A balance of form and function: Planar polarity and development of the vestibular maculae

The mechanosensory hair cells of the inner ear have emerged as one of the primary models for studying the development of planar polarity in vertebrates. Planar polarity is the polarized organization of cells or cellular structures in the plane of an epithelium. For hair cells, planar polarity is manifest at the subcellular level in the polarized organization of the stereociliary bundle and at the cellular level in the coordinated orientation of stereociliary bundles between adjacent cells. This latter organization is commonly called Planar Cell Polarity and has been described in the greatest detail for auditory hair cells of the cochlea. A third level of planar polarity, referred to as tissue polarity, occurs in the utricular and saccular maculae; two inner ear sensory organs that use hair cells to detect linear acceleration and gravity. In the utricle and saccule hair cells are divided between two groups that have opposite stereociliary bundle polarities and, as a result, are able to detect movements in opposite directions. Thus vestibular hair cells are a unique model system for studying planar polarity because polarization develops at three different anatomical scales in the same sensory organ. Moreover the system has the potential to be used to dissect functional interactions between molecules regulating planar polarity at each of the three levels. Here the significance of planar polarity on vestibular system function will be discussed, and the molecular mechanisms associated with development of planar polarity at each anatomical level will be reviewed. Additional aspects of planar polarity that are unique to the vestibular maculae will also be introduced.     

Sequential expression of Efhc1/myoclonin1 in choroid plexus and ependymal cell cilia

EFHC1 is a gene mutated in patients with idiopathic epilepsies, and encodes the myoclonin1 protein. We here report the distribution of myoclonin1 in mouse. Immunohistochemical analyses revealed that the myoclonin1 first appeared at the roof of hindbrain at embryonic day 10 (E10), and moved on to choroid plexus at E14. At E18, it moved to ventricle walls and disappeared from choroid plexus. From neonatal to adult stages, myoclonin1 was concentrated in the cilia of ependymal cells at ventricle walls. At adult stages, myoclonin1 expression was also observed at tracheal epithelial cilia in lung and at sperm flagella in testis. Specificities of these immunohistochemical signals were verified by using Efhc1-deficient mice as negative controls. Results of Efhc1 mRNA in situ hybridization were also consistent with the immunohistochemical observations. Our findings raise “choroid plexusopathy” or “ciliopathy” as intriguing candidate cascades for the molecular pathology of epilepsies caused by the EFHC1 mutations.     

Secreted frizzled-related protein disrupts PCP in eye lens fiber cells that have polarised primary cilia

Planar cell polarity (PCP) signaling polarises cells along tissue axes. Although pathways involved are becoming better understood, outstanding issues include; (i) existence/identity of cues that orchestrate global polarisation in tissues, and (ii) the generality of the link between polarisation of primary cilia and asymmetric localisation of PCP proteins. Mammalian lenses are mainly comprised of epithelial-derived fiber cells. Concentrically arranged fibers are precisely aligned as they elongate along the anterior-posterior axis and orientate towards lens poles where they meet fibers from other segments to form characteristic sutures. We show that lens exhibits PCP, with each fiber cell having an apically situated cilium and in most cases this is polarised towards the anterior pole. Frizzled and other PCP proteins are also asymmetrically localised along the equatorial-anterior axis. Mutations in core PCP genes Van Gogh-like 2 and Celsr1 perturb oriented fiber alignment and suture formation. Suppression of the PCP pathway by overexpressing Sfrp2 shows that whilst local groups of fibers are often similarly oriented, they lack global orientation; consequently when local groups of fibers with different orientations meet they form multiple, small, ectopic suture-like configurations. This indicates that this extracellular inhibitor disrupts a global polarising signal that utilises a PCP-mediated mechanism to coordinate the global alignment and orientation of fibers to lens poles.     

