Multiciliated cell development and ciliary resorption at the mammalian choroid plexus.

Ciliopathies are a group of diseases caused by defects in cilia, hair-like organelles that can have many functions ranging from regulating extracellular fluid flow to sensing mechanical or chemical stimuli. Multiciliated cells (MCCs) with motile cilia are found in locations that include the central nervous system, where they are critical for homeostasis. Specifically, ependymal MCCs line the brain ventricles and central canal of the spinal cord, while other specialized MCCs occupy highly vascularized structures known as the choroid plexuses (ChPs) and produce cerebrospinal fluid (CSF). Now, a recent study has shown that murine ChP MCCs develop nodal-like cilia. Interestingly, ChP cilia were found to undergo resorption during early postnatal stages in part through axoneme regression, and this phenomenon was mirrored in human postmortem ChP samples. Taken together, these findings reveal important new insights about the ultrastructure of MCCs that comprise the mammalian ChP, and may have ramifications for other MCC populations in health and disease states.

Neuroepithelial bodies and terminal bronchioles are niches for distinctive club cells that repair the airways following acute notch inhibition.

Lower airway club cells (CCs) serve the dual roles of a secretory cell and a stem cell. Here, we probe how the CC fate is regulated. We find that, in response to acute perturbation of Notch signaling, CCs adopt distinct fates. Although the vast majority transdifferentiate into multiciliated cells, a "variant" subpopulation (v-CCs), juxtaposed to neuroepithelial bodies (NEBs; 5%-10%) and located at bronchioalveolar duct junctions (>80%), does not. Instead, v-CCs transition into lineage-ambiguous states but can revert to a CC fate upon restoration of Notch signaling and repopulate the airways with CCs and multiciliated cells. The v-CC response to Notch inhibition is dependent on localized activation of ß-catenin in v-CCs. We propose that the CC fate is stabilized by canonical Notch signaling, that airways are susceptible to perturbations to this pathway, and that NEBs/terminal bronchioles comprise niches that modulate CC plasticity via ß-catenin activation to facilitate airway repair post Notch inhibition.

The FAM13A Long Isoform Regulates Cilia Movement and Coordination in Airway Mucociliary Transport.

Single nucelotide polymorphisms (SNPs) at the FAM13A locus are among the most commonly reported risk alleles associated with chronic obstructive pulmonary disease (COPD) and other respiratory diseases; however, the physiological role of FAM13A is unclear. In humans, two major protein isoforms are expressed at the FAM13A locus: "long" and "short," but their functions remain unknown, partly because of a lack of isoform conservation in mice. We performed in-depth characterization of organotypic primary human airway epithelial cell subsets and show that multiciliated cells predominantly express the FAM13A long isoform containing a putative N-terminal Rho GTPase-activating protein (RhoGAP) domain. Using purified proteins, we directly demonstrate the RhoGAP activity of this domain. In Xenopus laevis, which conserve the long-isoform, Fam13a deficiency impaired cilia-dependent embryo motility. In human primary epithelial cells, long-isoform deficiency did not affect multiciliogenesis but reduced cilia coordination in mucociliary transport assays. This is the first demonstration that FAM13A isoforms are differentially expressed within the airway epithelium, with implications for the assessment and interpretation of SNP effects on FAM13A expression levels. We also show that the long FAM13A isoform coordinates cilia-driven movement, suggesting that FAM13A risk alleles may affect susceptibility to respiratory diseases through deficiencies in mucociliary clearance.

The binding of LARP6 and DNAAF6 in biomolecular condensates influences ciliogenesis of multiciliated cells.

