Structural defects in cilia of the choroid plexus, subfornical organ and ventricular ependyma are associated with ventriculomegaly
© Swiderski et al.; licensee BioMed Central Ltd. 2012
Received: 11 July 2012
Accepted: 3 October 2012
Published: 9 October 2012
Hydrocephalus is a heterogeneous disorder with multiple etiologies that are not yet fully understood. Animal models have implicated dysfunctional cilia of the ependyma and choroid plexus in the development of the disorder. In this report, we sought to determine the origin of the ventriculomegaly in four Bardet Biedl syndrome (BBS) mutant mouse strains as models of a ciliopathy.
Evans Blue dye was injected into the lateral ventricle of wild- type and BBS mutant mice to determine whether obstruction of intra- or extra-ventricular CSF flow contributed to ventriculomegaly. Transmission electron microscopy (TEM) was used to examine the ultrastructure of the choroid plexus, subfornical organ (SFO), subcommisural organ (SCO), and ventricular ependyma to evaluate their ultrastructure and the morphology of their primary and motile cilia.
Results and discussion
No obstruction of intra- or extra-ventricular CSF flow was observed, implying a communicating form of hydrocephalus in BBS mutant mice. TEM analyses of the mutants showed no evidence of choroidal papillomas or breakdown of the blood:CSF barrier. In contrast, structural defects were observed in a subpopulation of cilia lining the choroid plexus, SFO, and ventricular ependyma. These included disruptions of the microtubular structure of the axoneme and the presence of electron-dense vesicular-like material along the ciliary shaft and at the tips of cilia.
Abnormalities in cilia structure and function have the potential to influence ciliary intraflagellar transport (IFT), cilia maintenance, protein trafficking, and regulation of CSF production. Ciliary structural defects are the only consistent pathological features associated with CSF-related structures in BBS mutant mice. These defects are observed from an early age, and may contribute to the underlying pathophysiology of ventriculomegaly.
KeywordsBardet-Biedl syndrome Cilia Hydrocephalus Ependyma Choroid plexus
Human hydrocephalus is a heterogeneous disorder with multiple etiologies including genetics, developmental defects, viral infection, tumors, hemorrhage and advanced age [1–3]. Congenital hydrocephalus is relatively common and affects 1 in 1,000 live births with a mortality rate of nearly 50% in the absence of shunt placement surgery. Hydrocephalus is characterized by enlarged ventricles resulting from an accumulation of cerebrospinal fluid (CSF) caused by obstruction of intra-ventricular CSF flow (non-communicating hydrocephalus); an imbalance of CSF synthesis and its resorption into the systemic circulation (communicating hydrocephalus), or atrophy of underlying brain tissue or incomplete brain development (hydrocephalus ex vacuo) .
CSF provides nutritional and metabolic support for the brain, waste removal for the central nervous system, and a protective cushion for the brain and spinal cord. It is produced primarily by epithelial cells of the choroid plexuses of the lateral, third and fourth ventricles and to a lesser degree by the ependyma and parenchyma . Beating of motile cilia on the ependymal lining of the ventricles is thought to facilitate intraventricular CSF circulation, particularly through the narrow aqueduct of Sylvius, as well as increase laminar flow across the ependymal surface . Animal models have implicated damage to or loss of the ependymal layer, reduction in number or loss of its cilia, impaired ependymal cilia motility, dysfunction of the subcommissural organ (SCO), and aqueduct stenosis in the development of hydrocephalus [[2, 5]; and references therein]. Further, primary cilia on the apical surface of the choroid plexus epithelium contribute to CSF homeostasis by acting as pressure sensors or as chemosensors that regulate CSF production, osmolarity, or CSF transcytosis from the choroid plexus epithelium into the ventricles via a cilia-based receptor and autonomic system of regulation [6–9].