Inversin, Wnt signaling and primary cilia

Mutations of the ankyrin-repeat protein Inversin, a member of a diverse family of more than 12 proteins, cause nephronophthisis (NPH), an autosomal recessive cystic kidney disease associated with extra-renal manifestations such as retinitis pigmentosa, cerebellar aplasia and situs inversus. Most NPH gene products (NPHPs) localize to the cilium, and appear to control the transport of cargo protein to the cilium by forming functional networks. Inversin interacts with NPHP1 and NPHP3, and shares with NPHP4 the ability to antagonize Dishevelled-stimulated canonical Wnt signaling, potentially through recruitment of the Anaphase Promoting Complex (APC/C). However, Dishevelled antagonism may be confined towards the basal body, thereby polarizing motile cilia on the cells of the ventral node and respiratory tract. Inversin is essential for recruiting Dishevelled to the plasma membrane in response to activated Frizzled, a crucial step in planar cell polarity signaling. During vertebrate pronephros development, the Inversin-mediated translocation of Dishevelled appears to orchestrate the migration of cells and differentiation of segments that correspond to the mammalian loop of Henle. Thus, defective tubule migration and elongation may contribute to concentration defects and cause cyst formation in patients with NPH.     

Planar cell polarity regulators in asymmetric organogenesis during development and disease

The phenomenon of planar cell polarity is critically required for a myriad of morphogenetic processes in metazoan and is accurately controlled by several conserved modules. Six “core” proteins, including Frizzled, Flamingo (Celsr), Van Gogh (Vangl), Dishevelled, Prickle, and Diego (Ankrd6), are major components of the Wnt/planar cell polarity pathway. The Fat/Dchs protocadherins and the Scrib polarity complex also function to instruct cellular polarization. In vertebrates, all these pathways are essential for tissue and organ morphogenesis, such as neural tube closure, left–right symmetry breaking, heart and gut morphogenesis, lung and kidney branching, stereociliary bundle orientation, and proximal–distal limb elongation. Mutations in planar polarity genes are closely linked to various congenital diseases. Striking advances have been made in deciphering their contribution to the establishment of spatially oriented pattern in developing organs and the maintenance of tissue homeostasis. The challenge remains to clarify the complex interplay of different polarity pathways in organogenesis and the link of cell polarity to cell fate specification. Interdisciplinary approaches are also important to understand the roles of mechanical forces in coupling cellular polarization and differentiation. This review outlines current advances on planar polarity regulators in asymmetric organ formation, with the aim to identify questions that deserve further investigation.     

The proteins encoded by the Drosophila Planar Polarity Effector genes inturned, fuzzy and fritz interact physically and can re-pattern the accumulation of “upstream” Planar Cell Polarity proteins

The frizzled/starry night pathway regulates planar cell polarity in a wide variety of tissues in many types of animals. It was discovered and has been most intensively studied in the Drosophila wing where it controls the formation of the array of distally pointing hairs that cover the wing. The pathway does this by restricting the activation of the cytoskeleton to the distal edge of wing cells. This results in hairs initiating at the distal edge and growing in the distal direction. All of the proteins encoded by genes in the pathway accumulate asymmetrically in wing cells. The pathway is a hierarchy with the Planar Cell Polarity (PCP) genes (aka the core genes) functioning as a group upstream of the Planar Polarity Effector (PPE) genes which in turn function as a group upstream of multiple wing hairs. Upstream proteins, such as Frizzled accumulate on either the distal and/or proximal edges of wing cells. Downstream PPE proteins accumulate on the proximal edge under the instruction of the upstream proteins. A variety of types of data support this hierarchy, however, we have found that when over expressed the PPE proteins can alter both the subcellular location and level of accumulation of the upstream proteins. Thus, the epistatic relationship is context dependent. We further show that the PPE proteins interact physically and can modulate the accumulation of each other in wing cells. We also find that over expression of Frtz results in a marked delay in hair initiation suggesting that it has a separate role/activity in regulating the cytoskeleton that is not shared by other members of the group.     

Planar cell polarity: intracellular asymmetry and supracellular gradients of Dachsous

The slope of a supracellular molecular gradient has long been thought to orient and coordinate planar cell polarity (PCP). Here we demonstrate and measure that gradient. Dachsous (Ds) is a conserved and elemental molecule of PCP; Ds forms intercellular bridges with another cadherin molecule, Fat (Ft), an interaction modulated by the Golgi protein Four-jointed (Fj). Using genetic mosaics and tagged Ds, we measure Ds in vivo in membranes of individual cells over a whole metamere of the Drosophila abdomen. We find as follows. (i) A supracellular gradient rises from head to tail in the anterior compartment (A) and then falls in the posterior compartment (P). (ii) There is more Ds in the front than the rear membranes of all cells in the A compartment, except that compartment''s most anterior and most posterior cells. There is more Ds in the rear than in the front membranes of all cells of the P compartment. (iii) The loss of Fj removes intracellular asymmetry anteriorly in the segment and reduces it elsewhere. Additional experiments show that Fj makes PCP more robust. Using Dachs (D) as a molecular indicator of polarity, we confirm that opposing gradients of PCP meet slightly out of register with compartment boundaries.     

