American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 435-437, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2004-0004PS
Genetic Regulation of Cilia Assembly and the Relationship to Human Disease
Steven L. Brody
Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri
Address correspondence to: Steven L. Brody, M.D., Washington University School of Medicine, Campus Box 8052, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail: brodys{at}msnotes.wustl.edu
Abbreviations: primary ciliary dyskinesia, PCD • polycystic kidney disease, PKD
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Introduction
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Ciliated cells of the airway are morphologically distinguished by an apical bristle of  200 cilia per cell that sweep the lumen. Simple in concept but biologically complex, these cells have important roles in maintaining airway health, and are also prone to injury and affected by genetic mutation. Substantial progress has recently been made in understanding the molecular and genetic basis of ciliated cell defects. Phenotype dissection of genetically deficient mice has facilitated the recognition that a surprising number of diseases are related to defects in ciliogenesis and ciliated cell function. Notably fueled by these observations is the development of human genetic analysis for two cilia-related diseases: primary ciliary dyskinesia (PCD) and polycystic kidney disease (PKD).
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Motile and Sensory Cilia
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Ciliated cells are not restricted to the airways of the sinuses and lung. The ependymal cells lining the ventricles of the brain and the spinal canal are equipped with motile cilia. Reproduction depends on ciliated cells in Fallopian tubes and flagellated sperm. These ciliated cells are typically identified by the presence of multiple axonemes on the same cell, each containing the distinctive 9+2 microtubule organization (Figure 1A). In contrast, sensory cells contain monocilia with a 9+0 organization. These cilia can detect a variety of signals including flow, motion, or neurochemical signals (1). This provides specific function for cilia in the tubules of the kidney, the organ of Corti, retina, olfactory neuroepithelium, and a wide number of other cell types throughout the body.
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Figure 1. Ciliated cell structure and assembly. (A) Ciliated cell types, localization, and structure. Epithelial cells with motile cilia are present in airways and other organs together with nonciliated cells (dark shade). Motile cilia can be identified by the ultrastructure of 9+2 microtubule pairs and heavy chain dyneins present as "arms" on microtubules shown in a diagram of the axoneme cross-section. Primary cilia are typically sensory cilia present in renal tubules or in sensory organs. Axoneme cross-section ultrastructure shows the 9+0 microtubule pair structure. The heavy chain dyneins are absent, but other axoneme proteins such as polycystins are expressed. The embryonic node contains both classes of ciliated cells; however, motile cilia are monocilium. These cilia generate directional flow in the node to signal sensory cilia and initiate left–right asymmetric axis development. (B) Ciliated cell differentiation and axoneme assembly. Unique programs direct motile and sensory ciliated cell differentiation to regulate expression of specific genes for diverse functions. In each tissue, a precursor cell must receive a signal to commit to the ciliated cell phenotype then produce centrioles that dock as basal bodies (mediated by foxj1 in motile cells) and axoneme proteins (e.g., microtubules, dyneins, and polycystins). Axoneme assembly proceeds by intraflagellar transports (IFT) through motor proteins moving outward (e.g., Kif3 complexes) or retrograde (e.g., IFT dyneins).
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Cilia and Situs Inversus
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Amazingly, interplay between motile and sensory cilia is required for determination of left–right axis in early vertebrate development. Several recent breakthroughs indicate that cilia direct left–right asymmetry by signaling within the embryonic node soon after anterior–posterior and proximal–distal axes are established. The node is a transiently appearing, bowl-like structure within the midline notochord containing an elegant cilia-powered apparatus. In the center of this bowl are cells with a single motile cilium (moving in an unusual vortical motion) that generate vectoral flow to transmit a signal received by primary cilia on the periphery (2). These sensory cilia bend in response to flow and generate a calcium-dependent response. This response triggers a program resulting in a left-sided, asymmetric heart and asymmetric patterning of visceral organs. Thus, left–right axis formation depends on proper node cell differentiation and cilia function. Accordingly, failure in node cilia function can result in randomization of left–right axis (half have situs inversus).
