, granulated, and cornified suprabasal layers.
The replacement of one epithelium with another during esophageal development has been documented in both mouse and human, and in 1993, Thorey et al.
proposed that the basal layer of the stratified squamous epithelium is derived from the columnar epithelium.10–12
However, direct evidence for this conversion was lacking due to the availability of suitable model systems.
We have developed an in vitro
model for studying esophageal development based on the culture of esophageal explants from embryonic day 11.5 (E11.5) mouse embryos. This culture model recapitulates the in vivo
development of the esophagus.7
Using immunohistochemical analysis of sections from developing mouse embryos, we initially demonstrated that the transition from columnar to stratified squamous epithelium is accompanied by a change in expression of the intermediate filament proteins cytokeratin 8 (K8, a marker of columnar epithelium) and 14 (K14, a marker for the basal epithelial layer). At E11.5 all the cells of the esophageal epithelium express K8 but not K14. Gradually, between E15.5 and E17.5, K8 expression is lost in the basal layer but is retained in the suprabasal layers until postnatal day 3 when it is completely lost. In contrast, K14 is absent in the esophageal epithelium at E11.5 but begins to be expressed around E17.5 in the basal cells, which are no longer expressing K8.
The explant culture model was established by placing esophageal tissue dissected from E11.5 embryos onto fibronectin-coated glass coverslips. Within 24 h, the explants attached and flattened onto the fibronectin substratum at which stage they are composed of a tube of epithelium surrounded by mesenchyme. The explants remain viable in culture for up to 20 days and differentiate as stratified squamous epithelium, exhibiting the same switch in expression of K8 to K14 as observed in vivo.
To determine the origin of the stratified squamous epithelium, we used our explant model to explore the possibility that basal cells arise through programmed cell death of the columnar cells, selective overgrowth of the squamous epithelium, or though a direct conversion of one cell type to another.
To distinguish the first of these possibilities, we separated the cells after 7, 11, or 15 days of culture by flow cytometry and then immunostained for either K8 or K14 and propidium iodide to identify dead cells. If the switch in cell type was due to a preferential death of the columnar cell population, the number of dead cells would be higher in the K8-expressing population. However, there was no observable difference in the proportion of dead cells in the K8- and K14-positive populations indicating that programmed cell death does not play a role in the conversion from columnar to stratified squamous epithelium.
To determine whether the switch from columnar to stratified squamous epithelium was due to selective overgrowth of the squamous epithelium we immunostained for the proliferation marker Ki-67. The embryonic esophageal epithelium in culture is highly proliferative up until the culture differentiates into multilayers after which time the majority of the proliferating cells become localized to the K14-positive cells of the basal layer. In order to determine whether cell division was required for conversion, we treated esophageal explants with mitomycin C, a DNA crosslinking agent that prevents the cells from entering the cell cycle. If the cell conversion is dependent on cell division, then we predicted it would not be able to occur in the presence of a cell cycle inhibitor. However, the transition from K8-positive columnar layer to the K14-expressing cells continued to occur in the presence of mitomycin C indicating that cell proliferation does not play a role in the epithelial cell conversion.
The possibility that the cells of the basal layer arise directly from the columnar epithelium was also investigated. Upon costaining for K8 and K14, we found some cells coexpressing both markers suggesting that K14-positive cells arose directly from K8-positive cells. To trace the lineage of the stratified squamous basal cells, we electroporated a reporter plasmid, the K14 promoter driving GFP expression, into esophageal explants obtained from E15.5 embryos. This timepoint was chosen since the basal phenotype starts to appear between E15.5 and E17.5. Costaining for GFP and K8 revealed that at least some of the cells that have an activate K14 promoter (GFP-positive) also express K8, indicating that the basal cells of the squamous epithelium arise through a direct conversion from K8-positive columnar cells.
The embryonic esophageal culture model has provided evidence of a direct conversion from columnar to stratified squamous cells in the developing esophagus (). Further analysis of the mechanisms involved in this conversion may also provide important insights underlying the mechanism(s) involved in Barrett's metaplasia in which the reverse conversion from stratified squamous epithelium to columnar epithelium occurs.13
Figure 2 Summary of the conversion of esophageal epithelium from simple columnar to stratified squamous tissue during development. At E11.5 the esophageal epithelium consists of a single layer of K8-expressing cells. Between E15.5 and E17.5, the epithelium starts (more ...)