Despite advances in the study of hydrocephalus, the molecular pathophysiology of this complex disorder, and communicating hydrocephalus in particular is not yet fully understood and requires further investigation. While intra- and extra-ventricular CSF flow in humans and rodents is comparable for the most part, they diverge at the point of resorption through arachnoid granulations. The human brain contains numerous arachnoid granulations while rodents have very few  and the choroid plexus plays a role in both the synthesis and resorption of CSF. In rodents, CSF is resorbed through fenestrated capillaries and venules of the choroid plexus that drain into the vein of Galen (vena cerebri interna and vena cerebri magna), in addition to the primary resorption route through the cerebral lymphatic system as well as the spinal cord [10, 11]. These differences make the use of animal models such as Bardet Biedl syndrome (BBS) mutant mice valuable tools for the study of non-arachnoid based communicating hydrocephalus and cilia dysfunction.
BBS is a rare autosomal recessive disorder that has become a model for cilia disorders based on a variety of dysfunctional phenotypes associated with the syndrome including retinal degeneration, lack of sperm flagella, obesity, polydactyly, anosmia, learning disabilities, and renal abnormalities [[12, 13]; and references therein]. A recent study of 21 BBS patients showed statistically significant increased CSF volume in both the surface of the brain and in the ventricles . BBS is caused by at least 17 genes, which, when individually mutated, give rise to common phenotypes [[13, 15–18]; and references therein]. Seven known BBS proteins (BBS1, BBS2, BBS4, BBS5, BBS7, BBS8, BBS9) are components of the BBSome, a coat complex involved in protein trafficking, including receptor trafficking to ciliary and plasma membranes [19–22]. BBS6, BBS10, and BBS12 form part of a chaperone complex required for BBSome assembly, BBS3 recruits the BBSome to the cilia [21, 23, 24] and BBS17 (Leucine-zipper transcription factor-like 1; LZTFL1) is a negative regulator of BBSome entry into cilia [18, 23]. BBS mutant mice have provided valuable insights into the underlying pathophysiology of the disorder by manifesting cardinal features of the human phenotype including obesity, retinal degeneration, male infertility, and olfactory deficits [[25–27]; and references therein].
In a previous report , we described a new neuroanatomical phenotype in Bbs2 −/− , Bbs4−/−,Bbs6 −/− and Bbs1 M390R/M390R knockin mutant mice (mice homozygous for the most common human BBS mutation that converts a methionine codon to an arginine codon). Each of the three BBS knockout strains as well as the Bbs1 M390R/M390R mice exhibit ventriculomegaly of the lateral and third ventricles of the brain, thinning of the cerebral cortex, and a reduction in the size of the hippocampus and corpus striatum. Unlike severe forms of hydrocephalus observed in other rodent models that result in embryonic or perinatal death or a dome-shaped cranium, the BBS mutant strains used in this study had no distortion of the cranium, the ventriculomegaly was not present at birth, and was progressive in nature . We hypothesized  that the ventriculomegaly is caused by atrophy or incomplete development of brain tissue resulting in a compensatory ex vacuo enlargement of the ventricles, or that compression of the cerebral cortex, hippocampus and corpus striatum are secondary effects of the enlarged ventricles caused by a yet unknown mechanism. Interestingly, transmission electron microscopy (TEM) analysis showed that a subpopulation of ependymal cilia lining the third ventricle of Bbs1M390R/M390R mice had swollen tips that contained vesicle-like inclusions and electron-dense material. These observations suggested that impaired flow of CSF without disruption of CSF production underlie the observed ventriculomegaly.
In the current report, we sought to investigate the potential contribution of structural defects in cilia of the central nervous system linked to hydrocephalus, particularly those of the choroid plexus, ependyma, SCO and subfornical organ (SFO) to the ventriculomegaly in BBS mutant mice. We examined CSF flow in vivo and performed a TEM analysis of tissue morphology and cilia structure of the choroid plexus, SCO, SFO, and ventricular ependyma.
Wild- type, Bbs1M390R/M390R, Bbs2−/−, Bbs4−/−, and Bbs6−/− mice were generated and maintained as described previously . All studies adhered to guidelines established for the care and use of experimental animals and were approved by the Animal Care and Use Committee of the University of Iowa.