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Cilia, Dyneins, Cilia, and Kartagener's Syndrome
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Cilia motility is produced by dyneins, proteins that are molecular motors. Dynein family members function within the cytoplasm for microtubule-based transport and within the axoneme of the cilia for transport and motility. Afzielius and coworkers recognized that these dyneins, visualized by electron microscopy as inner and outer arms on microtubules, were absent in individuals with impaired cilia beating as well as situs inversus, and hypothesized that this was the root of Kartagener's syndrome—bronchietasis, nasal polyps/chronic sinusitis, and situs inversus (3). It was over twenty-five years later that the molecular and genetic bases of Afzelius's important observations were uncovered.
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Molecular Basis of PCD
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Progress in molecular biology and production of genetically deficient mice has sped understanding the genetic basis of PCD (ciliary dysmotility associated with otitis, sinusitis, bronchietasis, and infertility, with or without situs inversus). The first firm molecular data linking cilia dysfunction and left–right asymmetry were established by (i) the identification of a spontaneously mutated heavy chain dynein gene (left–right dynein, lrd) in the embryonic node of a mouse known for almost half a century to have randomized situs (half the mice had situs inverusus), and (ii) the observation that mice engineered deficient in a subunit of the heterotrimeric kinesin motor protein KIF3 had absent nodal cilia and random situs (4, 5). These reports were rapidly followed by description of situs inversus in mice with target deletion of other dynein genes and characterization of mutations in the human homologs of the deleted mouse genes in patients with PCD. One of the best examples of this relationship between finding in mice and man is that interruption of the mouse axonemal dynein heavy chain 5 (Mdnah5) resulted in mice with a Kartagener's phenotype: situs inversus, ciliary dysmotility, recurrent respiratory infections (6). Similarly, mutations in the human dynein axonemal heavy chair DNAH5 have been isolated in individuals with PCD (7). To date, mutations in DNAH5 and in the axonemal dynein intermediate chain gene 1 (DNAI1) have been found to result in PCD (8). A mutation in the human LRD homolog axonemal heavy chain type 11 (DNAH11) is also associated with situs inversus and ciliary dyskinesia. Though this mutation is hypothesized to affect motor function, it does not result in the ultrastructural loss of microtubule arms (9). These findings emphasize the inadequacy of using only clinical symptoms and transmission electron microscopy for PCD diagnosis, and indicate the paucity of known genetic defects in PCD.
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Can an Unexpected Knockout Mouse Phenotype Uncover a New PCD Gene?
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The recognition that a DNA polymerase knockout mouse had an unexpected Kartagener's syndrome phenotype led to the cloning of a novel protein (named DPCD) reported in this issue of the AJRCMB by Zariwala and colleagues (10). The authors describe how the Dpcd gene was inadvertently deleted on the negative strand of DNA during engineered interruption of the DNA polymerase  gene. This study identifies another potential gene in PCD and also lends pause to consider how often other unexpected phenotypes might also be due to this type of problem! Screening by Ostrowski's group did not identify Dpcd mutations in families with PCD, but cilia and left–right asymmetry defects are genetically and clinically heterogeneous disorders. This is, in part, due to the relatively small number of well characterized affected families, the huge size of dynein genes, the large number of different dyneins that are present in the axoneme, and the abundance of other regulatory and structural proteins required for cilia function. Surely, dynein mutations will not be the only cause of PCD. Thus, understanding the biology of cilia and ciliated cells will lead to identification of the genetic basis of lung and other diseases.
