Chapter 13 Ciliary Dysfunction in Developmental Abnormalities and Diseases

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Abstract

Cilia are small microtubule‐based cellular appendages that are broadly classified as being either motile or immotile (primary cilia). Since their initial discovery several centuries ago, motile cilia have been of general interest to basic scientists and others who study the dynamics and physiological relevance of their motility. More recent discoveries have found that motile and immotile cilia, the later of which are present on nearly all cells in the mammalian body, also have major roles during development and in postnatal life. Dysfunction of the cilium is the basis for multiple human genetic disorders that have collectively been called the ciliopathies. The phenotypes associated with cilia dysfunction in mammals are diverse and include randomization of the left–right body axis, abnormalities in neural tube closure and patterning, skeletal defects such as polydactyly, cystic kidney, liver, and pancreatic diseases, blindness and anosmia, behavioral and cognitive defects, and obesity. The connection between disease and developmental defects due to the loss of ciliary function has brought the efforts of the biomedical research establishment to bear on this underappreciated and long overlooked organelle. Several groups have applied en silico, genetic, and biochemical approaches to identify the components of the cilia proteome. The resulting datasets have contributed to a remarkable increase in the rate at which human ciliopathy disease loci are being identified. This intense basic and clinical research interest has revealed that the cilium is a very complex sensory machine involved in transducing extracellular stimuli involved in many different signaling pathways into cellular responses. Although major advances have been made in understanding the importance of the cilium, it remains enigmatic how the cilium functions to coordinate signaling pathways and how loss of this organelle results in the severe defects observed in human ciliopathies.

Introduction

Although cilia and flagella and the processes responsible for their formation have been a great curiosity in the realm of cell biology for over a century, only recently has there been an insurgence of interest in the cilium as a clinically relevant organelle. In large part, the renaissance began with the discovery that cilia mutant mice exhibit random left–right body axis specification and that mutations in cilia localized proteins cause various forms of human renal cystic diseases. From these seminal studies, a new paradigm rapidly emerged in which the cilium serves a complex range of functions important during development and for maintaining normal tissue physiology. The dysfunction of this small microtubule‐based organelle, which was once considered to be a vestigial remnant of our evolutionary past, is now known to be a major contributor to a remarkable spectrum of diseases and developmental disorders referred to as the ciliopathies. Although the cilium is currently appreciated as an essential organelle, the challenge to elucidate its normal functions during development as well as in adult tissue homeostasis remains. Concomitant with this challenge is assessing how dysfunction of the cilium disrupts signaling events that contribute to the severe disease phenotypes associated with ciliopathies.

Cilia and flagella are highly complex structures that require more than 500 proteins for normal maintenance and function (Ostrowski et al., 2002, Pazour, 2004, Pazour et al., 2005). Since the cilium is devoid of ribosomes, cells must transport all cilia subcomponents from the cell body to the cilium. This is achieved through a process known as intraflagellar transport (IFT) which was first described in the laboratory of Dr. Rosenbaum (Kozminski et al., 1993, Kozminski et al., 1995, Kozminski et al., 1998). Briefly, IFT mediates the bidirectional transport of axonemal subunits and signaling machinery between the base and tip of the cilium (Cole, 2003, Pazour et al., 2002a, Scholey, 2003). A detailed description of IFT and its importance for cilia assembly and signaling is provided in Chapter 2 of this volume.

Cilia are found on most cells in the mammalian body (Olsen, 2005, Satir and Christensen, 2008). The ubiquitous nature of the cilium is in agreement with the wide spectrum of disease phenotypes associated with ciliary mutants (Afzelius, 2004, Eley et al., 2005). They extend from the surface of cells into the local environment and are classified as either motile or nonmotile. The primary cilium (9 + 0 microtubule architecture) is immotile and is present as a solitary structure on most cells including epithelia, fibroblasts, and neurons. However, there are also examples where a solitary cilium on a cell can be motile. This is observed on cells of the embryonic node where the rotational motility of the primary cilium is required for normal left–right body axis specification (for more detail on cilia and left–right axis formation, see Chapter 6, this volume) (Essner et al., 2002) and the flagella on sperm which are essential for cell movement (Inaba, 2003, Mitchell, 2007). In contrast, epithelial cells lining the ventricles of the brain (Worthington and Cathcart, 1963), the respiratory (Lansley et al., 1992) and reproductive tracts assemble hundreds of motile cilia (9 + 2 microtubule architecture) (Lyons et al., 2006) that beat in a synchronized manner to direct fluid movement. Disruption of normal cilia motility can result in a number of disease states in humans, such as hydrocephalus, chronic bronchiectasis, sinusitis, situs inversus, and infertility. Both motile and nonmotile cilia are thought to have important sensory functions that allow cells to receive and respond to extracellular stimuli (Huang et al., 2004, Josef et al., 2005, Pan and Snell, 2002).

