Signaling Control of Mucociliary Epithelia: Stem Cells, Cell Fates, and the Plasticity of Cell Identity in Development and Disease

Mucociliary epithelia are composed of multiciliated, secretory, and stem cells and line various organs in vertebrates such as the respiratory tract. By means of mucociliary clearance, those epithelia provide a first line of defense against inhaled particles and pathogens. Mucociliary clearance relies on the correct composition of cell types, that is, the proper balance of ciliated and secretory cells. A failure to generate and to maintain correct cell type composition and function results in impaired clearance and high risk to infections, such as in congenital diseases (e.g., ciliopathies) as well as in acquired diseases, including asthma, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF). While it remains incompletely resolved how precisely cell types are specified and maintained in development and disease, many studies have revealed important mechanisms regarding the signaling control in mucociliary cell types in various species. Those studies not only provided insights into the signaling contribution to organ development and regeneration but also highlighted the remarkable plasticity of cell identity encountered in mucociliary maintenance, including frequent trans-differentiation events during homeostasis and specifically in disease. This review will summarize major findings and provide perspectives regarding the future of mucociliary research and the treatment of chronic airway diseases associated with tissue remodeling.

A ‘tad’ of hope in the fight against airway disease

Xenopus tadpoles have emerged as a powerful in vivo model system to study mucociliary epithelia such as those found in the human airways. The tadpole skin has mucin-secreting cells, motile multi-ciliated cells, ionocytes (control local ionic homeostasis) and basal stem cells. This cellular architecture is very similar to the large airways of the human lungs and represents an easily accessible and experimentally tractable model system to explore the molecular details of mucociliary epithelia. Each of the cell types in the tadpole skin has a human equivalent and a conserved network of genes and signalling pathways for their differentiation has been discovered. Great insight into the function of each of the cell types has been achieved using the Xenopus model and this has enhanced our understanding of airway disease. This simple model has already had a profound impact on the field but, as molecular technologies (e.g. gene editing and live imaging) continue to develop apace, its use for understanding individual cell types and their interactions will likely increase. For example, its small size and genetic tractability make it an ideal model for live imaging of a mucociliary surface especially during environmental challenges such as infection. Further potential exists for the mimicking of human genetic mutations that directly cause airway disease and for the pre-screening of drugs against novel therapeutic targets.

A systematic, label-free method for identifying RNA-associated proteins in vivo provides insights into vertebrate ciliary beating

Cell-type specific RNA-associated proteins (RAPs) are essential for development and homeostasis in animals. Despite a massive recent effort to systematically identify RAPs, we currently have few comprehensive rosters of cell-type specific RAPs in vertebrate tissues. Here, we demonstrate the feasibility of determining the RNA-interacting proteome of a defined vertebrate embryonic tissue using DIF-FRAC, a systematic and universal (i.e., label-free) method. Application of DIF-FRAC to cultured tissue explants of Xenopus mucociliary epithelium identified dozens of known RAPs as expected, but also several novel RAPs, including proteins related to assembly of the mitotic spindle and regulation of ciliary beating. In particular, we show that the inner dynein arm tether Cfap44 is an RNA-associated protein that localizes not only to axonemes, but also to liquid-like organelles in the cytoplasm called DynAPs. This result led us to discover that DynAPs are generally enriched for RNA. Together, these data provide a useful resource for a deeper understanding of mucociliary epithelia and demonstrate that DIF-FRAC will be broadly applicable for systematic identification of RAPs from embryonic tissues.

ΔN-Tp63 mediates Wnt/β-catenin-induced inhibition of differentiation in basal stem cells of mucociliary epithelia

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 Are Required for the Development and Function of Vertebrate Mucociliary Epithelia

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.

Tissue Culture of Human Adult Adenoids and of Middle Ear Mucosa

The increased number of mucus producing cells as well as the presence of stratified squamous epithelium in pathological and experimental middle ear conditions, point towards the possibility of metaplastic changes of the middle ear mucosa, similar to the metaplastic capabilities of respiratory mucosae in general, as observed clinically or provoked experimentally. The purpose of this study was to develop a model of postembryonic human respiratory mucosae, in vitro, for the study of triggering or inducing factors involved in its normal and metaplastic differentiation. Explants from adenoids and middle ear mucosa were cultured, both as organ cultures and monolayers, for periods of up to two weeks, and their developmental characteristics were studied and described. Over 50% of the explants showed mitosis, epithelial and monolayer growth, ciliary activity and differentiation into ciliated and into mucus-producing cells. Adenoid explants were grown in air without and with added 5% CO2. Under the latter conditions, the proportion of explants and monolayers showing ciliary activity was 50% greater. It is concluded that this model might be suitable for further studies of the factors which control cyto-differentiation in mucociliary epithelia. Maintaining its growth for a longer period would, however, be desirable.