Axonemal positioning and orientation in three-dimensional space for primary cilia: What is known, what is assumed, and what needs clarification

Authors


Abstract

Two positional characteristics of the ciliary axoneme—its location on the plasma membrane as it emerges from the cell, and its orientation in three-dimensional (3D) space—are known to be critical for optimal function of actively motile cilia (including nodal cilia), as well as for modified cilia associated with special senses. However, these positional characteristics have not been analyzed to any significant extent for primary cilia. This review briefly summarizes the history of knowledge of these two positional characteristics across a wide spectrum of cilia, emphasizing their importance for proper function. Then the review focuses what is known about these same positional characteristics for primary cilia in all major tissue types where they have been reported. The review emphasizes major areas that would be productive for future research for understanding how positioning and 3D orientation of primary cilia may be related to their hypothesized signaling roles within different cellular populations. Developmental Dynamics 240:2405–2431, 2011. © 2011 Wiley Periodicals, Inc.

INTRODUCTION

Ciliary axonemal variation is an impressive example of a wide spectrum of modifications to this radially symmetrical, microtubular, plasma-membrane covered organelle, reflecting an apparent optimization for cell and tissue specific functions. Historically, the understanding of ciliary structure and function has been linked to advances in analytical techniques—from the first description by light microscopy more than a century ago (Zimmermann,1898), to detailed observations of ultrastructure 50 years ago, to complex molecular dissections today. Current literature contains a plethora of diagrammatic representations of axonemal ultrastructure coupled with decorative specific molecular motifs, illustrating the adaptive modifications of basic structure across the range of cilia from those whose function is tied to active rapid motility to those that remain stationary (such as found in rods and cones), to the most recent favored child of the ciliary family—the primary cilium. For any class of cilia, a complete description of its morphology linked to function requires knowledge of a diverse set of attributes that includes everything from axonemal and basal body structural biochemistry, IFT transport systems/motors, molecular machinery for sensing/signal transduction, length………a list that eventually should find a place for the approximately 2,500 putative genes and their associated proteins currently thought to be present in the ciliome (Gherman et al.,2006; Inglis et al.,2006; Thomas et al.,2010; Ocbina et al.,2011; Tasouri and Tucker,2011; Lai et al.,2011).

However, additional significant attributes relating to spatial relationships of the cilium also are critical for normal function. This review focuses on two positional characteristics of cilia—the location of emergence of the axoneme from the parent cell and the orientation of the axoneme in 3D space. The purpose is to summarize what is known about these two spatial characteristics of the axoneme of the primary cilium and their significance for function by presenting them within the broad conceptual framework of what is known to be significant about positional characteristics across the range of ciliary axonemal modifications seen in nature.

A half century after the first description of a solitary cilium in kidney tubular epithelial cells (Zimmermann, 1898), electron microscopical analyses became routine and the primary cilium rapidly was recognized as an organelle in cells from a range of different tissues (reviewed in del Cerro and Snider,1969; Wheatley,2005). Starting during the 2 decades between 1960 and 1980 and continuing to the present, primary cilia have been described not only in a wide range of epithelial cells, but also in several neuronal types (Barnes,1961; del Cerro and Snider,1969; Fuchs and Schwark,2004; Berbari et al.,2007; Green and Mykytyn,2010; Whitfield and Chakravarthy, 2009; Han and Alvarez-Bulla, 2010; Besse et al.,2011; Lee and Gleeson,2011; Yoshimura et al.,2011), myocytes (Sorokin,1962; Lu et al.,2008; Clement et al.,2009; Shi and Tarbell,2011), and cells of connective tissues (Scherft and Daems,1967; Garant et al.,1968; Federman and Nichols,1974; Wilsman,1978,1979; Couve,1986; Whitfield,2003; Malone et al.,2007; Donnelly et al.,2008; Thivichon-Prince et al.,2009; Temiyasathit and Jacobs,2010; Kwon et al.,2010; Lee et al.,2010). Although early electron microscopical studies clearly documented both the complex structure of these organelles and their incidence of one per cell in multiple cellular types, the potential function of primary cilia remained elusive, with a dominant suggestion that they were vestigial (Grillo and Palay,1963; Federman and Nichols,1974; Fawcett,1981; and recently reviewed by Wheatley,2005,2008; Bloodgood, 2009).

Early ultrastructural comparisons of axonemes of primary cilia, of sensory cilia (such as found in specialized organs for olfaction, hearing, and sight), and of motile cilia (such as found in the respiratory tract, reproductive tract, and lining the ependyma) demonstrated that primary cilia lacked specific components required for active motility. This perhaps also contributed to the concept that primary cilia were the poor cousins in the widely diverse family of cilia and flagella. Historically, the ability to seriously study potential functions of the primary cilium was limited by axonemal size (with a diameter just at the resolution of light microscopy [0.2 μm]) and its elusive nature even when viewed by transmission electron microscopy (TEM) where the probability of seeing a profile of a primary cilium on a given thin section, even in relatively small cells, is only approximately 1/100 profiles examined. Even though primary cilia could be shown by serial sectioning to have an incidence of essentially one per differentiated cell (Wilsman,1978; Wilsman and Fletcher,1978; Wilsman et al.,1980), papers continued to be published announcing that a “rare” or “occasional” cilium could be observed in a particular cell type. This contributed to the consensus view that the study of the function of primary cilia was a sideshow compared with the breakthroughs being made at the same time by Satir, Gibbons, and others demonstrating the remarkably complex and beautiful ultrastructure of motile cilia and cilia involved with special senses (Fawcett,1981; partially summarized in Sanderson, 1984; Satir and Christensen,2007).

However, over the past 15 years the primary cilium has been brought to center stage, and this can be attributed to at least two breakthroughs for the study of this organelle. First, with the development of specific antibodies for modified tubulins, it is now routine to visualize this organelle at the light microscopical level, using epifluorescence, confocal or multiphoton microscopy (Farnum et al.,2009). The extension of this technology and of associated imaging modalities has enabled analysis to even sub-regions of axonemal and basal body structure. Second, following the initial discovery of the association between aberrant primary cilium function and early onset polycystic kidney disease in humans, a wide range of ciliopathies has shown to be associated with loss or modification of primary cilia, and appropriate in vitro and in vivo models have been developed to study primary ciliary function within the context of disease (Hildebrandt and Otto,2005; Lehman et al.,2008; Menezes and Germino,2009; Deltas and Papagregoriu, 2010; Winyard and Jenkins,2011).

In the past 15 years, reviews have highlighted the literally hundreds of studies of the correlation between normal ciliary structure and signaling capabilities, and abnormalities in human disease (recent reviews include: Afzelius,2004; Davenport and Yoder,2005; Bandano et al., 2006; Bisgrove and Yost,2006; Marshall and Nonaka,2006; Fliegauf et al.,2007; Basu and Breuckner, 2008; Sharma et al.,2008; Berbari et al.,2009; Cardenas-Rodriguez and Bandano, 2009; Gerdes et al.,2009; Smith and Rohatgi,2011; Tasouri and Tucker,2011; Vincensini et al.,2011). It is clear that cilia are remarkably diverse in their signaling capabilities, and that both sensory and motile functions can be separate or mixed in various proportions across the spectrum of cilia currently being studied (Bloodgood,2010). Basic ciliary structure can be considered to have evolved to serve hybridized functions (entirely for sensory reception, for active motility, or for a combination of these) leading to the variable and highly evolved specializations seen in different cell types and at different developmental stages (Golinska,1982; Fuchs and Schwark,2004; Götz and Striker, 2006; Eggenschwiler and Anderson,2007; D'Angelo and Franco,2009,2010; Lehman et al.,2009; Gluenz et al.,2010; Rohatgi and Snell,2010; Satir et al.,2010; Logan et al.,2011). Analogously, it can be considered that the fundamental microtubular and membrane structure of cilia has the versatility to support a wide range of receptor/ signaling modifications, leading to the full spectrum of ciliary specializations seen (Reiter and Mostov,2006; McClintock et al.,2008; Reiter,2008; Scholey,2008; Thomas et al.,2010; Vincensini et al.,2011).

For cilia that have either a primary motile function (such as motile cilia that move mucus along the respiratory tract) or a primary sensory function (such as modified cilia of rods and cones in the retina), appropriate orientation in three-dimensional (3D) space is required for normal function. Also required is the appropriate positioning of the cilium in relation to the parent cell. In fact, ciliopathies have been described that are the direct result of inappropriate orientation and/or positioning of these kinds of cilia (De longh and Rutland, 1989; Rutland and De longh, 1990; Biggart et al.,2001; Bandano et al., 2006; Fliegauf et al.,2007; Liu et al.,2007; Sharma et al.,2008; Nigg and Raff,2009). In contrast, relatively little is known about the requirement for proper positioning and axonemal orientation in 3D space for primary cilia. Assumptions are made about positioning and orientation of primary cilia in epithelia associated with tubular organs, both in vivo and in experimental models in vitro, but these assumptions have not been examined critically in relationship to function. The situation is more complex for primary cilia associated with other cellular types, because the axonemes of these cilia do not emerge from the cell into the lumen of a tubular organ, but are either intimately associated with adjacent cells or with the extracellular matrix (ECM) of the specific tissue.

The purpose of this review is to examine critically the following questions concerning primary cilia over the range of cells/ tissues/ organs in which their function has been hypothesized/ studied: What is known about the position of emergence of the ciliary axoneme from the cell? What is known about the orientation of the axoneme in 3D space after emergence from the cell? What is known about either positioning or 3D orientation in relation to function, or in relation to the pathology of known diseases? What is known about how 3D axonemal orientation and positioning either differ or are the same from cell to cell within a population of cells? How does this potential variability relate to hypothesized functions? And what still needs to be known to relate position and 3D orientation to function? This review demonstrates that current understanding of the significance of positioning and 3D orientation of cilia varies widely across cell types. Consistent with the depth of our overall knowledge of primary ciliary function, much more is known for primary cilia of epithelia associated with tubular organs, than for primary cilia associated with any other tissue. Analysis of the complexity of axonemal positioning and 3D orientation in these other tissues presents significant challenges that may deepen the understanding of ciliary function during development and intercellular cellular communication in differentiated tissues.

POSITION VS. 3D ORIENTATION OF THE CILIARY AXONEME

Definitions

The broad issue of directionality associated with the ciliary axoneme requires consideration of both the position on the cell surface (or unicellular organism) where the cilium resides, as well as co-ordinates in relationship to a specific global or local reference frame for how the axoneme is oriented in 3D space. Degrees of freedom associated with positioning and 3D orientation are variables related to specific functions when considering the full spectrum of cilia, from actively motile cilia to non–actively motile primary cilia, and all modifications in between. The most trivial case would be one degree of freedom for both, i.e., the axoneme for all cells in a population always positioned at a specific site on a cell and all directed with the same (and constant) 3D orientation. In the most complex case, the position the axoneme emerges from the cell would vary among cells in a population, would be capable of changing over time for an individual cell, and the axoneme would be oriented differently in 3D space for cells of a given population, including different ranges of movement associated with the axonemal waveform. To the extent that it has been carefully analyzed, precision of both positioning and axonemal 3D orientation is an absolute requirement for appropriate ciliary function, and loss of controlled axonemal directionality is associated with motile and/or signaling deficiencies in ciliopathies (Sanderson, 1984; Rautiainen et al., 1990; Sharma et al.,2008; Gerdes et al.,2009; Satir et al.,2010; Vincensini et al.,2011).

