Temporal Notch signaling and Hes-mediated competitive de-repression regulate mucociliary cell fates in Xenopus

Abstract

Mucociliary epithelia are found across different organs in animals, where they release bioactive substances and generate extracellular fluid flows. One key function of mucociliary epithelia is the clearance of pathogens, e.g. in the vertebrate lung and the epidermis of amphibian tadpoles. Mucociliary clearance relies on the correct balance between secretory cells that release mucus, ciliated cells that generate fluid flow as well as specialized cell types, including pH-regulating ionocytes and basal stem cells. Notch signaling, Hes repressors and cell type-inducing transcription factors (e.g. Foxi1, Mcidas, Spdef and Tp63) regulate cell fates and cell type proportions across mucociliary systems. Lateral inhibition was proposed to control mucociliary cell fates, but current models cannot explain how more than two cell types are generated and how Hes genes are employed as mediators during patterning. Using the Xenopus tadpole epidermis, we addressed these open questions in mucociliary biology through a combination of in vivo and organoid experiments, time-resolved transcriptomic studies and mathematical modeling. This revealed that ionocytes, ciliated cells, secretory cells and basal cells are preferentially specified at different time points and Notch signaling levels via sequentially expressed Hes factors. We termed this mode of patterning "competitive de-repression", because cell fates are selected by suppression of alternative fate choices, and demonstrate that this relies on differential active repression of cell fate transcription factors. Mathematical modeling further indicated the need for a positively Notch-regulated patterning factor, and we provide evidence that Spdef mediates Notch input for secretory and basal cell specification. Collectively, this work presents a coherent model for Notch- and Hes-mediated mucociliary cell fate specification in a vertebrate tissue, which allows for the specification of more than two cell fates within the Notch lateral-inhibition paradigm.