Much of our current understanding of ciliary function and cilia mediated signaling activities have come from studies in model organisms such as Chlamydomonas, Trypanosome, Tetrahymena,Caenorhabditis elegans, zebrafish, and mice (Brown et al., 1999, Dentler, 2005, Evans et al., 2006, Hou et al., 2007, Krock and Perkins, 2008, Pan and Snell, 2005, Pan et al., 2006, Pedersen et al., 2005). In lower eukaryotes, disruption of cilia does not have a major impact on the development or survival of the organism, thus, these simpler model systems have had major utility. Their viability has allowed the identification of ciliary components, analysis of cilia assembly processes, and the study of cilia mediated regulation of signaling pathways. In contrast, disruption of cilia assembly in vertebrates, such as mice, is lethal at around embryonic day 9.5 (Murcia et al., 2000, Nonaka et al., 1998). This is due to severe developmental defects involving abnormal neural tube closure, neural tube patterning, and body axis specification. In humans, mutations that completely disrupt cilia formation (such as IFT null mutations) have yet to be identified which is likely due to lethality. However, there are now multiple examples where mutations in cilia proteins cause human diseases (see Table 13.1, Table 13.2, Table 13.3, Table 13.4, Table 13.5, Table 13.6) such as Primary Cilia Dyskinesia (PCD [OMIM 242650]), Polycystic Kidney Disease (PKD [OMIM 173900]), Nephronophthisis (NPHP [OMIM 256100]), Joubert syndrome (JBTS [OMIM 21330]), Meckel–Gruber syndrome (MKS [OMIM 249000]), Leber Congenital Amaurosis (LCA [OMIM 611755]), Senior–Loken syndrome (SLSN [OMIM 266900]), Kartagener syndrome (KS [OMIM 244400]), Alstrom syndrome (ALMS [OMIM 203800]), and Orofaciodigital type 1 syndrome (OFD1[OMIM 311200]) Asphyxiating thoracic Dystrophy (ATD [OMIM 208500]), Bardet–Biedl syndrome (BBS [OMIM 209900]) to mention a few. In this chapter, we will describe the functions attributed to the various forms of cilia and discuss the current understanding of cilia dysfunction and the associated disease and developmental phenotypes.

Section snippets

The Oak Ridge Polycystic Kidney (Orpk) Mouse: A Model For Human Ciliopathies

The best example of the spectrum of phenotypes associated with cilia dysfunction in mammals is seen in the oak ridge polycystic kidney mouse model (ORPK, ift88Tg737Rpw). The ORPK mouse was originally described as a model for human autosomal recessive polycystic kidney disease (ARPKD). ORPK mice have a hypomorphic mutation in a gene initially called Tg737 and now referred to as ift88. This model originated in the colony at the Oak Ridge National Laboratory though a large‐scale transgene

Functions and Phenotypes Associated with Abnormal Motile Cilia

Arguably, the best known cilia are the motile forms exemplified by those found on cells of the respiratory tract and ependymal cells lining the ventricles in the brain. In the respiratory tract, these cilia play important roles in mucus clearance; while the ependymal cilia mediate movement of cerebral spinal fluid. Motile cilia are also present in the female reproductive system facilitating the movement of the ovum. These motile cilia with 9 + 2 axoneme architecture, are normally present in

Functions and Diseases Associated with Immotile Cilium

The immotile primary cilium is present on most cells in the mammalian body with few exceptions such as bone marrow‐derived cells (Wheatley et al., 1996). For a recent listing of cell types known to have primary cilia, see the Primary Cilia Resource Site (http://www.primary-cilium.co.uk/). Until recently, primary cilia were thought to be of relative unimportance; however, this perception has changed. The organelle is now known to be critical for normal development as well as for tissue

Oligogenic Inheritance and Clinical Variability in the Ciliopathies

One fascinating aspect of the ciliopathies is their range of clinical variability. For example, the embryonic lethality observed in MKS as compared to the less severe multisystem disorders, such as BBS, or the more tissue specific disorder PKD. One can reconcile such a range of clinical outcomes when one thinks of the previously unappreciated complexity of the cilium.

In mouse models, it has become apparent that complete ablation of cilia formation and function leads to early embryonic lethality

Cilia Proteome and Genome Databases and the Identification of Human Ciliopathy Genes

An important resource that has helped propel the field of cilia biology from basic science into the realm of biomedical science has been the compilation of cilia/flagella proteomes from multiple organisms. Several different strategies have been utilized to define the protein makeup of cilia and each has its benefits and drawbacks. One of the initial approaches was based on the insightful observation of Swoboda et al. that multiple IFT and cilia genes in C. elegans are coregulated by the

Acknowledgments

This work was supported in part by the National Institutes of Health RO1 (NIDDK 65655, NIDDK 075996, HD056030) to BKY, National Kidney Foundation Postdoctoral award to NS, and a T32 award postdoctoral award (5T32HL007553, Dr. Janet Yother, UAB) to NFB. We thank Dr. Kathryn Anderson, Dr. Kirk Mykytyn for unpublished images. We also thank Mandy J. Croyle for technical assistance and Amber O'Connor and Sarah Mollo for critical reading of the manuscript.

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