Positioning of the Cilium on the Associated Cell

For polarized epithelial cells the cilia, whether solitary or multiple per cell, always locate to the luminal side of a tubular organ. The particularly interesting example in Figure 1A is from the uterine tube of a rabbit demonstrating examples of both multiciliated cells, as well as cells with a single cilium (Rumery and Eddy,1974). Solitary cilia most commonly are shown, either in micrographs or in diagrams, (essentially) centered on the apical surface of the associated epithelial cell (Fig. 1B). Similarly, nodal cilia are positioned on the luminal side of the cell (Fig. 1C,D). In the murine node pit, cells are found with the cilium in a posterior position relative to the cellular axes (Fig. 1E, arrow; Fig. 1F); this asymmetry of positioning becomes significant for directional flow generation (Tabin and Vogan,2003; Lee and Anderson,2008). Therefore, for cilia associated with tubular epithelia, defining the position of the axoneme is straightforward—always luminal and with different degrees of central positioning on the apical surface, depending upon the epithelial type. Cilia associated with special senses, despite the complexity of their overall structure, also are found always emerging on a luminal side of the associated organ, as exemplified by the multiple cilia of individual olfactory neurons of the murine olfactory mucosa (Fig. 1G; McEwen et al.,2008; and Fig. 1H; Menco,1997).

Figure 1.

Position of emergence of cilia from polarized epithelial cells. A: Scanning electron micrographs of the fimbriae of the uterine tube of a rabbit demonstrate the luminal positioning of axonemes emerging from both multiciliated cells, as well as from cells with a single solitary cilium (white arrowheads, Scale bar = 25 μm). B: As shown at higher magnification, primary cilia are positioned centrally on the luminal face of individual cells (Scale bar = 1 μm). C–E: Nodal cilia emerge from the exposed surface of the node and initially are positioned essentially centrally on each nodal cell, seen here in the ventral node of the medakafish. C is an image of the entire node (Scale bar = 50 μm; the area enclosed by the white box is seen at higher magnification in D (Scale bar = 5 μm). The transmission electron micrograph in the inset in D demonstrates the characteristic 9 + 0 microtubular organization of the axoneme of these cilia (Scale bar = 0.05 μm). For flow generation resulting in establishment of the left/right axis, a subset of nodal cilia in the murine ventral node are positioned posteriorly on individual cells (arrow, E; Scale bar = 1 μm). F: The relative shifting of the position of emergence of the cilium from individual nodal cells over time, changing from a central localization (F, left) to a posterior localization (F, right) is shown diagrammatically for the murine nodal pit. G: The schematic shows the positional relationships of the multiple cilia of individual olfactory neurons of murine olfactory epithelium. OSN, olfactory sensory neuron; SC, sustenacular cell (or supporting cell); BC, basal cell. H: The scanning electron micrograph of an olfactory receptor cell neuron in an E20 rat embryo shows multiple cilia (black arrow indicates one ciliary axoneme) radiating from a single neuron onto the nasal olfactory epithelium (Scale bar = 1 μm). I: Is an electron micrograph of the position of docking of the cilium to the chondrocytic plasma membrane in porcine growth plate cartilage. Note the complexity of structures (alar sheets) associated with the positioning of the basal body to the plasma membrane (Scale bar = 0.1 μm). Figure 1A,B from Rumery and Eddy,1974 (Figs. 7, 8) used with permission from John Wiley and Sons; Figure 1C,D from Okada et al., 2005 (Fig. 1K,L) used with permission from Elsevier; Figure 1E,F from Lee and Anderson,2008 (Fig. 2A,B) used with permission from John Wiley and Sons; Figure 1G from McEwen et al.,2008 (Fig. 12.1B) used with permission from Elsevier; Figure 1H from Menco,1997 (Fig. 4) used with permission from Oxford University Press.

The assumption is that positioning of the cilium relative to the cell is established at the time of initial docking of the mother centriole (future basal body of the cilium) to the plasma membrane after cellular replication (Dawe et al.,2007; Pearson et al.,2007; Marshall,2008, Hoyer-Fender,2010; Satir et al.,2010; Kobayashi and Dynlacht,2011). The molecular basis of basal body docking and how its specificity is carried through multiple cell cycles is an intense and important area of investigation, which is critical for understanding how cilia become positioned at specific and predictably consistent positions on the cellular plasma membrane. Specification of docking position is an example of cytotaxis—the ability of a pre-existing cellular structure to determine the position of a newly formed cellular structure postdivision—thus contributing instructions about future organelle positioning and cellular shape (Benzing and Walz,2006; Benzing et al.,2007; Feldman et al.,2007; Pearson et al.,2007; Marshall,2008; Park et al.,2008).

That docking occurs at an essentially constant position in epithelial cells of tubular organs contrasts with what is seen in neurons, myocytes, and cells of connective tissue origin. Docking of the basal body is a consistent feature of connective tissue cells (Fig. 1I), but for different cell types there is variability for where the basal body docks, and hence for where the axoneme emerges (Dawe et al.,2007; Pearson et al.,2007). For cells that have polarity associated with their shape with a clearly definable major and minor cellular axis (such as growth plate chondrocytes, articular cartilage chondrocytes, or tenocytes), the axoneme of the primary cilium characteristically emerges from approximately the center of one of the cellular long axes, and is found in a juxtanuclear position closely associated with the Golgi apparatus of the cell (Fig. 2A,B; Farnum et al.,2009; Gardner et al.,2011). Axonemes of adjacent cells may emerge from opposite sides of their respective cells, making the prediction of where to find the axoneme emerging from a given cell only half as certain as for epithelial cells (Fig. 2C; Ascenzi et al.,2007).

Figure 2.

Position of emergence of cilia from nonepithelial cells. Articular cartilage chondrocytes have clearly defined major and minor cellular axes. A: The primary cilium (arrowheads) emerges from the cell from a juxtanuclear position associated with the Golgi (Scale bar = 1 μm). B: Similarly, in tenocytes the ciliary axoneme emerges from a juxtanuclear position centered on one of the long axes of the cell (arrow; Scale bar = 2 μm). C: In growth plate chondrocytes, axonemes from cilia on adjacent cells emerge from either side of the long axis of the cell, so that adjacent chondrocytes may have axonemes pointing in opposite directions (Scale bar = 3 μm). D: Data from z-stacks were used used to reconstruct the position of the cilium (red) in a hippocampal neuron. Note its position adjacent to the axon. E: When cells have a complex irregular shape, the position of emergence of the ciliary axoneme is difficult to define, even in epithelia, as shown in a basal cell of the gingiva (Scale bar = 1 μm). The complexity of defining the position of emergence of the axoneme from cells with a highly irregular shape is demonstrated by the images in Figure 2F–H of sinus adventitial reticular cells of the bone marrow stroma. In Figure 2F, E, endothelial cell; R, nucleus of a reticular cell, and the arrow points to the ciliary axoneme (Scale bar = 0.2 μm). G Shows the ciliary axoneme emerging from a reticular cell in the hematopoietic parenchyma (Scale bar = 1 μm). At higher magnification (Fig. 2G; Scale bar = 0.2 μm), the complexity of the relationship of the axoneme to adjacent hematopoietic cells is shown. The series of images in Figure 2, by contrast to those in Figure 1, emphasize that axonemal positioning on the parent cell has multiple levels of complexity, depending upon the cell type and its shape. It is only in highly polarized epithelia as shown in Figure 1 that positioning of the cilium is highly predicable from one cell to the other in a cellular population. Figure 2A from Farnum et al. 2009 (Fig. 1A) used with permission from Elsevier; Figure 2B from Gardner et al., 2011 (Fig. 3B) used with permission from John Wiley and Sons; Figure 2C from Ascenzi et al.,2007 (Fig. 2b) used with permission from Elsevier; Figure 2D from Breunig et al.,2008 (Fig. 2C, 2C′) used with permission from the National Academy of Sciences; Figure 2E, from Warfvinge and Elofsson,1988 (Fig. 3) used with permission from Springer; Figures 2F,G,H from Yamazaki,1988 (Figures 3, 1, 2, respectively) used with permission from Elsevier.

In cells of complex and irregular shape, such as neurons and smooth muscle cells, the exact positioning of the axoneme as it emerges from the cell is not straightforward to predict. For neurons the axoneme often is depicted as emerging from the neuronal cell body adjacent to the axon, but how consistent this is for a population of neurons has not been described (Fig. 2D; Breunig et al.,2008). In cells of highly irregular shape, position of emergence in published micrographs lacks a definable consistency, even in cells of epithelia (Fig. 2E; Warfvinge and Elofsson,1988). This is emphasized by the micrographs of Figure 2F,G,H, showing the axoneme on bone marrow stromal cells (Yamazaki,1988). There is a complex and not easily definable relationship of axonemes both to their parent cell as well as to adjacent cells in this complex multicellular environment.

The point to emphasize comparing Figure 1 to Figure 2 is that axonemal positioning on a given cell has different levels of complexity, depending upon the cell type and its shape (Marshall,2008). Only in highly polarized epithelia of tubular organs is positioning of the ciliary axoneme as it emerges from the cell highly predicable from one cell to the other, as well as essentially predictable for the cellular population (Fig. 1). There is a degree of predictability for the position of axonemal emergence for cells with definable major and minor axes, given the constancy of location of the cilium in a juxtanuclear position along the long axis of the cell. When considering cells of highly complex and irregular shape, especially when associated in tissues and/or organs with multiple cell types, basal body positioning for primary cilia is difficult to define for either an individual cell or for the cellular population (Lehman et al.,2009; Logan et al.,2011).

Orientation of the Ciliary Axoneme in 3D Space

Defining the orientation of the axoneme in 3D space requires a co-ordinate system with multiple axes as in Figure 3 (left), showing the potential degrees of freedom of ciliary axonemal orientation (Marshall and Kintner,2008; Farnum and Wilsman,2011). Microscopical approaches through optical sectioning provide a method for having quantitative analyses replace qualitative impressions (Farnum et al.,2009). Theta (θ) describes orientation relative to the x/y plane, and ϕ describes orientation relative to the x/z plane. Currently, it has not been established if the axoneme can rotate around its own long axis, indicated by Ψ in Figure 3A, as has been shown for bacterial flagella (Macnab,1999); indeed, there is some evidence that the basal body, because of attachments to the actin cytoskeleton, is constrained from making this kind of movement (Weinbaum et al.,2010).

Figure 3.

Orientation of the axoneme in three-dimensional (3D) space. The diagram at the left in Figure 3 demonstrates co-ordinates for defining the position of the axoneme (x,y,z), and two angles of tilt: θ relative to the x/y plane; ϕ relative to the x/z plane. Theoretically the axoneme could reorient by rotating around its own long axis (Ψ), but this has not been demonstrated in any biological system for primary cilia. A–H: This kind of co-ordinate system has been used to make a quantitative characterization of centriolar orientation in Chlamydomonas mutants. The differential interference contrast images in B,C,D compare the morphology of the wild-type (B) to two mutants (C,D). Using 3D co-ordinates for centriolar orientation (E) and then plotting the data specifically for θcentriole demonstrates that the mean θcentriole for the wild-type (F, black line) is significantly lower than that seen in the two mutants (black line in G,H). Thus, the use of 3D co-ordinates allows one to make a quantitative analysis of what was previously a qualitative description (mother-daughter centriole pairs randomly localized on the surface (C); mother-daughter centriole pairs positioned independently on the surface and no longer found in pairs [D]). Figure (left panel) from Marshall and Kintner,2008 (Fig. 1) used with permission from Elsevier; Figure 3A–H from Feldman et al.,2007 (Fig. 1) used with permission of the Public Library of Science.

