Interkinetic nuclear migration (INM) is a cell polarity-based phenomenon in which progenitor cell nuclei migrate along the apico-basal axis of the pseudostratified epithelium in synchrony with the cell cycle. INM is suggested to be at least partially cytoskeleton-dependent and to regulate not only the proliferation/differentiation of stem/progenitor cells but also the localized/overall size and shape of organs/tissues. INM occurs in all three of the germ-layer derived epithelia, including the endoderm-derived gut. However, INM has not been documented in the esophagus and respiratory tube arising from the anterior foregut. Esophageal atresia with or without trachea-esophageal fistula (EA/TEF) is a relatively common developmental defect. Transcription factors and signaling molecules have been implicated in EA/TEF, but the etiology of EA/TEF-which has been suggested to involve cell polarity-related mechanisms-remains highly controversial. In the present study, we first examined whether INM exists in the trachea and esophagus of mouse embryos at embryonic day 11.5 (E11.5), just after separation of the two tubes from the anterior foregut. By labeling the DNA-synthesizing stem cell nuclei with 5-ethynyl-2'-deoxyuridine, a nucleotide analogue, and statistically analyzing chronological changes in the distribution pattern of the labeled nuclei by using multidimensional scaling, we showed the existence of INM in both the esophagus and trachea, with differences in the INM magnitude and cycle pattern. We further showed morphological changes from the INM-based pseudostratified single layer to the stratified multilayer in the esophageal epithelium in association with a temporal loss/perturbation of AB polarity, suggesting a possible relation with the pathogenesis of EA/TEF.

The apico-basal (AB) polarity of epithelial cells is maintained by organized arrays of the cytoskeleton and adhesion apparatus. We previously reported that mouse embryonic esophageal epithelium exhibits interkinetic nuclear migration (INM), an AB-polarity-based regulatory mechanism of stem-cell proliferation, and suggested that the pseudostratified single columnar epithelium, a hallmark of INM, is converted to stratified squamous epithelium via rearrangement of the cytoskeleton and cell-adhesion apparatus. Here, we chronologically examined morphological changes in the cytoskeleton and adhesion apparatus in the mouse esophageal epithelium at embryonic day (E) 11.5, E13.5, E14.5, and E15.5, during which epithelial conversion has been suggested to occur. We used phalloidin to examine the apical terminal web (ATW), immunofluorescent anti-zonula occludens protein (ZO-1) antibody to reveal ZO-1, and anti-gamma tubulin antibody to detect primary cilia (PC). At E11.5, a thick ATW, apically oriented ZO-1 and apical PC were observed, indicating a pseudostratified single columnar structure. At E13.5 and E14.5, the phalloidin-staining, ZO-1, and PC distribution patterns were not apically localized, and the epithelial cells appeared to have lost the AB polarity, suggesting conversion of the epithelial structure and cessation of INM. At E15.5, light and transmission electron microscope observations revealed the ATW, ZO-1, PC, and tight junction which were localized into two-1ayers: the apical and subapical layers of the epithelium. These findings suggest that dynamic remodeling of the cytoskeleton and adhesion apparatus is involved in the conversion from pseudostratified single columnar to stratified squamous morphology and is closely related with temporal perturbation of the AB-polarity and cessation of INM.