Motile cilia on the cell surface produce fluid flows in the body and abnormalities in motile cilia cause primary ciliary dyskinesia. Dynein axonemal assembly factor 6 (DNAAF6), a causative gene of primary ciliary dyskinesia, was isolated as an interacting protein with La ribonucleoprotein 6 (LARP6) that regulates ciliogenesis in multiciliated cells (MCCs). In MCCs of Xenopus embryos, LARP6 and DNAAF6 were colocalized in biomolecular condensates termed dynein axonemal particles and synergized to control ciliogenesis. Moreover, tubulin alpha 1c-like mRNA encoding α-tubulin protein, that is a major component of ciliary axoneme, was identified as a target mRNA regulated by binding LARP6. While DNAAF6 was necessary for high α-tubulin protein expression near the apical side of Xenopus MCCs during ciliogenesis, its mutant, which abolishes binding with LARP6, was unable to restore the expression of α-tubulin protein near the apical side of MCCs in Xenopus DNAAF6 morphant. These results indicated that the binding of LARP6 and DNAAF6 in dynein axonemal particles regulates highly expressed α-tubulin protein near the apical side of Xenopus MCCs during ciliogenesis.

Planar Cell Polarity in the Multiciliated Epithelial Lining of the Mouse Eustachian Tube.

OBJECTIVES: Planar cell polarity (PCP) signaling, essential for uniform alignment and directional beating of motile cilia, has been investigated in multiciliated epithelia. As a complex structure connecting the middle ear to the nasopharynx, the eustachian tube (ET) is important in the onset of ear-nose-throat diseases. However, PCP signaling, including the orientation that is important for ciliary motility and clearance function in the ET, has not been studied. We evaluated PCP in the ET epithelium. STUDY DESIGN: Morphometric examination of the mouse ET. METHODS: We performed electron microscopy to assess ciliary polarity in the mouse ET, along with immunohistochemical analysis of PCP protein localization in the ET epithelium. RESULTS: We discovered PCP in the ET epithelium. Motile cilia were aligned in the same direction in individual and neighboring cells; this alignment manifested as ciliary polarity in multiciliated cells. Additionally, PCP proteins were asymmetrically localized between adjacent cells in the plane of the ET. CONCLUSIONS: The multiciliated ET epithelium exhibits polarization, suggesting novel structural features that may be critical for ET function. LEVEL OF EVIDENCE: NA Laryngoscope, 134:3795-3801, 2024.

Dual-color live imaging unveils stepwise organization of multiple basal body arrays by cytoskeletons.

For mucociliary clearance of pathogens, tracheal multiciliated epithelial cells (MCCs) organize coordinated beating of cilia, which originate from basal bodies (BBs) with basal feet (BFs) on one side. To clarify the self-organizing mechanism of coordinated intracellular BB-arrays composed of a well-ordered BB-alignment and unidirectional BB-orientation, determined by the direction of BB to BF, we generated double transgenic mice with GFP-centrin2-labeled BBs and mRuby3-Cep128-labeled BFs for long-term, high-resolution, dual-color live-cell imaging in primary-cultured tracheal MCCs. At early timepoints of MCC differentiation, BB-orientation and BB-local alignment antecedently coordinated in an apical microtubule-dependent manner. Later during MCC differentiation, fluctuations in BB-orientation were restricted, and locally aligned BB-arrays were further coordinated to align across the entire cell (BB-global alignment), mainly in an apical intermediate-sized filament-lattice-dependent manner. Thus, the high coordination of the BB-array was established for efficient mucociliary clearance as the primary defense against pathogen infection, identifying apical cytoskeletons as potential therapeutic targets.

Crb3 is required to organize the apical domain of multiciliated cells.

Cell shape changes mainly rely on the remodeling of the actin cytoskeleton. Multiciliated cells (MCCs) of the mucociliary epidermis of Xenopus laevis embryos, as they mature, dramatically reshape their apical domain to grow cilia, in coordination with the underlying actin cytoskeleton. Crumbs (Crb) proteins are multifaceted transmembrane apical polarity proteins known to recruit actin linkers and promote apical membrane growth. Here, we identify the homeolog Crb3.L as an important player for the migration of centrioles or basal bodies (collectively centrioles/BBs) and apical domain morphogenesis in MCCs. Crb3.L is present in cytoplasmic vesicles close to the ascending centrioles/BBs, where it partially colocalizes with Rab11a. Crb3.L morpholino-mediated depletion in MCCs caused abnormal migration of centrioles/BBs, a reduction of their apical surface, disorganization of their apical actin meshwork and defective ciliogenesis. Rab11a morpholino-mediated depletion phenocopied Crb3.L loss-of-function in MCCs. Thus, the control of centrioles/BBs migration by Crb3.L might be mediated by Rab11a-dependent apical trafficking. Furthermore, we show that both phospho-activated ERM (pERM; Ezrin-Radixin-Moesin) and Crb3.L are recruited to the growing apical domain of MCCs, where Crb3.L likely anchors pERM, allowing actin-dependent expansion of the apical membrane.