These co-ordinates can be used in the context of any given global or local reference frame of biological interest, such as in relationship of the axoneme to a local cellular feature, i.e. θaxoneme,cell (long axis of the axoneme relative to the long axis of the cell) or to an organ feature, i.e. θaxoneme,joint (long axis of the axoneme relative to the articular surface of a joint). An example of the use of these co-ordinates is given in a recent study of mutants in Chlamydomonas, replacing qualitative descriptions with quantitative values for changes of orientation of the centriole relative to the body axis (Fig. 3A–H; Feldman et al.,2007). This figure also demonstrates one of the conventions used for presenting orientation data in graphic form. Although presented here for motile cilia in a unicellular organism, the same quantitative approach can be used for describing axonemal 3D orientation of primary cilia in any cellular population.

There are two other important considerations when defining axonemal orientation in 3D space. First, while this co-ordinate system is useful for providing a snapshot of the 3D coordinates of the axoneme, a complete description of 3D orientation needs to include the potential for waveform changes, including bending. Second, it is important to describe the extent to which axonemal 3D orientation is consistent or variable across cells of a population and the extent to which orientation on either a cellular or a population basis changes over time and/or under different environmental or experimental conditions. The latter is assumed to be quite different between considerations of position of emergence and axonemal 3D orientation after emergence: position of emergence of a given axoneme is assumed to be temporally fixed, while 3D orientation of the axoneme in space is assumed to range from highly stable to temporally transient, depending on the specific function of a given cilium.

SIGNIFICANCE OF AXONEMAL POSITIONING AND 3D ORIENTATION: WHAT MIGHT BE CONSIDERED RELEVANT FOR PRIMARY CILIA BASED ON WHAT IS KNOWN FROM OTHER CILIARY TYPES?

Cilia Associated With Special Senses

Modified cilia associated with signal transduction for the special senses of hearing, sight and olfaction are perhaps the best examples of how precision of both positioning and 3D orientation are critical for establishing appropriate directionality both for receiving signals from the environment and for relaying them to other cells within the organ. The highly modified axonemal structure of the light-sensitive photoreceptor sensory cilium complex of rods and cones (Fig. 4A), containing almost 2000 proteins, provides alignment between the retinal pigmented epithelium (RPE, i.e., a monolayer of cells with tight junctions separating the neural retina from the choroid) and the synaptic terminal. This is an excellent example of the significance of the positioning of the cilium in relation to the ciliated cell and adjacent cells of the population (Liu et al.,2007; Insinna and Besharse,2008; Kennedy and Malicki,2009; Wright et al.,2010; Louvi and Grove, 2011). As an aside, despite the widespread use of RPE cells as an experimental in vitro system for studying ciliary vesicular and protein transport and, in particular, the BBSome complex (Loktev et al.,2008; Gibbs et al.,2010; Jin et al.,2010; Mukhopadhyay et al.,2010; Westlake et al.,2011, as very recent examples), the relationship between the primary cilium of RPE cells and the modified cilia of sight receptors is largely conjectural and based on morphological time sequenced observations. The ciliary axoneme of RPE cells emerges from the apical side of the one-cell layer retinal pigmented epithelium, and extends into the interphotoreceptor matrix (Nishiyama et al.,2002). In the rat, there is an incidence of one cilium per RPE cell during pre- and postnatal development, but RPE cells lack cilia by postnatal day 30. This suggests that the cilium of the RPE cell may be significant only during development through this postnatal period (Norrander et al.,1998; Nishiyama et al.,2002). Axonemal orientation in 3D space in RPE cells has not been studied. The primary cilium of RPE cells has been hypothesized to play a role in positioning of photoreceptor cells during development, although there seems to be no experimental data to specifically support this hypothesis (Fisher and Steinberg,1982).

Figure 4.

Requirement of precise three-dimensional (3D) orientation of cilia associated with special senses. A: The structure of the mammalian retina is highly polarized with multiple discrete cellular layers. Rods and cones are highly modified cilia in the photoreceptor layer requiring precise 3D orientation for normal functioning, shown in both a histological image and diagrammatically in A. The special sense of hearing also relies on modified cilia with a precise 3D orientation. B: A schematic of hair bundle orientation is shown, demonstrating the alignment of stereocilia (red, labeled with phalloidin) relative to a kinocilium (green, labeled with anti-acetylated tubulin), sitting on a discoid cuticular plate. C: As can be seen, the specific 3D orientation of the hair bundles differs in the saccule (hair bundles facing away from each other) and the utricle (hair bundles facing toward each other). This precise and complex spatial variability of 3D orientation is required for normal function. D: Olfactory receptor neurons have a small cluster of 6–10 cilia positioned to bind odorant molecules from the olfactory mucosa. Although they have a 9+2 microtubular configuration, they are not actively motile. E: They have a characteristic alignment relative to each other, as shown, where the basal feet of the basal bodies of the cilia of one cell can be seen in a circular configuration relative to each other (Scale bar = 0.4 μm). F: Olfactory sensory information received by means of the multiple cilia of a given cell relays through a single axon to be received by second order olfactory neurons. Figure 4A from Wright et al.,2010 (Fig. 1b,c) used with permission from Nature Publishing Group; Figure 4B,C from Wang et al.,2006 (Figs. 4D and 2, respectively) modified and used with permission from Society for Neuroscience; Figure 4D from Louvi et al., 2011 (Fig. 2D) used with permission from Elsevier; Figure 4E from Reese,1965 (Fig. 46) used with permission from Rockefeller University Press, originally published in J Cell Biol 25:209–230; Figure 4F from Menini et al.,2004, Physiology (Fig. 1) used with permission from the American Physiological Society.

Similar to the highly ordered orientation of the ciliated region of rods and cones, precise 3D arrangement of ciliated cells of the sensory epithelium of mammalian inner-ear ducts relies on proper juxtaposition of actin-filled stereocilia in relationship to a microtubular kinocilium on the apical surface of the epithelium (Fig. 4B,C; Dabdoub and Kelley,2005; Perozo,2006; Wang et al.,2006; Hu and Corwin,2007; Vladar et al.,2009). In the cochlea, precise positioning and 3D orientation of the basal body, the kinocilium and the stereocilia bundle are achieved through planar cell polarity (PCP) signaling pathways (Wang et al.,2005; Jones and Chen,2008; Goetz and Anderson,2010). Initial basal body docking is at the cellular center; as the kinocilium becomes eccentrically positioned, final polarization is established (Cotanche and Corwin,1991; Denman-Johnson and Forge,1999; Jones et al.,2008). Orientation of the hair bundles differs in the saccule and the utricle, so appropriate positioning and 3D orientation are required both within an individual ciliated cell and also among ciliated cells of a population (Fig. 4C; Denman-Johnson and Forge,1999; Wang et al.,2006; Gillespie and Müller,2009). Thus, functional polarity is achieved through morphological polarity.

Olfactory sensory neurons demonstrate similar highly defined positioning and 3D orientation of their associated cilia (Bannister,1965; Cushieri and Bannister, 1975; Menco,1997; McEwen et al.,2008; Jenkins et al.,2009). A small cluster of cilia emerges from individual olfactory sensory neurons on the epithelial surface (Fig. 4D; Louvi and Grove,2011). These cilia are highly aligned in their positioning on the plasma membrane, as shown by the circular arrangement of their basal bodies within the cytoplasm (Fig. 4E; Reese,1965). However, axonemes of individual cilia have multiple degrees of freedom of their 3D spatial orientation, and the cluster as a whole covers a wide range of angles as they extend in multiple directions (DeMaria and Nagi, 2010; Green and Mykytyn,2010). Olfactory input from all the cilia of a given cell is then transmitted to one axon, and axonal input from a given cell is integrated with input from multiple cells carrying different odor receptors, for final input to the olfactory bulb and to the olfactory cortex (Fig. 4F; Zou et al.,2001; Menini et al.,2004; Mayer et al.,2009; McEwen et al.,2008). Olfactory perception is an excellent example of the significance of both positioning of cilia on a cell, and differential 3D orientation of axonemes of a given cell as they extend into the environment. It also is an elegant example of the complexity of integration of input from multiple ciliated cells within a population of cells.

Actively Motile Cilia, Including Nodal Cilia

The coordinated beating of actively motile cilia, be they in unicellular organisms (Fig. 5A) or lining the ependyma, the respiratory tract, or the reproductive tract (Fig. 5B), has been studied for several decades, using combinations of video-based microscopy, mathematical modeling, and computer simulations (Hines and Blum,1982; Marino and Giello, 1982; Sleigh et al.,1988; Guerin and Liron, 1992; Schwartz et al.,1997; Guerin and Levit-Gurevich, 2001; Brokaw,2002; Hilfinger and Jülicher, 2008; Chen et al.,2009,2010). Uniform alignment of basal bodies deep to the apical border of epithelial cells mirrors the uniform positioning of ciliary axonemes, which also can be identified by the essentially parallel alignment of the two central microtubule singlets within axonemes of adjacent cilia (Fig. 5C; De longh and Rutland, 1989; Rautiainen et al.,1990; Boisvieux-Ulrich and Sandoz,1991). Positioning of cilia always is on the apical surface and often covers this apical surface with as many as 200 axonemes. Orientation of axonemes in 3D space is coordinated among adjacent cilia of a given cell and among cilia of the entire field of cells. Orientation is temporally transient, with the effective stroke usually planar and the recovery stroke non-planar; there is obvious complexity in achieving the characteristic metachronal synchrony of the ciliary wave (Fig. 5D; Gueron and Liron,1992; Sanderson, 1984; Sleigh et al.,1988; Mitchison and Mitchison,2010). Therefore both proper positioning and axonemal 3D orientation are essential to achieve the appropriate directionality of movement of the fluid. For almost four decades, lack of precision in generating this waveform has been considered a salient clinical feature of primary ciliary dyskinesia (PCD; Afzelius,1985; Rutland and De longh, 1990; Afzelius,2004; Bisgrove and Yost,2006; Vincensini et al.,2011). (As an aside, the “primary” in PCD modifies dyskinesia of motile cilia, and does not refer to primary cilia; PCD encompasses diseases with clinical signs directly referable to an abnormality of motile cilia, as opposed to motile ciliary dysfunction secondary to some other cause; see also Wheatley,2005.)

Figure 5.