Tissue polarity and PCP protein function: C. elegans as an emerging model

Polarity is the basis for the generation of cell diversity, as well as the organization, morphogenesis, and functioning of tissues. Studies in Caenorhabditis elegans have provided much insight into PAR-protein mediated polarity; however, the molecules and mechanisms critical for cell polarization within the plane of epithelia have been identified in other systems. Tissue polarity in C. elegans is organized by Wnt-signaling with some resemblance to the Wnt/planar cell polarity (PCP) pathway, but lacking core PCP protein functions. Nonetheless, recent studies revealed that conserved PCP proteins regulate directed cell migratory events in C. elegans, such as convergent extension movements and neurite formation and guidance. Here, we discuss the latest insights and use of C. elegans as a PCP model.     

Wnt and planar cell polarity signaling in cystic renal disease

Cystic kidney diseases can cause end stage renal disease, affecting millions of individuals worldwide. They may arise early or later in life, are characterized by a spectrum of symptoms and can be caused by diverse genetic defects. The primary cilium, a microtubule-based organelle that can serve as a signaling antenna, has been demonstrated to have a significant role in ensuring correct kidney development and function. In the kidney, one of the signaling pathways that requires the cilium for normal development is Wnt signaling. In this review, the roles of primary cilia in relation to canonical and non-canonical Wnt/PCP signaling in cystic renal disease are described. The evidence of the associations between cilia, Wnt signaling and cystic renal disease is discussed and the significance of planar cell polarity-related mechanisms in cystic kidney disease is presented. Although defective Wnt signaling is not the only cause of renal disease, research is increasingly highlighting its importance, encouraging the development of Wnt-associated diagnostic and prognostic tools for cystic renal disease.     

Wnt and planar cell polarity signaling in cystic renal disease

Cystic kidney diseases can cause end stage renal disease, affecting millions of individuals worldwide. They may arise early or later in life, are characterized by a spectrum of symptoms and can be caused by diverse genetic defects. The primary cilium, a microtubule-based organelle that can serve as a signaling antenna, has been demonstrated to have a significant role in ensuring correct kidney development and function. In the kidney, one of the signaling pathways that requires the cilium for normal development is Wnt signaling. In this review, the roles of primary cilia in relation to canonical and non-canonical Wnt/PCP signaling in cystic renal disease are described. The evidence of the associations between cilia, Wnt signaling and cystic renal disease is discussed and the significance of planar cell polarity-related mechanisms in cystic kidney disease is presented. Although defective Wnt signaling is not the only cause of renal disease, research is increasingly highlighting its importance, encouraging the development of Wnt-associated diagnostic and prognostic tools for cystic renal disease.     

Planar cell polarity genes and neural tube closure

Closure of the neural tube is essential for normal development of the brain and spinal cord. Failure of closure results in neural tube defects (NTDs), common and clinically severe congenital malformations whose molecular mechanisms remain poorly understood. On the other hand, it is increasingly well established that common molecular mechanisms are employed to regulate morphogenesis of multicellular organisms. For example, signaling triggered by polypeptide growth factors is highly conserved among species and utilized in multiple developmental processes. Recent studies have revealed that the Drosophila planar cell polarity (PCP) pathway, which directs position and direction of wing hairs on the surface of the fly wing, is well conserved, and orthologs of several genes encoding components of the pathway are also found in vertebrates. Interestingly, in vertebrates, this signaling pathway appears to be co-opted to regulate "convergent extension" cell movements during gastrulation. Disruption of vertebrate PCP genes in Xenopus laevis or zebrafish causes severe gastrulation defects or the shortening of the trunk, as well as mediolateral expansion of somites. In Xenopus, in which the neural tube closes by elevation and fusion of neural folds, inhibition of convergent extension can also prevent neural tube closure causing a "spina bifida-like" appearance. Furthermore, several of the genes involved in the PCP pathway have recently been shown to be required for neural tube closure in the mouse, since mutation of these genes causes NTDs. Therefore, understanding the mechanisms underlying the establishment of cell polarity in Drosophila may provide important clues to the molecular basis of NTDs.     