Visualization of intra-ventricular and extra-ventricular CSF flow
Seven month-old wild- type and Bbs4 −/− mice (n = 3 for each genotype) were anesthetized with a mixture of ketamine (91 mg/kg) and xylazine (9.1 mg/kg) intraperitoneally (i.p.). The fur, skin, membrane and musculature on the surface of the skull were removed and a small hole was drilled into the skull at Bregma level 33. Evans Blue dye (5–10 μl, 2% in 1X PBS; phosphate-buffered saline minus Ca+2 and Mg+2) was injected slowly into the lateral ventricle using a 25 μl Hamilton syringe to visualize the movement of CSF. The syringe was left in the ventricle post-injection to prevent CSF loss and the dye was allowed to circulate with the CSF for 20 min. Mice were euthanized by an overdose of ketamine and xylazine followed by cervical dislocation and whole mice were immediately frozen at −20°C. The next day, frozen heads were cut in the axial, coronal and sagittal planes and photographed with an Olympus SZX12 stereomicroscope while the tissue was still frozen to prevent dye diffusion.
To examine the ultrastructure of ependyma from the lateral and third ventricles and choroid plexuses from the lateral ventricles prior to the onset of ventriculomegaly seen at P9, newborn (P0) and P2 wild- type and Bbs1M390R/M390R animals (n = 3 for each age and genotype) were euthanized and the skin surrounding the skull was removed. The intact skull was placed in 4% paraformaldehyde in 1X PBS for 4–5 hr at 4°C, and transferred to 2.5% glutaraldehyde-0.1 M cacodylate buffer overnight at 4°C. The next day, intact skulls were stabilized in 2% agarose and 100 μm-thick sections were cut with a vibratome, post-fixed with 1% OsO4, rinsed, dehydrated in a series of alcohol and flat embedded in Eponate-12 epoxy resin (Ted Pella, Redding, CA). Tissues were sectioned (85 nm thickness) with a Leica UC-6 ultramicrotome (Wein, Austria). Sections were counterstained with uranyl acetate and lead citrate, and photographed with a JEOL JEM-1230 (Tokyo, Japan) transmission electron microscope. Electron microscopic images were taken with a Gatan UltraScan 1000 (Pleasonton, CA) 2kx2k CCD digital camera.
To study the progression of ventriculomegaly in P9 Bbs1 M390R/M390R and Bbs2 −/− , Bbs4 −/− and Bbs6 −/− mice up to 2 years of age (n = 3 for each age and genotype), the protocol was modified to include transcardial perfusion of anesthetized mice with 1X PBS, followed by a solution of 4% paraformaldehyde-0.25% glutaraldehyde. Brains were removed and post-fixed overnight with 2.5% glutaraldehyde-0.1 M cacodylate buffer. The next day, 100 μm-thick sections were cut with a vibratome and processed for TEM analysis of the lateral and third ventricles, choroid plexus, SCO and SFO as described above.
MRI of brain ventricles
Sagittal MRI images of wild- type and Bbs1 M390R/M390R , Bbs2−/−, Bbs4−/−, and Bbs6−/− mouse brains (n = 3 for wild type and each BBS mutant strain) was performed as described . All images were collected from 6 month-old mice with the exception of Bbs1M90R/M390R mice which were 3.5 months old.
Intra-ventricular and extra-ventricular CSF flow
Choroid plexus ultrastructure
Ultrastructural analysis of BBS mutant choroid plexus cilia
When clusters of choroid plexus cilia from P0, P2, and P10 mutant animals were viewed longitudinally, a sub-population of cilia contained vesicle-like inclusions and electron-dense material along the ciliary shaft distal to the transition zone. This material was larger than the IFT-like particles typically seen in the choroid plexus cilia (Figure 4F-H). The mutant cilia also exhibited disruption of the ciliary axoneme that was not seen in comparably aged wild- type mice (Figure 4 E-H). In some cilia, the electron-dense material was found between the peripheral microtubule doublets of the axoneme and the ciliary plasma membrane where IFT particles are typically located (Figure 4F, G), while in others it was found in the ciliary lumen, surrounded by the peripheral microtubule doublets (Figure 4H).