Requirement of precise three-dimensional (3D) orientation of actively motile cilia. A: Ciliated unicellular organisms move by means of coordinated cillary beating of hundreds of cilia; controlled movement, including turning and phototaxis, depends upon precision of orientation of the 3D beating pattern of adjacent cilia. B: A similar co-ordination of waveform in 3D space is required for the metachronal pattern of movement of cilia lining the respiratory tract (Scale bar = 10 μm). C: In cross-section, the precision of ciliary alignment can be seen both in the parallel positioning of inner singlets of adjacent axonemes (black arrows) and identical orientation of basal feet of adjacent basal bodies within the cytoplasm (C, white arrows; Scale bar = 1 μm). D: The details of the complexity of the 3D waveform remain a subject of analysis, but the need for precise co-ordination of movement of adjacent axonemes is known to be critical for appropriate function. E: A similar precision of positioning and coordinated oriented movement in 3D space is required for cilia in nodal cells (Scale bar = 5 μm). There is a ventro-posterior emergence of individual cilia from the basal body, which itself is positioned posteriorly on the cell (Fig. 4F). F,G: Modeling of the relationship of these two kinds of nodal cilia to each other has demonstrated that the counterclockwise fluid movement by actively motile cilia generates flow of significant magnitude to cause bending of nonmotile primary cilia (Fig. 4H,I), resulting in signal transduction that is critical for establishing left–right asymmetry. This is an elegant example of the critical importance of both precise positioning of the cilium on the cell as well as generation of a precise axonemal waveform in 3D space for normal functioning. Laterality defects manifested in ciliopathies can result either from disruption of positioning of the cilia or from inappropriate waveform of the motile cilia in 3D space. Figure 5A: from http://.genome.gov/Images/press_photos/lowres/85-72.jpg; Figure 5D from Sanderson, 1984 (Fig. 1a) used with kind permission from Springer Science+Business Media: Biology of the Integument, Vol. 1 Invertebrates, Chapter 3, Cilia, by Michael J. Sanderson p. 21; Figures 5E,F from Okada et al., 2005 (Figures 4c,d) used with permission from Elsevier; Figures 5G,H from Chen et al.,2009 (Figures 2, 5) used with permission from Elsevier; Figure 5I, Chen et al.,2010 (Fig. 12) used with permission from Elsevier.

Positioning and 3D orientation of nodal cilia have been an intense area of investigation, with recent studies demonstrating the complexity of organization of potentially two different types of ciliated cells in the node, with both participating in the establishment of laterality. One set of cilia is thought to generate fluid movement; the second set of cilia is thought to respond to the fluid flow. Important for the current discussion is that, for proper generation of fluid movement, motile nodal cilia must not only be positioned apically on the cell but also must be at a proper posterior angle of tilt (Fig. 5E,F; McGrath and Brueckner,2003; Tabin and Vogan,2003; Buceta et al.,2005; Nonaka et al.,2005; Tanaka et al.,2005; Fliegauf et al.,2007; Basu and Brueckner,2008; Hirokawa et al.,2009; Borovina et al.,2010). Modeling has demonstrated that movement of the axonemes of these cilia in a counterclockwise direction generates fluid flow sufficient to be detected by mechanically sensitive primary cilia (also referred to as passive cilia) that respond to the flow and generate signaling through bending (Figs. 5G,H,I; Cartwright et al.,2008; Lee and Anderson,2008; Chen et al.,2009,2010). Although the specifics of the interactions involved between these two types of nodal cilia warrant further study, what is clear is that precise positioning and 3D axonemal orientation of both sets of cilia are required. Again, laterality defects in ciliopathies have been associated with defects in one or the other of the nodal cilia (Nonaka et al.,1998; McGrath and Brueckner,2003; Badano et al.,2006; Marshall and Nonaka,2006; Basu and Breuckner, 2008; Sharma et al.,2008).

A common theme is that, for both modified cilia associated with special senses and for actively motile cilia, appropriate positioning of the basal body on the plasma membrane and a defined orientation of the axoneme in 3D space (be it fixed or temporally transient), are required for normal function; loss of appropriate positioning and/or of 3D orientation results in some loss of function associated with ciliopathies (Rautiainen et al.,1990; Rutland and De longh, 1990; Marshall,2008; Gerdes et al.,2009; Goetz and Anderson,2010; Hoyer-Fender,2010). Depending upon the tissue, cilia on adjacent cells may mirror their neighbors; for others, positioning and axonemal orientation are different on different cells, but definable for the population of cells within the tissue. Also, the site of basal body insertion on the apical surface of the cell (translational orientation) as well as the angle of the basal body with respect to its long axis (rotational orientation), affects ciliary function (Marshall,2008). For the remainder of this review, these same concepts of positioning and 3D axonemal orientation will be examined for nonactively motile primary cilia. The theme that emerges is that, for primary cilia in many tissues, little is known about positioning and 3D axonemal orientation; however, these attributes of the axoneme are likely critical for normal function of the primary cilium and therefore worthy of persistent study.

SIGNIFICANCE OF AXONEMAL POSITIONING AND 3D ORIENTATION FOR PRIMARY CILIA IN POLARIZED EPITHELIA

Axonemal positioning for emergence from the cell and axonemal orientation in 3D space are considerably less complex in primary cilia associated with polarized epithelial cells of tubular organs than for actively motile cilia or sensory cilia. Indeed, positioning and 3D orientation for axonemes of these primary cilia can be described with straightforward generalities that cover a wide range of tubular epithelial types. These generalities do not appear to have been rigorously established experimentally; however, there also is no indication from the literature that would challenge the validity of these assumptions. This is perhaps one reason why functional studies of primary cilia of epithelial tissues have advanced at a significantly faster pace than seen to date for primary cilia in other cellular types.

Axonemes of primary cilia in a wide variety of highly polarized epithelial tissues follow a basic structure in which the relationship of microtubules to each other is consistent from cell type to cell type, whereas signaling capabilities associated with the ciliary membrane reflect cell-specific functions (Fig. 6A; Reiter,2008; Bloodgood,2010; Rohatgi and Snell,2010; Seeley and Nachury,2010; Evron et al.,2011; Francis et al.,2011; Tasouri and Tucker,2011; Vincensini et al.,2011). Whether shown in scanning electron micrographs (Fig. 6B; Mykytyn et al.,2004), confocal images, or diagrammatically (Fig. 6C; Boletta and Germino,2003), the axoneme of the primary cilium is depicted as emerging from the center of the luminal side of the cell of tubular organs (Fig. 6F; Seeley and Nachury,2010). Again, as depicted diagrammatically, essentially all axonemes in a field of cells are positioned identically. This means the issue of defining axonemal 3D orientation for a population of cells is a trivial exercise (Fig. 6C). Apical axonemal positioning has been studied in relationship to the establishment of PCP for motile nodal cilia and motile cilia associated with multiciliated cells of the ependyma and respiratory tract (Goodrich,2008; Goetz and Anderson,2010; Marshall,2008,2010). In these motile ciliary populations, one hypothesis is that cilia, initially positioned secondary to planar cell polarity signaling, have the ability to self-organize in response to their own cilia-generated fluid flow. The degree to which the specific positioning and later orientation of nonmotile primary cilia populations on epithelia result from an interaction of planar cell polarity signaling and passive bending in response to fluid flow within the environment is an area requiring further study (Fig. 6E; Mitchell et al.,2007; Jones et al.,2008; Marshall and Kintner,2008; Marshall,2008,2010; Santos and Reiter,2008; Satir et al.,2010).

Figure 6.

Axonemal positioning and three-dimensional (3D) orientation for primary cilia in polarized epithelia. A: Axonemal and basal body structure of the primary cilium are remarkably similar across a wide range of polarized epithelia; primary differences in signaling capabilities are associated with specialization of receptors on the axonemal membrane. B,C,F: Both in scanning microscopy images (B, primary cilia of renal tubule cells; Scale bar = 0.5 μm) or in diagrams (C), primary cilia in polarized epithelial cells are shown as emerging from essentially the geometrical center of the apical surface of the cell, with a capability of responding to fluid flow within the lumen (F). D: The response to fluid flow is depicted in diagrams as occurring only in two dimensions, with all cells responding in the same manner and with the axoneme lacking any complex 3D waveform. Although it is assumed that bending can occur in all possible directions, this has not been studied rigorously. This is in contrast to actively motile cilia. E: It is hypothesized that, for motile nodal cilia and for actively motile cilia on multiciliated cells, ciliary organization within the population of cells results in response both to planar cell polarity signaling as well as cilia-generated fluid flow (demonstrates ciliary orientation in multiciliated (a) and nodal (b) cells, and axonemal orientation in relation to fluid flow directions (large arrowheads)). G: Diagrammatically, bending of primary cilia in response to fluid flow almost always is shown with the point of maximal bending positioned centrally on the axoneme (D). However, in published videos of the primary cilium bending in response to fluid flow, the axoneme does not appear to have either the same degree of curvature or the same positioning of the point of maximal curvature as usually diagrammatically (D) (Scale bar = 5μm). Figure 6A,F from Seeley and Nachury,2010 (Figures 1, 4) used with permission from the Company of Biologists; Figure 6B from Mykytyn et al.,2004 (Fig. 5c) used with permission from the National Academy of Scientists; Figure 6C from Boletta and Germino,2003 (Fig. 5) used with modification with permission from Elsevier; Figure 6D from Janmey and McCulloch,2007 (Fig. 6) used with permission from Annual Reviews; Figure 6E from Marshall,2010 (Fig. 1) used with permission of the Nature Publishing Group; Figure 6G from Schwartz et al.,1997, Am. J. Physiol. Renal Physiol 272 (Fig. 2) used with permission of American Physiological Society.

Axonemal orientation in 3D space in response to fluid flow for primary cilia in polarized epithelial cells essentially always is depicted in 2D (Fig. 6D, as an example; Janmey and McCulloch,2007); bending is assumed to be planar with respect to the direction of fluid flow. Axonemal movement is passive, and a waveform in 3D space has not been described. The basic question of how many degrees of freedom are associated with axonemal bending in response to fluid flow for these primary cilia is fundamental. Is the axoneme, because of its radial symmetry, equally capable of making a bending response to fluid flow equally from all possible directions, and are the same signaling pathways activated in response to fluid flow from different directions?

For primary cilia of polarized epithelia, the hypothesis is that fluid flow bends the axoneme, with a response by strain-sensitive proteins leading to signaling (Resnick and Hopfer,2008; Rydholm et al.,2010; Weinbaum et al.,2010). Diagrammatically the position of bending of the axoneme in epithelial cells most frequently is depicted with a gradient of curvature with maximal bending at the midpoint of the axoneme (Fig. 6D). This contrasts with the few published video frames of bending of the axoneme in response to fluid flow in vitro, where the axoneme appears quite straight, with maximal bending at the junction of the axoneme with the cell surface (Fig. 6G; Roth et al.,1988; Schwartz et al.,1997; Praetorius and Spring,2001). The position of bending may be dependent upon the length of the axoneme, but the localization of strain-sensitive proteins is at the ciliary base, meaning that, when bending occurs specifically at the base, signaling is activated (Resnick and Hopfer,2008; Gardner et al.,2011; Rydholm et al.,2010). The base also is the point at which maximum membrane stresses are developed (Weinbaum et al.,2010). Whether there is significance to other positions along the axoneme where bending occurs is not clear, and is an active area of study through modeling (Shiba et al.,2005; Rydholm et al.,2010; Weinbaum et al.,2010). Given that the position of bending is an important consideration in all other motile and nonmotile cilia (be it in an active waveform, or in restricted locations), it is likely that the position of bending along the axoneme also is important for signal transduction by primary cilia. Additional unanswered questions regarding the bending response of primary cilia in highly polarized epithelia associated with tubular organs include the following: (1) Is a certain threshold of bending required to activate signal transduction? (2) Are the same pathways activated at different degrees of bending? and (3) Is the system toggled and/or does it undergo a refractory period after bending? (Whitfield,2008; Lee et al.,2010).