An amino acid-resolution interactome for motile cilia illuminates the structure and function of ciliopathy protein complexes.

Motile cilia are ancient, evolutionarily conserved organelles whose dysfunction underlies motile ciliopathies, a broad class of human diseases. Motile cilia contain myriad different proteins that assemble into an array of distinct machines, so understanding the interactions and functional hierarchies among them presents an important challenge. Here, we defined the protein interactome of motile axonemes using cross-linking mass spectrometry (XL/MS) in Tetrahymena thermophila. From over 19,000 XLs, we identified 4,757 unique amino acid interactions among 1,143 distinct proteins, providing both macromolecular and atomic-scale insights into diverse ciliary machines, including the Intraflagellar Transport system, axonemal dynein arms, radial spokes, the 96 nm ruler, and microtubule inner proteins, among others. Guided by this dataset, we used vertebrate multiciliated cells to reveal novel functional interactions among several poorly-defined human ciliopathy proteins. The dataset therefore provides a powerful resource for studying the basic biology of an ancient organelle and the molecular etiology of human genetic disease.

Transcriptional Regulation of Airway Epithelial Cell Differentiation: Insights into the Notch Pathway and Beyond.

The airway epithelium is a critical component of the respiratory system, serving as a barrier against inhaled pathogens and toxins. It is composed of various cell types, each with specific functions essential to proper airway function. Chronic respiratory diseases can disrupt the cellular composition of the airway epithelium, leading to a decrease in multiciliated cells (MCCs) and an increase in secretory cells (SCs). Basal cells (BCs) have been identified as the primary stem cells in the airway epithelium, capable of self-renewal and differentiation into MCCs and SCs. This review emphasizes the role of transcription factors in the differentiation process from BCs to MCCs and SCs. Recent advancements in single-cell RNA sequencing (scRNAseq) techniques have provided insights into the cellular composition of the airway epithelium, revealing specialized and rare cell types, including neuroendocrine cells, tuft cells, and ionocytes. Understanding the cellular composition and differentiation processes within the airway epithelium is crucial for developing targeted therapies for respiratory diseases. Additionally, the maintenance of BC populations and the involvement of Notch signaling in BC self-renewal and differentiation are discussed. Further research in these areas could provide valuable insights into the mechanisms underlying airway epithelial homeostasis and disease pathogenesis.

Immunomodulatory effects of inactivated Ligilactobacillus salivarius CECT 9609 on respiratory epithelial cells.

The microbiota in humans and animals play crucial roles in defense against pathogens and offer a promising natural source for immunomodulatory products. However, the development of physiologically relevant model systems and protocols for testing such products remains challenging. In this study, we present an experimental condition where various natural products derived from the registered lactic acid bacteria Ligilactobacillus salivarius CECT 9609, known for their immunomodulatory activity, were tested. These products included live and inactivated bacteria, as well as fermentation products at different concentrations and culture times. Using our established model system, we observed no morphological changes in the airway epithelium upon exposure to Pasteurella multocida, a common respiratory pathogen. However, early molecular changes associated with the innate immune response were detected through transcript analysis. By employing diverse methodologies ranging from microscopy to next-generation sequencing (NGS), we characterized the interaction of these natural products with the airway epithelium and their potential beneficial effects in the presence of P. multocida infection. In particular, our discovery highlights that among all Ligilactobacillus salivarius CECT 9609 products tested, only inactivated cells preserve the conformation and morphology of respiratory epithelial cells, while also reversing or altering the natural immune responses triggered by Pasteurella multocida. These findings lay the groundwork for further exploration into the protective role of these bacteria and their derivatives.