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Human Diseases of Sensory Cilia
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Not all mice with left–right asymmetry defects due to deficient proteins in node cilia have defects in microtubule "arms" or even in motile cilia function. Cilia diseases may also be due to isolated defects in sensory cilia: PKD, retinal disease, deafness, growth syndromes (Bardet Biedel syndrome) are now associated with ciliogenesis defects (11–13). In fact, work relating ciliogenesis defects to PKD has accelerated faster than PCD investigation. Epithelial cells of the renal tubules have primary cilium extending into the lumen that sense flow by bending, then signaling through a calcium-dependent channel. It is not fully understood how a mutation in polycystin genes (polycystin-1 or -2) or inversin (in infantile nephronophthisis) results in abnormal ciliogenesis and renal cyst formation (reviewed in Ref. 14). Interruption of inversin in mice results in cystic kidneys, but also pancreatic islet cell dysplasia, and situs inversus. Whereas polycystins are localized within the ciliary axoneme, inversin localizes within basal bodies, suggesting the potential that this protein (and others that localize in the basal body) may also have a role in cell mitosis.
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Central Questions Regarding Ciliated Cell Differentiation, Ciliogenesis, and Intraflagellar Transport
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Little is understood about differences in cell programs that are responsible for differentiation of sensory and motile ciliated cells. Ciliated cells arise in the airway from undifferentiated cells in the trachea during the canalicular stage, and as lung development proceeds, cilia progressively appear in large, then small airways. The preciliated cell initiates ciliogenesis through an unknown signal (Figure 1B). Over 250 proteins are then required for ciliogenesis. The stages of ciliogenesis are conceived to be different for motile and sensory ciliated cells, but much of this is inferred from ultrastructural analysis of electron micrographs in developing and repairing airway cells and in Chamydomonas (15, 16). These studies support a multistep paradigm by which precursor structures give rise to centrioles that subsequently transform to basal bodies. In sensory ciliogenesis, the lone basal body arises from the same centriole that organizes the mitotic spindle. The basal body migrates apically, then docks at the apical membrane to initiate axoneme formation. Axoneme assembly occurs as cargo in large multiprotein complexes moves along microtubules bidirectionally in a process called intraflagellar transport (IFT) (17). Transport from the cell to the distal axoneme is driven by a heterotrimeric complex of kinesin kif3 and retrograde movement is driven by cytoplasmic dynein (Figure 1B). IFT cargoes include polypeptides for assembly, motor, and sensory functions. Genetic defects in IFT motors or cargo have severe consequences. Failed IFT is related to not only PKD, but also the genetic disorder retinitis pigmentosa. The relationship between IFT defects and PCD is also postulated, but the molecular defect is yet to be identified; however, some PCD patients are reported to have eye, hearing, and renal disorders.
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More than Sweeping the Airways—Other Unique Functions of Ciliated Cells
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Much as renal tubule cells have importance in salt and water transport, the ciliated cell is also equipped with specialized tools for transport and signaling. The apical membrane of the ciliated cell is uniquely organized by scaffolding proteins not present on Clara cells. These scaffold and adapter proteins such as ezrin and EBP50/NHERF bind the apical membrane proteins cystic fibrosis transmembrane conductance regulator (CFTR) and ß2-adrenergic receptor in macromolecular complexes (18, 19). Within the airway, these proteins are only expressed on ciliated, but not Clara cells (20). Thus, the ciliated cell controls airway liquid in a process that is paired with cilia transport, possibly to optimize regulation of airway clearance. In addition, growth factor receptors, drug transporters (p-glycoprotein), and Toll-like receptors have been reported to be uniquely present in the airway on ciliated cells. Future efforts related to the molecular analysis of ciliated cell differentiation as well as the process of ciliogenesis will likely uncover the genetic root of other airway diseases and may reveal the pathophysiology of acquired lung diseases.
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Acknowledgments
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Supported, in part, by the National Institutes of Health (HL56244 and HL63988), and Career Investigator Award from the American Lung Association.
Received in original form January 5, 2004
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Copyright © 2004 American Thoracic Society.
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Introduction
Motile and Sensory Cilia