Because axonemes of primary cilia in a field of epithelial cells are positioned similarly and respond as a field of axonemal bending without a complex waveform, in vivo studies of signaling responses to fluid flow are not only possible, but are assumed to directly reflect the in vivo system (Shiba et al.,2005; Rydholm et al.,2010). This means that in vitro analysis is a powerful approach for analyzing specific signaling pathways, as has been shown for the rapid advances in understanding signal transduction through passive axonemal bending for the primary cilia of kidney epithelial cells. As will be seen, the complexity of studying primary ciliary signaling in neurons, myocytes and connective tissue cells is particularly challenging, both in vitro and in vivo, because axonemal positioning, bending and orientation in 3D space cannot be described with generalizations that apply uniformly for all cells within a population in vivo.

GENERALIZATIONS: AXONEMAL POSITIONAL CHARACTERISTICS FOR PRIMARY CILIA IN ADDITIONAL TISSUE TYPES

Studies completed decades ago concluded that for neurons, chondrocytes, osteocytes, and smooth muscle cells (among others) the incidence of primary cilia was essentially one per cell (examples include Wilsman,1978; Wilsman and Fletcher,1978; Wilsman et al.,1980). Collection of incidence data required analysis by serial sections at the electron microscopical (EM) level, and most studies of this type were done well before functional analyses of primary cilia at the light microscopical level were possible. In conjunction with incidence studies, investigators often commented on positional characteristics of the axoneme relative to the cell, but no studies systematically attempted to characterize positional information for axonemes within a population of cells within the tissue. It is important to emphasize that, because of the heterogeneity associated with axonemal cellular positioning among different cells in these cellular populations, even purely descriptive data are rarely provided for most tissues and generalizations for axonemes within populations of cells have been lacking.

However, based on observations made over the last five decades, it is clear that there are multiple significant ways that positional characteristics of the axoneme of primary cilia differ from what has been discussed either for primary cilia associated with highly polarized tubular epithelia, or for motile and/or sensory cilia found in multiple organs. First, primary cilia in neurons, muscle cells, and connective tissue cells characteristically are found with the axoneme at least partially embedded in what is currently referred to as a ciliary pit or pocket (Fig. 7A,B; Sorokin,1962; Garant et al.,1968; Haust,1987; Jensen et al.,2004; Ghossoub et al.,2011). The ciliary pocket is an invagination of the plasma membrane, which, in different cells within a population, may surround either part of the axoneme before it emerges into the surrounding matrix, or may surround essentially the entire axoneme before its emergence (Fig. 7A–E; Albrecht-Buehler and Bushnell,1980; Poole et al.,1985; Breunig et al.,2008; Molla-Herman et al.,2010; Rattner et al.,2010; Rohatgi and Snell,2010). The proximity of the axonemal membrane to the plasma membrane of the pocket varies from micrograph to micrograph, and has been referred to as the ciliary reservoir (Moser et al.,2010). It is clear that the pocket invagination is formed by a deep curvature of the plasma membrane at the docking site of the basal body and is usually symmetrically positioned surrounding the axoneme for a variable fraction of the total axonemal length (Rohatgi and Snell,2010). Within a given population of cells, axonemes can be found fully embedded within a pit in some cells and fully extended from other cells, and at the whole range of partially embedded levels in between.

Figure 7.

Additional observations of positional relationships of axonemes of primary cilia in a variety of cellular types. A: In human arterial endothelial cells, primary cilia can be found emerging from a deep ciliary pit, on the luminal side of the cell (v = ciliary pit, small arrows = transitional fibers, large arrow = basal foot; Scale bar = 0.1 μm). B: Similarly, murine odontoblasts have primary cilia, again with the axoneme surrounded almost entirely with the microenvironment of the ciliary pit (arrow = ciliary axoneme, Ce = centriole; Scale bar = 0.1 μm). C: The full extent of the ciliary pit associated with an articular chondrocyte is seen (Scale bar = 0.4μm), where only the axonemal tip actually extends into the extracellular matrix. D: Similarly, in quiescent 3T3 cells grown to confluence, the ciliary axoneme is surrounded for 3/4 of its length by the ciliary pit, before emerging at an angle (Scale bar = 0.4 μm). E: In hippocampal neurons, a ciliary pit surrounds over 2/3 of the ciliary axoneme, before it emerges, as seen in this reconstruction from 12 serial sections. F: In chondrocytes, odontoblasts and other cells of connective tissues, the axoneme extends not into a fluid filled lumen, but into a dense extracellular matrix (ECM), and there is evidence of direct attachments between the axoneme and components of the ECM. This has led to hypotheses that deflection of the cilium may occur during joint movement causing deformation of the ECM and resulting in signal transduction through the primary cilium (Fig. 7G). Figure 7A from Haust 1987 (Fig. 6) used with permission of Springer; Figure 7B from Garant et al.,1968 (Fig. 15) used with permission of Elsevier; Figure 7D from Albrecht-Buehler and Bushnell,1980 (Fig. 3) used with permission from Elsevier; Figure 7E from Breunig et al.,2008 (Fig. 2F) used with permission from the National Academy of Sciences; Figure 7F from Satir and Christensen,2007 (Fig. 4) used with permission from Annual Reviews; Figure 7G from Whitfield,2008 (Fig. 2) used with permission from Elsevier.

Hypotheses have been developed about the significance of the pocket for forming a specialized microenvironment surrounding the ciliary axoneme associated with vesicular trafficking for endocytosis and/or exocytosis; with creating a microenvironment for specific sensory functions; with temporally controlling exposure of the axoneme to soluble signaling factors and/or selective sorting of proteins and lipids to cilia; or with providing a stable actin-cilium interface for ciliary positioning (Molla-Herman et al.,2010; Rohatgi and Snell,2010; Ghossoub et al.,2011). This relates ultimately to how trafficking is regulated within the cilium and if there is a diffusion barrier at the ciliary base (Breslow and Nachury,2011; Ocbina et al.,2011). Given the potential for an interactive effect between axonemal length and sensitivity, it also has been suggested that in-pocketing of the axoneme to different degrees may relate to variations in axonemal receptivity to, and/or amplification of, the signal (Gardner et al.,2011; Kim et al.,2010; Weinbaum et al.,2010; Abdul-Majeed et al.,2011; Rondanino et al.,2011). However, the extent of dynamic movement by the axoneme of a given cell from the extremes of being essentially fully surrounded by a pocket to being fully extended from the plasma membrane is not known (Moser et al.,2010). Sorokin (1962) hypothesized that, in nonpolarized cells, the relationship of the axoneme to the pocket is established during ciliogenesis. However, currently it is not known how the extent of the pocket varies over time for a given cell or for cells within a population, or why some cells have a pocket while adjacent cells do not.

Second, while the axonemes of primary cilia in polarized epithelia emerge from the cell into a fluid-filled tubular environment, the axonemes of primary cilia in other tissues most commonly emerge directly into an ECM, which, although fluid filled, also is characterized by complex associations of structural molecules (Fig. 7F; Satir and Christensen,2007). The nature of, and extent of, direct attachments of the ciliary membrane to the ECM is being investigated and may differ in different cell types (Poole et al.,2001; Jensen et al.,2004; McGlashan et al.,2006; Christensen et al.,2008; Knight et al.,2009). Bending of the axoneme in these cells also may be passive as the axoneme is moved in conjunction with the ECM under compression/tension during movement (Fig. 7G), perhaps by means of an integrin-dependent signaling cascade (Shyy and Chien,1997; Grashoff et al.,2003; Terpstra et al.,2003; Millward-Sadler and Salter,2004; Whitfield,2008; Lee et al.,2010; McGlashan et al.,2010). Similar mechanotransduction pathways involving sensing through primary cilia may also act in vascular smooth muscle cells (Wang and Li,2010; Shi and Tarbell,2011). An important question would be, does bending of the axoneme in tissues such as cartilage or tendon reflect the compressive/tensile nature of the environment of these tissues? Even though it has not been studied experimentally, it is intuitive that freedom of movement of the ciliary axoneme must be quite restricted when surrounded by an ECM, and not on the same temporal scale as seen in epithelial-based primary cilia in tubular organs with a fluid environment.

Different degrees of bending of the ciliary axoneme as it emerges from the ciliary pocket are seen in Figure 8A–C. In all these, the axoneme has some level of confinement. In Figure 8A, confinement of the axoneme extending from this articular chondrocyte is by the ECM and also by direct touching of the axoneme to the plasma membrane of the adjacent cell. In Figure 8B, the axoneme of the chondrocyte emerges from a shallow ciliary pocket but immediately curves to directly follow the curve of the plasma membrane, suggesting a very restricted capability of being moved, even passively (Wilsman and Fletcher,1978; Poole et al.,1985). In the fibroblast in Figure 8C, the axoneme emerges into a space confined by collagen fibrils of the ECM (Brooker et al.,1971). In these nonepithelial cells, sharp bends, almost kinks, appear to confine the axoneme in space, and are in contrast to what has been presumed to be the temporally flexible passive movement of axonemes of primary cilia of polarized epithelia of tubular organs. In chondrocytes, a serial section EM analysis demonstrated the nonuniform termination of axonemal microtubules (Fig. 8D), which could be interpreted to lead to unequal structural rigidity along the axoneme (Wilsman,1978; Wilsman et al.,1980). This could, in part, be involved with allowing acute bending of the axoneme as it leaves either the plasma membrane or the ciliary pocket, with the point of bending being different from cell to cell depending on the degree of local axonemal stiffness, as has been modeled for actively motile cilia (Mitchison and Mitchison,2010). It also has been suggested, as an alternative to axonemal bending as a requirement for sensing, that pressure sensing might be the primary way ciliary tips receive signals from the external environment to be relayed through the axoneme to the cell to activate signal transduction pathways (Bell,2007,2008). Presumably cilia with quasi-permanent or permanent bends in them due to confinement by the ECM would be able to sense environmental cues through this kind of pressure sensing mechanism.

Figure 8.

Potential constraints on axonemal movement for nonepithelial primary cilia. A: The axoneme from the cilium of one articular chondrocyte appears to directly contact the plasma membrane of the adjacent chondrocyte, potentially restricting even passive movement of the axoneme in three-dimensional (3D) space. B,C: Two examples of close confinement of the ciliary axoneme, essentially sandwiched between the cellular plasma membrane and collagen fibrils of the ECM, are seen in an articular chondrocyte (B) and fibroblast (C). D: The nonuniform termination of outer microtubular doublets in these axonemes may result in unequal structural rigidity at different levels of the axoneme, facilitating the sharp axonemal bending seen in these cilia. E–G: Demonstrate examples of the complexity of axonemal positioning in cells of irregular shape. The cilium (C) emerging from the myoepithelial cell in E appears to make direct contact with a neighboring endothelial cell. In F, the ciliary axoneme (C) of an irregularly shaped endothelial cell (E) emerges into the lumen (L) of the alveolus adjacent to a macrophage (MA). G is of a type II interstitial cell (IC type II) in the rat duodenal enteric plexus. This cell, which serves as a pacemaker within the enteric nervous system, is shown with a centriole (ct) and its associated ciliary axoneme (arrow). The latter emerges from the Golgi (g) region of the cell, in close proximity to a nerve trunk (nt). The basal body of the cilium docks on the plasma membrane in a position such that the axoneme directly touches the cellular plasma membrane. The positional relationships of primary cilia in these cells are difficult to describe, yet may be significant for understanding potential function of the cilium in pacemaker activity. Scale bar = 1 μm in all images. Figure 8B from Wilsman and Fletcher,1978 (Fig. 9) used with permission from John Wiley and Sons; Figure 8C from Brooker et al.,1971 (Fig. 1) used with permission of Wiley-Blackwell; Figure 8D from Wilsman et al.,1980 (Fig. 3) used with modification and with permission of John Wiley and Sons; Figures 8E,F from Nickerson,1989 (Figures 1, 2 used with permission from Springer; Figure 8G from Escribano et al.,2011 (Fig. 4C) used with permission from Histology and Histopathology.

