SUMMARY Mucociliary epithelia provide a first line of defense against pathogens. Impaired regeneration and remodeling of mucociliary epithelia are associated with dysregulated Wnt/β-catenin signaling in chronic airway diseases, but underlying mechanisms remain elusive, and studies yield seemingly contradicting results. Employing the Xenopus mucociliary epidermis, the mouse airway, and human airway Basal cells, we characterize the evolutionarily conserved roles of Wnt/β-catenin signaling in vertebrates. In multiciliated cells, Wnt is required for cilia formation during differentiation. In Basal cells, Wnt prevents specification of epithelial cell types by activating ΔN-TP63, a master transcription factor, which is necessary and sufficient to mediate the Wnt-induced inhibition of specification and is required to retain Basal cells during development. Chronic Wnt activation leads to remodeling and Basal cell hyperplasia, which are reversible in vivo and in vitro, suggesting Wnt inhibition as a treatment option in chronic lung diseases. Our work provides important insights into mucociliary signaling, development, and disease.
SummaryMucociliary epithelia provide a first line of defense against pathogens in the airways and the epidermis of vertebrate larvae. Impaired regeneration and remodeling of mucociliary epithelia are associated with dysregulated Wnt/β-catenin signaling in chronic airway diseases, but underlying mechanisms remain elusive and studies of Wnt signaling in mucociliary cells yield seemingly contradicting results. Employing the Xenopus mucociliary epidermis, the mouse airway, and human airway basal stem cell cultures, we characterize the evolutionarily conserved roles of Wnt/β-catenin signaling in mucociliary cells in vertebrates. Wnt signaling is required in multiciliated cells for cilia formation during differentiation stages, but in Basal cells, Wnt signaling prevents specification and differentiation of epithelial cell types by activating ΔN-TP63 expression. We demonstrate that ΔN-TP63 is a master transcription factor in Basal cells, which is necessary and sufficient to mediate the Wnt-induced inhibition of differentiation and is required to retain basal stem cells during development. Chronic stimulation of Wnt signaling leads to mucociliary remodeling and Basal cell hyperplasia, but this is reversible in vivo and in vitro, suggesting Wnt inhibition as an option in the treatment of chronic lung diseases. Our work sheds light into the evolutionarily conserved regulation of stem cells and differentiation, resolves Wnt functions in mucociliary epithelia, and provides crucial insights into mucociliary development, regeneration and disease mechanisms.
Na+/H+ exchangers (NHEs) represent a highly conserved family of ion transporters that regulate pH homeostasis. NHEs as well as other proton transporters were previously linked to the regulation of the Wnt signaling pathway, cell polarity signaling, and mucociliary function. Furthermore, mutations in the gene SLC9A3 (encoding NHE3) were detected as additional risk factors for airway infections in cystic fibrosis patients. Here, we used the Xenopus embryonic mucociliary epidermis as well as human airway epithelial cells (HAECs) as models to investigate the functional roles of NHEs in mucociliary development and regeneration. In Xenopus embryos, NHEs 1–3 were expressed during epidermal development, and loss of NHE function impaired mucociliary clearance in tadpoles. Clearance defects were caused by reduced cilia formation, disrupted alignment of basal bodies in multiciliated cells (MCCs), and dysregulated mucociliary gene expression. These data also suggested that NHEs may contribute to the activation of Wnt signaling in mucociliary epithelia. In HAECs, pharmacological inhibition of NHE function also caused defective ciliation and regeneration in airway MCCs. Collectively, our data revealed a requirement for NHEs in vertebrate mucociliary epithelia and linked NHE activity to cilia formation and function in differentiating MCCs. Our results provide an entry point for the understanding of the contribution of NHEs to signaling, development, and pathogenesis in the human respiratory tract.
Multiciliated cells (MCCs) are extremely highly-differentiated, presenting >100 cilia and basal bodies. We analyzed how MCCs are lost from the airway-like Xenopus embryonic epidermis during developmental tissue remodeling. We found that some MCCs undergo apoptosis, but that the majority trans-differentiate into secretory cells. Trans-differentiation involves loss of ciliary gene expression, cilia retraction and lysosomal degradation. Apoptosis and trans-differentiation are both induced by a changing signaling environment through Notch, Jak/STAT, Thyroid hormone and mTOR signaling, and trans-differentiation can be inhibited by Rapamycin. This demonstrates that even cells with extreme differentiation features can undergo direct fate conversion. Our data further suggest that the reactivation of this developmental mechanism in adults can drive tissue remodeling in human chronic airway disease, a paradigm resembling cancer formation and progression.
Tissue functions are determined by the types and ratios of cells present, but little is known about self-organizing principles establishing correct cell type compositions. Mucociliary airway clearance relies on the correct balance between secretory and ciliated cells, which is regulated by Notch signaling across mucociliary systems. Using the airway-like Xenopus epidermis, we investigate how cell fates depend on signaling, how signaling levels are controlled, and how Hes transcription factors regulate cell fates. We show that four mucociliary cell types each require different Notch levels and that their specification is initiated sequentially by a temporal Notch gradient. We describe a novel role for Foxi1 in the generation of Delta-expressing multipotent progenitors through Hes7.1. Hes7.1 is a weak repressor of mucociliary genes and overcomes maternal repression by the strong repressor Hes2 to initiate mucociliary development. Increasing Notch signaling then inhibits Hes7.1 and activates first Hes4, then Hes5.10, which selectively repress cell fates. We have uncovered a self-organizing mechanism of mucociliary cell type composition by competitive de-repression of cell fates by a set of differentially acting repressors. Furthermore, we present an in silico model of this process with predictive abilities.
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