The mouse embryonic oesophagus is initially lined with a simple columnar epithelial layer which changes during the course of development to a stratified squamous tissue. To study the mechanism of this transition, we developed an in vitro model, based on oesophageal explants isolated from E11.5d mouse embryos, which fully recapitulates the normal in vivo development. In this system, the columnar epithelial markers cytokeratins 8 and 18 (K8, 18) were strongly expressed at the beginning of the culture period and decreased in the basal layer of the epithelium at around 5 days of culture. Expression of K8 + 18 persisted in the suprabasal layers of the stratified epithelium for several more days. In contrast, the stratified squamous epithelial marker cytokeratin 14 (K14) was absent at the beginning, and cells expressing it progressively appeared within the basal layer from day 5 to day 9 of culture. The two possible mechanisms for the change are (1) a direct conversion of columnar cells to the basal layer cells of the squamous epithelium; (2) an overgrowth of columnar by squamous cells. Our results show that the first mechanism is operative. Firstly, co-staining for K8 and K14 demonstrates that some cells express both markers during the transition period. Secondly, after electroporation of a construct containing the K14 promoter driving nuclear GFP into the epithelium of E15.5 oesophagus, some cells expressed both K8 and GFP. Thirdly, there is no preferential loss of the columnar cells by apoptosis. Fourthly, inhibitors of apoptosis do not affect the process. Finally, inhibitors of cell division do not affect the process. In terms of the molecular mechanism, inhibitor studies suggest that de novo DNA methylation is required for the loss of the K8 expression but not for the acquisition of the K14 expression. The results show that, in normal development, the squamous epithelium arises from the columnar epithelium by a direct conversion process.

Timing of organ development during embryogenesis is coordinated such that at birth, organ and fetal size and maturity are appropriately proportioned. The extent to which local developmental timers are integrated with each other and with the signaling interactions that regulate morphogenesis to achieve this end is not understood. Using the absolute requirement for a signaling pathway activity (bone morphogenetic protein, BMP) during a critical stage of tooth development, we show that suboptimal levels of BMP signaling do not lead to abnormal morphogenesis, as suggested by mutants affecting BMP signaling, but to a 24-h stalling of the intrinsic developmental clock of the tooth. During this time, BMP levels accumulate to reach critical levels whereupon tooth development restarts, accelerates to catch up with development of the rest of the embryo and completes normal morphogenesis. This suggests that individual organs can autonomously control their developmental timing to adjust their stage of development to that of other organs. We also find that although BMP signaling is critical for the bud-to-cap transition in all teeth, levels of BMP signaling are regulated differently in multicusped teeth. We identify an interaction between two homeodomain transcription factors, Barx1 and Msx1, which is responsible for setting critical levels of BMP activity in multicusped teeth and provides evidence that correlates the levels of Barx1 transcriptional activity with cuspal complexity. This study highlights the importance of absolute levels of signaling activity for development and illustrates remarkable self-regulation in organogenesis that ensures coordination of developmental processes such that timing is subordinate to developmental structure.

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