A third observation is that the relationship of axonemes on adjacent cells of the same population in situ in many cellular populations is difficult to define, but clearly is less predictable than that found for the axonemes of primary cilia on highly polarized epithelia. An example of this is in Figure 8E of a myoepithelial cell (M) in the bovine mammary gland (Nickerson,1989). The cilium (C) emerges in close proximity to, apparently touching, an endothelial cell (E). This same complexity can be seen in a highly irregularly shaped epithelial cell (E, Fig. 8F), also in the mammary gland, where the cilium (C) emerges into the lumen (L) in close proximity to a macrophage (MA; Nickerson,1989). Another example is shown for the cilium of an interstitial cell type II in the rat duodenal enteric plexus (Fig. 8G). The axoneme emerges to touch an adjacent nerve trunk (nt) in a confined space (Escribano et al.,2011; Sasano,1986). As shown in these examples, axonemal positioning of the cilium for a given cell relative to the cilia of other cells of the population is difficult to define and would be laborious to attempt to characterize when axonemes emerge in close proximity to other cells and are confined near to, or even surrounded by, adjacent cells. Positioning of the axoneme relative to the cell for primary cilia of polarized epithelia, as well as for motile and sensory ciliary populations, is known to be critical for optimal function. The complexity and varied nature of axonemal positioning on irregularly shaped epithelial cells, neurons, smooth muscle cells, and cells of connective tissue origin is well documented. An important area requiring significant further research is the degree to which specific axonemal positioning is required for normal ciliary functioning in these various cellular types (Goodrich,2008).

A final observation relative to axonemal positioning in nonepithelial cells is that the ciliary basal body and the associated centriole characteristically remain associated with the Golgi of the cell in a juxtanuclear position. While the relationship of the centriolar apparatus to the Golgi during the cell cycle, including mitosis, is well described (Santos and Reiter,2008; Gerdes et al.,2009; Nigg and Raff,2009; Gonçalves et al.,2010; Satir et al.,2010; Seeley and Nachury,2010; Gibson et al.,2011; Kim et al.,2011), the polarity of epithelial cells postdivision is such that the close intracellular proximity of these organelles is somewhat lost during ciliogenesis, when the mother centriole docks on the plasma membrane forming a basal body (Dawe et al.,2007; Pearson et al.,2007; Pitaval et al.,2010). However, in cells of connective tissue origin such as ameloblasts, chondrocytes (Fig. 2A), and osteocytes and tenocytes (Fig. 2B), the close positioning of the primary cilium and its centriole to the juxtanuclear Golgi is a characteristic feature that at least partially defines positioning of the cilium with reference to the cell (Poole et al.,1997).

GENERALIZATIONS: AXONEMAL ORIENTATION IN 3D SPACE FOR PRIMARY CILIA IN MULTIPLE TISSUE TYPES

The only consistent generalization to be made about the orientation in 3D space of the axoneme of primary cilia in tissues other than the highly polarized epithelia of tubular organs is that, from cell to cell, 3D axonemal orientation is highly variable. Figure 9 shows electron micrographs of articular chondrocytes, presented in a consistent orientation relative to the bone. The orientation of the axoneme relative to the cell is different for all three cells. In Figure 9A the axoneme is seen in transverse section, therefore oriented orthogonal to the plane of the section. In Figure 9B, the axoneme emerges at an angle of ∼45° relative to the long axis of the chondrocyte; in Figure 9C the axoneme emerges at an angle essentially parallel to the long axis to the cell. In Figure 9D of cells in bronchiolar smooth muscle of the human airway, ciliary axonemes are seen by light microscopy by reaction with antibodies to α-tubulin. Different degrees of bending of the axoneme and different angles relative to the cell are interpreted to represent different orientations of the ciliary axoneme in 3D space after emerging from the cell (Wu et al.,2009). Similarly, in the field of hypothalamic neurons in Figure 9E, ciliary axonemes (green) emerge from neuronal cell bodies (red) in multiple directions (Fuchs and Schwark,2004). An apparent exception to this generalization is the preferential orientation of axonemes of primary cilia of vascular smooth muscle cells, positioned at ∼60° in relation to the cross-sectional plane of the artery as they project into the ECM (Lu et al.,2008).

Figure 9.

Axonemal orientation in three-dimensional (3D) space in nonepithelial primary cilia. A–C: The ciliary profiles show that, in a given population of articular chondrocytes, the 3D orientation of the ciliary axoneme relative to the cell is highly variable. A,C: The sectioning plane of these three cells is the same, but the angle of the axoneme varies from essentially orthogonal to the section plane (A) to parallel to the sectioning plane (C). Note that in all three images, the cilium and its associated centriole are closely associated with the Golgi of the cell. In studies of axonemal positioning in a cell, it is critical to follow any given axonemal profile in adjacent serial sections to demonstrate definitive connection of the axoneme to the parent cell. Thus, while the association of the axoneme to the parent cell is clear in Figure 9B and 9C, it would be necessary to examine serial sections to verify the relationship of the axonemal profile in Figure 9A to its associated cell. D: The nonuniformity of axonemal orientation in 3D space is demonstrated in human bronchiolar smooth muscle cells. These axonemes project into the ECM with a level of deflection and curvature that varies from cell to cell. E: Similarly, in this field of hypothalamic neurons, axonemes (green) emerge from the associated cells (red) in multiple directions and with multiple degrees of curvature. This is in contrast to the stereotypic uniformity of 3D orientation of axonemes of primary cilia described in polarized epithelial cells, such as in the kidney tubule. Scale bar = 0.2 μm in A,B,C, 10 μm in D,E. Figure 9D from Wu et al.,2009 (Fig. 2A) used with permission from American College of Chest Physicians; Figure 9E from Fuchs and Schwark 2004 (Fig. 1) used with permission from Elsevier.

Quantitative descriptive data of the orientation of ciliary axonemes in 3D space requires following axonemal profiles through all adjacent serial sections in which they appear, be that through serial sections at the EM level or by optical sectioning with confocal or multiphoton microscopy. Therefore, from single images the only conclusion to draw is that there is an apparent variability of 3D orientation of axonemes of different cells in a population, which is in contrast to the conformity of axonemal orientation in 3D space of primary cilia in a population of cells in polarized epithelia of tubular organs. However, even the snapshots in Figure 9A–C of ciliary axonemes in different cells of a population at one point in time raise questions that are important for understanding ciliary function in these cells. For a given cell, is the 3D orientation of the axoneme fixed, or is it capable of being changed over time? On the assumption that, based on their microtubular 9+0 structure, these primary cilia are not capable of active movement, can the axoneme be moved passively, and is this required for signal transduction? For a given cell in a proliferative cell population, such as actively dividing cells in growth plate cartilage, will the axoneme of at least one of the daughter cells mirror the original 3D orientation of the axoneme of the mother cell before division, as has been suggested for migrating fibroblasts (Albrecht-Buehler,1977; Albrecht-Buehler and Bushnell,1979; Katsumoto et al.,1994)? Because many connective tissues demonstrate high tissue anisotropy, is the 3D orientation of the ciliary axoneme of a given cell established through mechanisms related to the establishment of planar cell polarity in epithelial cells?

AXONEMAL POSITIONING AND 3D ORIENTATION: QUANTITATIVE DESCRIPTIVE DATA FOR A POPULATION OF CONNECTIVE TISSUE CELLS

For axonemes associated with primary cilia in nonepithelial tissues, there is variability of both basal body cellular positioning and axonemal 3D orientation from cell to cell within a population. The question can be asked: Despite the cell-to-cell variability, is there a consistency that can be defined for all axonemes within a given cellular population? The hypothesis is that variability of directional positioning and 3D axonemal orientation are significant for coordinated integration of responses from the population of cells in a tissue, especially for tissues such as growth plate or articular cartilage where tissue cellularity is low and cells have no direct connections to each other. These tissues are exquisitely sensitive to their mechanical environment and are highly anisotropic in both matrix and cellular organization. One speculation is that information about the mechanical environment of the tissue is received by individual cells and is unique to the specific position of the cell within the tissue. Communication among cells for a coordinated population response might be through the ECM, allowing a higher order intercellular communication as has been demonstrated for neuronal networks and bone cell networks (Banes et al.,2001; Turner et al.,2002; Bray et al.,2005; Bonewald and Johnson,2008; Goodrich,2008; Green and Mykytyn,2010; Vladar et al.,2009; and recently discussed in Farnum and Wilsman,2011). This also is consistent with the hypothesis that connective tissue might function as a body-wide mechanosensitive signaling network (Langevin,2006).

Results from a recently published study demonstrate that quantitative descriptive data for articular chondrocytes from different regions (weight-bearing and non–weight-bearing) and from different cellular zones (superficial zone chondrocytes and radial zone chondrocytes) can describe axonemal cellular positioning and orientation in 3D space with more predictive power for the population of cells than for any given cell within the population (Farnum and Wilsman,2011). Figure 10 shows both a superficial zone chondrocyte (Fig. 10A) and a radiate zone chondrocyte (Fig. 10B), demonstrating the size and shape differences for cells in these two populations. EM serial sections were followed, and co-ordinates for ϕaxoneme and θaxoneme relative to the articular surface of the distal femur were calculated for over 400 cilia and presented as polar plots (as explained in the caption for Fig. 10). The plots in Figure 10C–F allow the following conclusions:

Figure 10.

Axonemal orientation in three-dimensional (3D) space in articular chondrocytes and tenocytes. A,B: The orientation of the major axis of the cell to the articular surface varies in superficial zone (A, Scale bar = 1 μm) and radiate zone (B, Scale bar = 1 μm) chondrocytes, making this an interesting contrasting orientation of cells within a population for the study of axonemal orientation in 3D space. C: Co-ordinates for this study followed the convention seen in Figure 3A, with ϕaxoneme representing the angle of the ciliary axoneme relative to the plane from the articular cartilage to the subchondral bone, varying from 0° to 180°. θaxoneme is the angle of the axoneme relative to the cranial/caudal and medial/lateral planes, varying from 0° to 360°. D,E: The equatorial plots in Figure 10D–F are as if the viewer were looking at the articular cartilage with the surface uppermost. In the weight-bearing region, the patterns of orientation are strikingly different in the superficial zone (D) compared with the radiate zone (E). F: In addition, orientation is more uniform for superficial zone cells in the weight-bearing region (D) compared with the non–weight-bearing region (F). These three plots demonstrate that, although it is not possible to predict with precision the 3D orientation of a given cilium, there is a pattern to the 3D orientation within the population of chondrocytes studied. G: The rose plots confirm the more consistent alignment in both zones of weight-bearing regions compared with non–weight-bearing regions. H: Tenocytes form a synctium of cells, highly aligned with the collagen fibers of the tissue (Scale bar = 15 μm). J: Analysis through serial-z sections by multiphoton microscopy with axonemes identified by staining with anti-acyl-tubulin demonstrated that axonemes for these cells are highly aligned in 3D space. θaxoneme parallels the alignment of the collagen in the proximal/distal and medial/lateral planes, and ϕaxoneme has a very restricted range, meaning minimal elevation of the axoneme in the anterior/posterior plane (Fig. 10I). Again, although it is not possible to predict the 3D orientation of the ciliary axoneme for a given cell, the 3D orientation of axonemes for the population of tenocytes can be defined. Figures 10A,B,C,D,E,F,G from Farnum and Wilsman,2011 (Figures 3c,3f, 2a5a,3c,6a,7 respectively); Figure 10I from Donnelly et al.,2008 (Figures 7c, 1 respectively); Figure 10J from Donnelly et al., 2010 (Fig. 1).