RBL2 represses the transcriptional activity of Multicilin to inhibit multiciliogenesis.

A core pathophysiologic feature underlying many respiratory diseases is multiciliated cell dysfunction, leading to inadequate mucociliary clearance. Due to the prevalence and highly variable etiology of mucociliary dysfunction in respiratory diseases, it is critical to understand the mechanisms controlling multiciliogenesis that may be targeted to restore functional mucociliary clearance. Multicilin, in a complex with E2F4, is necessary and sufficient to drive multiciliogenesis in airway epithelia, however this does not apply to all cell types, nor does it occur evenly across all cells in the same cell population. In this study we further investigated how co-factors regulate the ability of Multicilin to drive multiciliogenesis. Combining data in mouse embryonic fibroblasts and human bronchial epithelial cells, we identify RBL2 as a repressor of the transcriptional activity of Multicilin. Knockdown of RBL2 in submerged cultures or phosphorylation of RBL2 in response to apical air exposure, in the presence of Multicilin, allows multiciliogenesis to progress. These data demonstrate a dynamic interaction between RBL2 and Multicilin that regulates the capacity of cells to differentiate and multiciliate. Identification of this mechanism has important implications for facilitating MCC differentiation in diseases with impaired mucociliary clearance.

Bidirectional multiciliated cell extrusion is controlled by Notch-driven basal extrusion and Piezo1-driven apical extrusion.

Xenopus embryos are covered with a complex epithelium containing numerous multiciliated cells (MCCs). During late-stage development, there is a dramatic remodeling of the epithelium that involves the complete loss of MCCs. Cell extrusion is a well-characterized process for driving cell loss while maintaining epithelial barrier function. Normal cell extrusion is typically unidirectional, whereas bidirectional extrusion is often associated with disease (e.g. cancer). We describe two distinct mechanisms for MCC extrusion, a basal extrusion driven by Notch signaling and an apical extrusion driven by Piezo1. Early in the process there is a strong bias towards basal extrusion, but as development continues there is a shift towards apical extrusion. Importantly, response to the Notch signal is age dependent and governed by the maintenance of the MCC transcriptional program such that extension of this program is protective against cell loss. In contrast, later apical extrusion is regulated by Piezo1, such that premature activation of Piezo1 leads to early extrusion while blocking Piezo1 leads to MCC maintenance. Distinct mechanisms for MCC loss underlie the importance of their removal during epithelial remodeling.

The MIR34B/C genomic region contains multiple potential regulators of multiciliogenesis.

The MIR449 genomic locus encompasses several regulators of multiciliated cell (MCC) formation (multiciliogenesis). The miR-449 homologs miR-34b/c represent additional regulators of multiciliogenesis that are transcribed from another locus. Here, we characterized the expression of BTG4, LAYN, and HOATZ, located in the MIR34B/C locus using single-cell RNA-seq and super-resolution microscopy from human, mouse, or pig multiciliogenesis models. BTG4, LAYN, and HOATZ transcripts were expressed in both precursors and mature MCCs. The Layilin/LAYN protein was absent from primary cilia, but it was expressed in apical membrane regions or throughout motile cilia. LAYN silencing altered apical actin cap formation and multiciliogenesis. HOATZ protein was detected in primary cilia or throughout motile cilia. Altogether, our data suggest that the MIR34B/C locus may gather potential actors of multiciliogenesis.

IL-4 drastically decreases deuterosomal and multiciliated cells via alteration in progenitor cell differentiation.