Ultrastructural analysis of BBS mutant ventricular ependymal cilia
Ultrastructural analysis of BBS mutant SCO and SFO
Discussion and conclusion
Animal models of hydrocephalus have demonstrated a relationship between cilia defects of the choroid plexus and ependyma and the development of the disorder [[2, 5, 7–9, 25, 33, 39–41] and references therein]. In this report, we used functional assays and ultrastructural analyses to examine ventriculomegaly in four BBS mutant mouse strains widely used as models of ciliopathies. We hypothesized that the ventriculomegaly in BBS mice might be due to ultrastructural damage to the epithelia or cilia of the choroid plexus, ependyma, SCO, or SFO. We demonstrated that a subpopulation of primary and motile cilia in the choroid plexus, SFO and the ependymal lining of the lateral and third ventricles had axonemal defects and in some instances contained electron-dense vesicle-like material along the ciliary shaft and at the tips of cilia. Notably, the mutant mice exhibited no physical obstruction of intra- or extra-ventricular flow implying the ventriculomegaly is associated with a communicating form of hydrocephalus.
It is possible that CSF resorption through the choroid plexus vasculature into the vein of Galen in the BBS mutant animals may be physically impaired due to compression of the vein as a secondary effect of ventricular enlargement or that an impediment of CSF flow across the cribriform plate into the lymphatics of the olfactory turbinates could lead to hydrocephalus . These obstructions could exacerbate the phenotype and will require further examination.
Ventriculomegaly due to increased CSF volume
The most common causes of communicating hydrocephalus in humans are increased CSF synthesis due to choroid plexus papillomas and impaired CSF resorption by the arachnoid granulations . To evaluate possible choroidal anomalies, we analyzed the ultrastructure of the choroid plexuses of the lateral and third ventricles in BBS mutant mice using TEM and found no evidence of choroidal papillomas. The normal appearance of the choroid plexus epithelia with intact junctions between cells argues against the possibility of passive diffusion of molecules from the choroid plexus vasculature into the CSF that could have led to breakdown of the blood:CSF barrier and a loss of CSF homeostasis. Still, we cannot rule out the possibility that the mice produce an increased volume of CSF as a compensatory response to hydrocephalus ex vacuo due to atrophy of underlying brain tissue or incomplete brain development. Future studies of the temporal appearance of ventriculomegaly by MRI analysis in conjunction with cell proliferation and cell death assays will address this issue. Another avenue of future research would be to examine the presence and localization of the aquaporin channels in the choroid plexus that control water transfer [; and references therein].
Ventriculomegaly and impaired cilia function in the SCO and SFO
Dysfunction of the SCO, a specialized zone of ependyma located on the roof of the third ventricle at the entrance to the aqueduct of Sylvius, has been shown to result in hydrocephalus in a number of animal models . The ependymal cells of the SCO secrete glycoproteins that aggregate to form the threadlike Reissner’s fibers that maintain patency of the aqueduct and central canal of the spinal cord [5, 44]. In the absence of a correctly functioning SCO, patency of the aqueduct is compromised, leading to aqueductal stenosis and non-communicating hydrocephalus . Given the apparent intact SCO ultrastructure and patency of the aqueduct of Sylvius and fourth ventricle in BBS mutant mice as old as 2 years of age, it appears unlikely that abnormalities in the SCO contribute to ventriculomegaly. Further, aqueductal stenosis does not appear to be a secondary effect of ventriculomegaly in the BBS mutant mice. Still, the lack of evidence to support the presence of Reissner’s fibers in the mutant mice remains to be addressed.
The SFO, which protrudes into the midline anterior wall of the third ventricle at the junction of the intraventricular foramina of Munro, is anatomically well positioned for its role in osmosensation and the regulation of body and CNS water balance. Although there is currently no known role for the primary and motile cilia that line the SFO [37, 38], we noted structural defects in some of these cilia in BBS mutant mice. They exhibited the axonemal abnormalities and electron-dense vesicle-like material as seen in primary cilia of the choroid plexus and motile ventricular ependymal cilia.