  • 1Position that the axoneme emerges from the cell: This differs for superficial and radiate zone cells. Essentially all axonemes in superficial zone cells emerge from the side of the cell facing away from the articular surface (Fig. 10D). In radiate zone cells, axonemes emerge from the cell in roughly equal proportions facing toward or away from the articular surface (Fig. 10E). In non–weight-bearing regions, this general pattern becomes more random in both cellular populations (shown here only for the superficial zone cells, Fig. 10F).
  • 2Axonemal orientation in 3D space: For superficial zone chondrocytes in weight-bearing regions, essentially all axonemes point away from the articular surface (ϕaxoneme restricted to 90° to 180°); in the medial/lateral and cranial/caudal planes θaxoneme covers the full range of values from 0° to 360° (Fig. 10D). Radiate zone chondrocytes have a fuller range of ϕaxoneme and a more restricted range of θaxoneme (data not shown; see Farnum and Wilsman,2011).

While the specifics of the data are interesting for interpretations of the possible functional role of the primary cilium in articular cartilage, it is the generalization that is important for this review. This study clearly demonstrated that, although for any given cell in the population of chondrocytes being analyzed it would not be possible to predict with total accuracy either the position of emergence of the axoneme from the cell or the 3D orientation of the axoneme in 3D space after emerging from the cell, it was possible to describe population characteristics that allow predictions for a population of chondrocytes in different weight-bearing or non–weight-bearing regions, and in different cellular zones. The predictive power was different for each of these subpopulations and for the two different parameters, varying from the extremes of essentially certainty (axonemes in superficial weight-bearing cells emerge from the cell in a position on the nonarticular side of the cell) to essentially random, or unable to make a prediction for a given cell (axonemes in superficial weight-bearing cells are orientated at all possible angles of θaxoneme in the direction facing the subchondral bone). These data are summarized in the Table in Figure 10G, where the rose diagrams indicate the relative degree of alignment of axonemes within a given population of chondrocytes of articular cartilage. The high resultant value for superficial zone chondrocytes in weight-bearing regions is a reflection of the restriction of values of ϕaxoneme to only 1/2 of its potential values. The equally high resultant value for radiate zone chondrocytes in weight-bearing regions is a reflection of the interaction of an equal division of axonemes pointing toward and away from the articular surface with a slightly restricted value of θaxoneme in both of these directions.

As an interesting comparison, the orientation of axonemes in the superficial weight-bearing areas conceptually is similar to that of axonemes of olfactory epithelia: emerging from one side of the associated cell, but then oriented in 3D space in all possible directions (Fig. 1G; Mayer et al.,2009; DeMaria and Nagi, 2010; Green and Mykytyn,2010). The difference is, of course, that olfactory sensory neurons have a cluster of 6–8 cilia per cell, while superficial zone chondrocytes have only one cilium per cell. The result achieved is the same: total 3D “coverage” of the extracellular space of the population of cells. For olfaction this specificity of organization ultimately leads to integrated olfactory perception within the CNS (Zou et al.,2001; Menini et al.,2004; McEwen et al.,2008; Mayer et al.,2009). One could speculate that the comparable 3D organization of ciliary axonemes in superficial zone chondrocytes might result in a similar level of coordinated sensory input and integration within articular cartilage.

A second study of axonemal orientation in 3D space in tenocytes from rat extensor tendon used immunohistochemistry for identification of ciliary axonemes and multiphoton microscopy as an approach for analyzing optical serial sections (Fig. 10J; Ascenzi et al.,2007; Donnelly et al.,2008,2010; Farnum et al.,2009). Tenocytes differ from articular chondrocytes in that they form a synctium of cells, with the potential for cells throughout the tissue to communicate with each other (Fig. 10H; Bray et al.,2005). Within the tenocyte population, θaxoneme had values falling in two restricted ranges, demonstrating alignment essentially paralleling the direction of the type I collagen fibrils, with essentially equal numbers of axonemes pointing in the proximal and distal directions in relationship to the long axis of the limb (Fig. 10I). ϕaxoneme had a very narrow range, demonstrating minimal elevation of the axoneme relative to the anterior/posterior axis of the limb (Fig. 10I). Therefore, the same generalization holds in tendon as was seen for articular cartilage: the 3D orientation of axonemes differs among individual cells but is definable for a population of cells, with predictable ranges for θaxoneme and ϕaxoneme for any given cell within the population of cells (Donnelly et al., 2008, 2010; Farnum and Wilsman,2011).

AXONEMAL POSITIONING AND 3D ORIENTATION IN CILIOPATHIES

Changes in both axonemal positioning and axonemal orientation in 3D space are a hallmark of ciliopathies that affect actively motile cilia. Compromised waveform, in the most extreme resulting in totally immotile cilia, is associated with a wide range of ciliary dysfunction under the broad name of primary ciliary dyskinesia (De longh and Rutland, 1989; Biggart et al.,2001; Afzelius,2004; Badano et al.,2006; Bisgrove and Yost, 2006; Sharma et al.,2008; Winyard and Jenkins,2011; and see also Wheatley,2005). The triad of symptoms in Kartagener's syndrome—chronic bronchiectasis, male infertility, and situs inversus totalis in half the patients—was known for almost 75 years before being linked to defects in ciliary/flagellar motility. Early hypotheses also proposed links between ciliary dysfunction and laterality defects. It currently is understood that precise positioning and 3D orientation of nodal cilia (including the appropriate tilt angle) are required for the break of body axis symmetry involved in establishment of laterality (Fig. 5F; Fliegauf et al.,2007; Basu and Breuckner, 2008; Borovina et al.,2010). Similarly, for cilia associated with special senses, it is well established that compromise to either position or to 3D orientation leads to sensory defects, a common manifestation in ciliopathies (Hildebrandt and Otto,2005; Jenkins et al.,2009; D'Angelo and Franco,2009; Nachury et al.,2010).

Positioning and 3D axonemal orientation are precisely defined for actively motile cilia, nodal cilia, and cilia associated with special senses, and any loss of this precision compromises function. An excellent example is the loss of orientation of stereocilia bundles in the cochlea in PCP mutants that never establish initial cell polarity, as well as in intraflagellar transport (IFT) mutants, where the basal body is not appropriately repositioned after initial docking. Both defects are associated with increased randomness of orientation of the stereocilia bundle, even though they act at different stages associated with basal body positioning and 3D orientation (Fig. 11A; Goetz and Anderson,2010). Studies also have focused on molecular aspects of motility compromise found in ciliopathies associated with inappropriate positioning and 3D orientation of cilia due to docking abnormalities. An excellent example is the loss of orientation of cilia of the mucociliary epithelium of amphibian embryos in the disheveled mutant (Fig. 11B; Park et al.,2008). The conclusion is: loss of appropriate axonemal positioning characteristics compromises function (Hoyer-Fender,2010).

Figure 11.

Axonemal positioning and three-dimensional (3D) orientation in ciliopathies. Loss of appropriate positioning and/or 3D orientation is a characteristic of ciliopathies in sensory and motile cilia, including nodal cilia. A: Shows the resulting lack of appropriate positioning of basal bodies in PCP and intraflagellar transport (IFT) murine mutants, even though disruption occurs at different points along the pathway (red boxes). Improper positioning disrupts signaling through the mechanosensory hair cells of the cochlea. The requirement for precision of positioning of the basal body for appropriate axonemal orientation and hence appropriate function also is seen in motile cilia of the mucociliary epidermis of amphibian embryos. In B, controls show the polarized morphology of labeled basal bodies, all oriented in the same direction and parallel to each other (white arrows). In disheveled mutants, this organized parallelism is lost (right hand frame). Plots of the angular measurement of individual basal bodies emphasize the difference in degree of alignment in the controls (left) compared with randomness in the mutants (right). Randomized docking leads to inability to generate a proper waveform and loss of motility. C (left) shows that, in normal growth plates, the differentiation of chondrocytes is visualized spatially as a highly ordered column of cells and axonemes emerge from adjacent cells along a virtual columnar axis, which is thought to be established post proliferation. The loss of columnation and appropriate shape of chondrocytes in a growth plate chondroma (C, right) correlates with randomization of the position of emergence of the primary cilium from cells. An important line of investigation is to test the hypothesis that this correlation of loss of tissue anisotropy with loss of axonemal positioning relates to the function of the primary cilium in the establishment of tissue anisotropy in growth plates. An example of loss of tissue anisotropy in growth plate ciliary mutants is seen in D, contrasting the highly organized columnar arrangement of chondrocytes in a wild-type growth plate (top), with the rounding of cells and relative loss of columnation in polaris mutants (bottom). Ciliary positioning and 3D orientation have not been studied in these mutants, but this would be an appropriate experimental system to analyze the relationship between cellular shape, tissue anisotropy and ciliary positioning and 3D orientation characteristics. Figure 11A from Goetz and Anderson,2010 (Fig. 4) used with permission of the Nature Publishing Group; Figure 11B from Park et al.,2008 (Fig. 7a–d) used with permission of the Nature Publishing Group; Figure 11C from de Andrea et al.,2010 (Fig. 6) used with permission of the Nature Publishing Group; Figure 11D from Ochiai et al.,2009 (Fig. 4A,B) used with permission of Sage Publishing.

Given that inappropriate positioning and/or 3D orientation of cilia is a characteristic of ciliopathies of sensory and motile cilia, including nodal cilia, it is not surprising that a similar loss of orientation/position is found in ciliopathies of the primary cilium (referred to as primary primary ciliary dyskinesia [sic] by Wheatley,2005) that have a skeletal phenotype (McGlashan et al.,2007; Ruiz-Perez et al.,2007; Haycraft and Serra,2008; Nigg and Raff,2009; Ochiai et al.,2009; Retting et al.,2009; Zaghloul and Katsanis,2009; Lee et al.,2010; Qiu et al.,2010; Ascenzi et al.,2011; Zaghloul and Brugman, 2011). The best studied of these at the cellular level have been in growth plate cartilage, which functions during both prenatal and postnatal life as the tissue responsible for elongation of bones. Chondrocytes in growth plate cartilage undergo a differentiation cascade of proliferation and cellular enlargement, before dying and being replaced by bone forming cells. Growth plate chondrocytes are arranged in columns in the direction of elongation of the bone and this highly anisotropic tissue arrangement is required for efficient elongation (Wilsman et al.,1996). Any skeletal abnormality leading to compromised length of bones, such as the more than 300 dwarfisms and chondrodysplasias, will be manifested morphologically in the growth plate by loss of cellular shape (tending to rounded rather than elongated cells) and loss of chondrocytic columnation (tending to loose disorganized columns) (Terpstra et al.,2003; Leighton et al.,2007; Ahrens et al.,2009; Retting et al.,2009; and reviewed in Farnum and Wilsman,2001,2002; Wuelling and Vortkamp,2010).