BACKGROUND: Allergic inflammation affects the epithelial cell populations resulting in goblet cell hyperplasia and decreased ciliated cells. Recent advances in single-cell RNA sequencing (scRNAseq) have enabled the identification of new cell subtypes and genomic features of single cells. In this study, we aimed to investigate the effect of allergic inflammation in nasal epithelial cell transcriptomes at the single-cell level. METHODS: We performed scRNAseq in cultured primary human nasal epithelial (HNE) cells and in vivo nasal epithelium. The transcriptomic features and epithelial cell subtypes were determined under IL-4 stimulation, and cell-specific marker genes and proteins were identified. RESULTS: We confirmed that cultured HNE cells were similar to in vivo epithelial cells through scRNAseq. Cell-specific marker genes were utilized to cluster the cell subtypes, and FOXJ1+ -ciliated cells were sub-classified into multiciliated and deuterosomal cells. PLK4 and CDC20B were specific for deuterosomal cells, and SNTN, CPASL, and GSTA2 were specific for multiciliated cells. IL-4 altered the proportions of cell subtypes, resulting in a decrease in multiciliated cells and loss of deuterosomal cells. The trajectory analysis revealed deuterosomal cells as precursor cells of multiciliated cells and deuterosomal cells function as a bridge between club and multiciliated cells. A decrease in deuterosomal cell marker genes was observed in nasal tissue samples with type 2 inflammation. CONCLUSION: The effects of IL-4 appear to be mediated through the loss of the deuterosomal population, resulting in the reduction in multiciliated cells. This study also newly suggests cell-specific markers that might be pivotal for investigating respiratory inflammatory diseases.

Structures and functions of cilia during vertebrate embryo development.

Cilia are hair-like structures that project from the surface of cells. In vertebrates, most cells have an immotile primary cilium that mediates cell signaling, and some specialized cells assemble one or multiple cilia that are motile and beat synchronously to move fluids in one direction. Gene mutations that alter cilia structure or function cause a broad spectrum of disorders termed ciliopathies that impact virtually every system in the body. A wide range of birth defects associated with ciliopathies underscores critical functions for cilia during embryonic development. In many cases, the mechanisms underlying cilia functions during development and disease remain poorly understood. This review describes different types of cilia in vertebrate embryos and discusses recent research results from diverse model systems that provide novel insights into how cilia form and function during embryo development. The work discussed here not only expands our understanding of in vivo cilia biology, but also opens new questions about cilia and their roles in establishing healthy embryos.

Spef1/CLAMP binds microtubules and actin-based structures and regulates cell migration and epithelia cell polarity.

During migration, cells invade, repair, and create barriers leading to the formation of new cellular contacts in target tissues. Cell migration requires many proteins that collectively form the cytoskeleton. The main cytoskeletal elements are actin filaments, microtubules (MTs), and intermediate filaments. These structures work in concert with a large number of accessory proteins that contribute in a variety of ways to regulate filament assembly and turnover, to alter the configuration or arrangement of filaments by bundling or crosslinking, to link the cytoskeleton to other structures in the cell, such as membranes and junctions, and to transport cargo along the filaments. Sperm flagella protein-1 (Spef1), also designated calponin homology and microtubules-associated protein (CLAMP), is a multifunctional protein that interacts with cytoskeletal structures, including MTs, actin filaments, and focal adhesions in epithelia. In this review, we outline Spef1/CLAMP structure and expression in several cellular models. The function of Spef1/CLAMP in flagellar and ciliary motility, MT-binding and stability, regulation of planar cell polarity, and potential contribution to the maintenance of actin-based structures, such as lamellipodia and filopodia during cell migration, are also discussed.

Aberrant Multiciliogenesis in Idiopathic Pulmonary Fibrosis.

We previously identified a novel molecular subtype of idiopathic pulmonary fibrosis (IPF) defined by increased expression of cilium-associated genes, airway mucin gene MUC5B, and KRT5 marker of basal cell airway progenitors. Here we show the association of MUC5B and cilia gene expression in human IPF airway epithelial cells, providing further rationale for examining the role of cilium genes in the pathogenesis of IPF. We demonstrate increased multiciliogenesis and changes in motile cilia structure of multiciliated cells both in IPF and bleomycin lung fibrosis models. Importantly, conditional deletion of a cilium gene, Ift88 (intraflagellar transport 88), in Krt5 basal cells reduces Krt5 pod formation and lung fibrosis, whereas no changes are observed in Ift88 conditional deletion in club cell progenitors. Our findings indicate that aberrant injury-activated primary ciliogenesis and Hedgehog signaling may play a causative role in Krt5 pod formation, which leads to aberrant multiciliogenesis and lung fibrosis. This implies that modulating cilium gene expression in Krt5 cell progenitors is a potential therapeutic target for IPF.