Planar cell polarity in the larval epidermis of Drosophila and the role of microtubules

We investigate planar cell polarity (PCP) in the Drosophila larval epidermis. The intricate pattern of denticles depends on only one system of PCP, the Dachsous/Fat system. Dachsous molecules in one cell bind to Fat molecules in a neighbour cell to make intercellular bridges. The disposition and orientation of these Dachsous–Fat bridges allows each cell to compare two neighbours and point its denticles towards the neighbour with the most Dachsous. Measurements of the amount of Dachsous reveal a peak at the back of the anterior compartment of each segment. Localization of Dachs and orientation of ectopic denticles help reveal the polarity of every cell. We discuss whether these findings support our gradient model of Dachsous activity. Several groups have proposed that Dachsous and Fat fix the direction of PCP via oriented microtubules that transport PCP proteins to one side of the cell. We test this proposition in the larval cells and find that most microtubules grow perpendicularly to the axis of PCP. We find no meaningful bias in the polarity of microtubules aligned close to that axis. We also reexamine published data from the pupal abdomen and find no evidence supporting the hypothesis that microtubular orientation draws the arrow of PCP.     

Revisiting planar cell polarity in the inner ear

Since the first implication of the core planar cell polarity (PCP) pathway in stereocilia orientation of sensory hair cells in the mammalian cochlea, much has been written about this subject, in terms of understanding how this pathway can shape the mammalian hair cells and using the inner ear as a model system to understand mammalian PCP signaling. However, many conflicting results have arisen, leading to puzzling questions regarding the actual mechanism and roles of core PCP signaling in mammals and invertebrates. In this review, we summarize our current knowledge on the establishment of PCP during inner ear development and revisit the contrast between wing epithelial cells in Drosophila melanogaster and sensory epithelia in the mammalian cochlea. Notably, we focus on similarities and differences in the asymmetric distribution of core PCP proteins in the context of cell autonomous versus non-autonomous role of PCP signaling in the two systems. Additionally, we address the relationship between the kinocilium position and PCP in cochlear hair cells and increasing results suggest an alternate cell autonomous pathway in regulating PCP in sensory hair cells.     

Atrial natriuretic peptides elevate cyclic GMP levels in primary cultures of rat ependymal cells

The aim of this study was to examine the effect of atrial natriuretic peptides on primary cultures of ependymal cells, as measured by changes in intracellular levels of cyclic GMP. Incubation of ependymal cells with rat atrial natriuretic peptide-(1-28) (rANP) elicited a 30-fold increase in ependymal cGMP content within 1 min and more than a 100-fold increase within 10 min to a plateau value of approximately 30 pmol/mg protein. The C-type natriuretic peptide (CNP) elicited a similar increase in cGMP levels; however the maximal effect was observed within 1 min and the levels subsequently dropped by 90% to a low plateau within 10 min. A comparison of the concentration-response curves for rANP, human ANP-(1-28) (hANP) and CNP showed that rANP, hANP and CNP had similar effects, with regards to elevation of cGMP levels at high concentrations, but with differing EC50 values. These results demonstrate the presence of a heterogenous population of functional ANP receptors in cultured ependymal cells suggesting that ANP may regulate specific ependymal cell activity.     

Uptake of peroxidase from the third ventricle by ependymal cells of the median eminence

Summary Peroxidase injected into the subarachnoid space in mice is absorbed by ependymal cells of the median eminence. The ependymal cells of the median eminence of the rat and Japanese quail absorb peroxidase injected into the third ventricle. The processes of these ependymal cells terminate at the capillaries of the primary plexus or those surrounding the ventromedial nucleus of the hypothalamus. In all three species, peroxidase is absorbed by the ependymal cells of the paraventricular organ and by those in close proximity to it. Some ependymal cells send processes to the capillaries in the lateral nucleus of the hypothalamus. These phenomena are discussed in relation to adenohypophysial function.A part of this investigation was effected while the senior author held a Visiting Professorship at the University of Giessen [Department of Anatomy (Professor A. Oksche, Director)].