To the extent it has been studied, growth plate phenotypes in ciliopathies of the primary cilium are characterized by cellular rounding and loss of columnation, resulting in shorter and wider bones than normal (Zhang et al.,2003; Haycraft and Serra,2008; Lehman et al.,2008; Li and Dudley,2009; Ochiai et al.,2009; Retting et al.,2009; Bimonte et al.,2010; Walczak-Sztulpa et al.,2010). The primary cilium has been hypothesized to be involved in the directed cellular movements postdivision that are required for reestablishing growth plate cellular columns after mitosis, with primary cilia positioned in a virtual axis through the column, emerging from opposite sides of postmitotic daughter cells (Fig. 11C, left), therefore essentially pointing along the proximal–distal axis of the bone (Dodds,1930; Leighton et al.,2007; Morales,2007; Haycraft and Serra,2008; Ahrens et al.,2009: Li and Dudley,2009; Farnum and Wilsman,2011). In growth plate osteochondromas, orientation of columns is lost, as is the positioning of axonemal emergence. Growth plate chondrocytes also appear more variable in shape and more rounded than normal (Fig. 11C, right; de Andrea et al.,2010).

A similar tendency toward loss of tissue anisotropy is seen in the diagrams of Figure 11D, in a mutant (polaris, synonymous with IFT88) directly associated with ciliary involvement in the establishment of PCP in epithelial cells (Haycraft et al., 2007; Haycraft and Serra, 2008; Ochiai et al.,2009). Compared with the organization of the control (Fig. 11D, top), the polaris mutant shows columnar disorganization and cellular rounding (Fig. 11D, bottom), as well as loss of cellular organization in articular cartilage. This same morphological phenotype also is characteristic of Kif3a, Smad1/5CKO, and Evc (Ellis-von Crevell) mutants (Zhang et al.,2003; McGlashan et al.,2007; Ruiz-Perez et al.,2007; Retting et al.,2009; Tasouri and Tucker,2011).

A definitive study of positioning and 3D axonemal orientation has not been done in either normal or abnormal growth plate cartilage in these ciliary mutants. What needs to be studied is the molecular basis of how the characteristic growth plate phenotype seen in these mutants is related to ciliary dysfunction, specifically the connection between loss of ciliary function and the loss of columnation accompanied by cellular rounding. However, the same initial conclusion appears to hold as seen in ciliopathies with inappropriate positioning and/or axonemal 3D orientation in motile cilia and cilia of the special senses. That is, there is an interrelationship between loss of appropriate axonemal positioning characteristics and compromised ciliary function. One approach to address this question is through modeling axonemal positional characteristics in normal mice compared with mutants with skeletal phenotypes (Ascenzi et al.,2011). The larger challenge is to develop experimental systems to analyze the molecular mechanisms linking compromised function of primary cilia in connective tissues to inappropriate axonemal positioning characteristics, and to establish the relationship of compromised ciliary function to loss of both tissue anisotropy and appropriate cellular shape.

SIGNIFICANCE OF AXONEMAL POSITIONING AND ORIENTATION IN 3D SPACE FOR PRIMARY CILIA: PERSPECTIVES AND AREAS FOR FUTURE STUDY

From an historical perspective, the extent of specific knowledge of axonemal positional and spatial relationships varies widely as one considers actively motile cilia of tubular organs, nodal cilia, highly modified cilia of the special senses, and primary cilia in both polarized epithelia as well as in tissues with complex tissue organization and irregular cellular shape. In the best studied systems—such as actively motile cilia with complex waveforms in a metachronal wave, nodal cilia positioned at specific tilt angles with synchronous circular movement, and sensory cilia associated with sight, smell and hearing—it is clear that appropriate axonemal positioning on the cell as well as appropriate axonemal orientation in 3D space are positional attributes essential for normal function. Although current data are sparse, it is reasonable to assume that these two positional characteristics are essential also for normal function of the primary cilium.

Primary Cilia in Polarized Cells of Tubular Epithelia: Are They Outliers in Their Simplicity Relating to Axonemal Positional Characteristics?

Primary cilia occur with an incidence of one per cell; the axoneme is thought to change 3D orientation only through passive movement. Those best understood at the molecular level are found in highly polarized cells of the epithelium of tubular organs. For these primary cilia, the central position of emergence from the apical surface of the cell and the 2D planar orientation of the axoneme in 3D space as it is moved by fluid flow appear to be invariant across a wide range of tubular epithelia, making this entire group of cilia among the most homogeneous with respect to positional characteristics of the axoneme. Basic questions of ciliary biology are being actively addressed in this relatively simple system, because it lends itself well to in vitro manipulation; assessment of normal positioning and 3D orientation are straightforward, given their constancy from cell to cell throughout the tissue.

Areas of active research relating directly to positional characteristics of the axoneme of these primary cilia focus on very fundamental questions that will likely prove to be relevant for understanding positioning and 3D axonemal orientation in all ciliary types. These include: What are the molecular mechanisms involved with positional docking of the mother centriole and its maturation into a basal body (Reiter and Mostov,2006; Dawe et al.,2007; Feldman et al.,2007; Pearson et al.,2007; Jonassen et al.,2008; Goetz and Anderson,2010; Gonçalves et al.,2010; Mirzadeh et al.,2010; Kobayashi and Dynlacht,2011)? How is ciliary positioning on the parent cell related to pathways that control oriented cell divisions and establish planar cell polarity (Jones et al.,2008; Dai et al.,2010; Quyn et al.,2010; Shah,2010; Croyle et al.,2011; Gibson et al.,2011; Wansleeben and Meijlink,2011)? Once centrally positioned after initial basal body docking, is there additional migration of the basal body/axonemal complex with final positioning being less central? This would be analogous to what has been demonstrated for ciliary positioning in the inner ear (Cotanche and Corwin,1991; Denman-Johnson and Forge,1999; Jones et al.,2008). How is axonemal length controlled and what is the relationship of axonemal length to axonemal bending and signal transduction (Gardner et al.,2011; Kim et al.,2010; Abdul-Majeed et al.,2011; Rondanino et al.,2011)? Finally, what is the evidence that signaling proteins can localize asynchronously to primary cilia of sister cells, allowing the possibility of segregation of differently aged centrioles leading to variability of signaling responses of primary cilia to environmental signals within a population of cells (Anderson and Stearns,2009)?

Axonemal Positional Relationships of Primary Cilia in Nonepithelial Cells: Might Heterogeneity at the Cellular Level Be Significant for Integrated Function at the Tissue Level?

Dissecting these same questions as they relate to primary cilia in nonepithelial cells such as neurons, myocytes, and cells of connective tissues (and in epithelial cells of irregular shape) is a challenge. However, these kinds of tissues may offer opportunities for examining the relationship between tissue organization and cellular shape, especially in highly anisotropic connective tissues, (Bornens,2002; Benzing and Walz,2006; Jones and Chen,2008; Blitzer et al.,2011; Minc et al.,2011). Results for axonemal positioning on the cell and axonemal 3D spatial orientation as found for articular chondrocytes and tenocytes are intriguing, because they clearly indicate that: (1) For cells of a defined shape, the position of emergence of the axoneme from the cell often has mirror-image symmetry from cell to cell (Figs. 2C, 10I); and (2) Although axonemal orientation in 3D space varies from cell to cell, it can be defined at a population level.

This suggests that axonemal positioning may be critical, not specifically for individual cells, but rather for the population of cells residing in a tissue. Important questions to address include: (1) Does the primary cilium have a role in establishing tissue organization in highly anisotropic tissues? (Chang et al.,2003; Li and Dudley,2009; Tao et al.,2009; Vladar et al.,2009); (2) In tissues where cells are of irregular shape or not highly polarized, what is the relationship (if any) between the primary cilium, cellular shape and pathways usually associated with the establishment of planar cell polarity? (Wang and Nathans,2007; Zallen,2007; Goodrich,2008; Jones et al.,2008; Spassky et al.,2008; Vladar et al.,2009; Blitzer et al.,2011); (3) In these tissues does the position of the basal body on the plasma membrane reflect its initial docking position, or is there the capability of lateral movement postdocking that might be critical for establishing cellular polarization, as has been demonstrated both in unicellular organisms (Absalon et al.,2007), as well as in establishment of cellular polarity in the inner ear (Cotanche and Corwin,1991; Denman-Johnson and Forge,1999; Jones et al.,2008; Jones and Chen,2008); and (4) In tissues where cells are isolated from each other, can experiments be designed to test the hypothesis that 3D orientation of the ciliary axoneme is related to intercellular communication (with individual cilia potentially involved with both sending and receiving information) through the extracellular matrix? (Wu et al.,2001; Whitfield,2004; Ingber,2006; Berzat and Hall,2010; Wheatley,2010).

In several ways spatial and positional relationship of the axonemes of primary cilia in nontubular organs are unique and lead to questions that may help in understanding the function of this subset of cilia. How are the cilia in cells that are surrounded by a dense ECM actually responding? Is it by bending translated through the ECM, by actual physical contact with adjacent cells, or through sensing pressure at the axonemal tip? (Whitfield,2003,2008; Janmey and McCulloch,2007; Bell,2007,2008; Chakravarthy et al.,2010; Wang and Li,2010). What is the significance of the ciliary pocket and the potential diffusion barrier at the base of the cilium in these cells? (Hu et al.,2010; Nachury et al.,2010; Breslow and Nachury,2011; Francis et al.,2011; Ghossoub et al.,2011). What is the significance of the apparently fixed angles of bending seen in axonemes of primary cilia of connective tissues? For these primary cilia is signal transduction activated continuously or through toggling, and can axonemal bending in these cells be modeled as it has been for other ciliary types? (Whitfield,2008; Lee et al.,2010; Rydholm et al.,2010).

Historically, primary cilia have been considered the poor cousins of motile cilia and modified cilia associated with special senses. Indeed, for decades the primary cilium was considered to have a simplified structure and composition compared with its actively motile relatives; only recently has the depth of its true complexity been realized, if not fully understood (reviewed by Wheatley,2005; Thomas et al.,2010). In the last fifteen years the primary cilium has moved to the spotlight, but still most investigations have focused on the primary cilium of polarized epithelia of tubular organs. A challenge is to extend the same kind of experimental versatility at the molecular level to an understanding of the primary cilium in multiple other tissues and organs of the vertebrate body. For these tissues, this includes a need for additional descriptive quantitative studies to define axonemal positional and spatial characteristics. Although the primary cilium has often been described as the cell's antenna (Poole et al.,1985; Benzing and Walz,2006; Marshall and Nonaka,2006; Singla and Reiter,2006; Gros et al.,2009; Vladar et al.,2009; Gupta et al., 2009), for most cells of the vertebrate body we do not yet understand where the antenna is positioned and where it is pointing.

Acknowledgements

The authors thank Jennifer Patterson for her expert help with the images and for obtaining copyright permissions.