Motile Cilia on Kidney Proximal Tubular Epithelial Cells Are Associated With Tubular Injury and Interstitial Fibrosis.

It is well established that mammalian kidney epithelial cells contain a single non-motile primary cilium (9 + 0 pattern). However, we noted the presence of multiple motile cilia with a central microtubular pair (9 + 2 pattern) in kidney biopsies of 11 patients with various kidney diseases, using transmission electron microscopy. Immunofluorescence staining revealed the expression of the motile cilia-specific markers Radial Spoke Head Protein 4 homolog A, Forkhead-box-protein J1 and Regulatory factor X3. Multiciliated cells were exclusively observed in proximal tubuli and a relative frequent observation in human kidney tissue: in 16.7% of biopsies with tubular injury and atrophy (3 of 18 tissues), in 17.6% of biopsies from patients with membranous nephropathy (3 of 17 tissues) and in 10% of the human kidney tissues derived from the unaffected pole after tumour nephrectomy (3 of 30 tissues). However, these particular tissues showed marked tubular injury and fibrosis. Further analysis showed a significant relation between the presence of multiciliated cells and an increased expression of alpha-smooth-muscle-actin (p-value < 0.01) and presence of Kidney-injury-molecule-1 (p-value < 0.01). Interestingly, multiciliated cells co-showed staining for the scattered tubular cell markers annexin A2, annexin A3, vimentin and phosphofructokinase platelet but not with cell senescence associated markers, like (p16) and degradation of lamin B. In conclusion, multiciliated proximal tubular cells with motile cilia were frequently observed in kidney biopsies and associated with tubular injury and interstitial fibrosis. These data suggest that proximal tubular cells are able to transdifferentiate into multiciliated cells.

Essential Roles of Efferent Duct Multicilia in Male Fertility.

Cilia are microtubule-based hair-like organelles on the cell surface. Cilia have been implicated in various biological processes ranging from mechanosensation to fluid movement. Ciliary dysfunction leads to a plethora of human diseases, known as ciliopathies. Although non-motile primary cilia are ubiquitous, motile multicilia are found in restricted locations of the body, such as the respiratory tract, the oviduct, the efferent duct, and the brain ventricles. Multicilia beat in a whip-like motion to generate fluid flow over the apical surface of an epithelium. The concerted ciliary motion provides the driving force critical for clearing airway mucus and debris, transporting ova from the ovary to the uterus, maintaining sperm in suspension, and circulating cerebrospinal fluid in the brain. In the male reproductive tract, multiciliated cells (MCCs) were first described in the mid-1800s, but their importance in male fertility remained elusive until recently. MCCs exist in the efferent ducts, which are small, highly convoluted tubules that connect the testis to the epididymis and play an essential role in male fertility. In this review, we will introduce multiciliogenesis, discuss mouse models of male infertility with defective multicilia, and summarize our current knowledge on the biological function of multicilia in the male reproductive tract.

Diversity and function of motile ciliated cell types within ependymal lineages of the zebrafish brain.

Motile cilia defects impair cerebrospinal fluid (CSF) flow and can cause brain and spine disorders. The development of ciliated cells, their impact on CSF flow, and their function in brain and axial morphogenesis are not fully understood. We have characterized motile ciliated cells within the zebrafish brain ventricles. We show that the ventricles undergo restructuring through development, involving a transition from mono- to multiciliated cells (MCCs) driven by gmnc. MCCs co-exist with monociliated cells and generate directional flow patterns. These ciliated cells have different developmental origins and are genetically heterogenous with respect to expression of the Foxj1 family of ciliary master regulators. Finally, we show that cilia loss from the tela choroida and choroid plexus or global perturbation of multiciliation does not affect overall brain or spine morphogenesis but results in enlarged ventricles. Our findings establish that motile ciliated cells are generated by complementary and sequential transcriptional programs to support ventricular development.