Defective cilia maintenance
BBS has been described as a degenerative disease of the cilium in which certain proteins accumulate in the cilia over time, leading to progressive ciliary dysfunction . Signaling proteins have been shown to accumulate in BBS mutant Chlamydomonas reinhartii flagella  and in zebrafish the absence of BBS proteins leads to delayed retrograde transport [47, 48]. The BBSome functions in the trafficking of membrane proteins between the plasma and ciliary membranes. Several G-protein coupled receptors including MCHR, SSTR3 and dopamine receptor 1 accumulate abnormally within neuronal cilia of BBS mutant mice [49, 50] and ciliary trafficking of the hedgehog signal transducer Smoothened is controlled by the BBSome [18, 22, 51]. Recently, the BBSome has been shown to control IFT assembly and IFT turnaround at the ciliary tip .
It is possible that the electron-dense vesicle-like material found in BBS mutant mouse choroid plexus, SFO and ependymal cilia are the result of defective cilia maintenance. The dimensions of the electron-dense, vesicle-like material are too large to have entered the cilia though the transition zone . A more likely scenario is that the material accumulates in the cilia due to defective anterograde and/or retrograde IFT caused by damage to the axoneme that has been observed in some of the defective cilia or that turnaround at the ciliary tip is impaired in the mutants .
The presence, at birth, of electron dense vesicle-like inclusions in some of the BBS mutant mouse choroid plexus primary cilia may result from defective ciliary maintenance could lead to impaired signaling between the choroid plexus cilia and epithelium and cause an ionic imbalance in the CSF resulting in its overproduction. The choroid plexuses develop during the early stages of mammalian embryogenesis and are fully formed, ciliated and functional at birth . Interestingly, studies of the Tg737orpk mouse and in vitro primary cultures of choroid plexus epithelia have underscored the importance of the choroid plexus and its primary cilia in the regulation of CSF production. It has been proposed that protein mislocalization caused by defective IFT in the Tg737orpk mouse choroid plexus primary cilia impairs their ability to signal to the underlying epithelium via a cAMP-regulated mechanism in order to modulate CSF production [7, 8]. Furthermore, clusters of primary cilia present on the apical surface of porcine choroid plexus primary epithelial cell cultures have been shown to act as negative regulators of fluid transcytosis by decreasing intracellular cAMP levels. These cilia also express neuropeptide FF (NPFF) receptor 2 thought to play a role in chemosensory function and regulation of fluid transport .
There has been a resurgence of interest in the study of higher vertebrates for the role of motile cilia in chemosensation or mechanosensation, functions thought to be ascribed solely to primary, immotile cilia [55–58] following the initial studies in Paramecium and Chlamydomonas reviewed in . Taste receptors have been localized in the ciliary membranes of mouse tracheal epithelial cells , progesterone receptors are present in the motile cilia of the mouse oviduct , and the Px2 receptor has been localized in rabbit tracheal motile cilia . The appearance of structurally defective ependymal cilia in BBS mutant mice as early as P9, which is shortly after the replacement of primary cilia by motile cilia in mice [32, 33], may impair a sensory mechanism that is relayed to the choroid plexus to regulate CSF synthesis, resulting in a loss of CSF homeostasis and ventriculomegaly.
In conclusion, abnormalities in BBS mutant mouse cilia structure and function have the potential to influence ciliary intraflagellar transport (IFT), ciliary beat frequency, cilia maintenance, protein trafficking, and regulation of CSF production. Ciliary structural defects are the only consistent pathological features associated with CSF-related structures in BBS mutant mice. These defects are observed from an early age, and may contribute to the underlying pathophysiology of ventriculomegaly. Additional research is necessary to establish a causal relationship between the ciliary abnormalities and the development of ventriculomegaly in BBS mutant mice. Continued study of the these mice will add to the growing body of knowledge of the roles played by cilia of the choroid plexus, SFO, and ventricular ependyma in CSF homeostasis as well as in understanding the underlying pathophysiologies of BBS.
We thank Gretel Beck, John Beck and Valerie Buffard for genotyping analysis, Chantal Allamargot (University of Iowa Central Microscopy Facility) for photographing the dye-injected brains, Dr. Daniel Thedens for assistance with the MRIs, Shawn Roach for preparing the figures, and Drs. Val Sheffield, Robert Mullins, and Seongjin Seo for helpful discussions.
Funding for this project was supported by a grant from the National Institutes of Health (GM067002).
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