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First published online November 10, 2005
doi: 10.1242/10.1242/dev.02153

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Dysfunctional cilia lead to altered ependyma and choroid plexus function, and result in the formation of hydrocephalus

¹ Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
² Department of Medicine, Division of Cardiovascular Disease, University of Alabama at Birmingham, Birmingham, AL 35294, USA
³ High Resolution Imaging Facility, University of Alabama at Birmingham, Birmingham, AL 35294, USA
⁴ Department of Medicine, Division of Nephrology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
⁵ Nephrology Research and Training Center, University of Alabama at Birmingham, Birmingham, AL 35294, USA
⁶ Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL 35294, USA

View larger version (79K): [in a new window]   Fig. 1. Tg737orpk mutant mice develop hydrocephalus. (A) Comparison of lateral views of 10-day-old wild-type and Tg737orpk mice indicates that the mutants exhibit a bulging forehead (arrow), characteristic of hydrocephalus. (B) Gross analysis of the brains from mutants shows signs of compression at the olfactory bulb and the frontal pole of the cerebrum (black arrowhead). Also, the cerebellum is more prominent in mutant animals (white arrowhead) than in the wild-type control. (C) Hematoxylin and Eosin-stained coronal sections through identical regions of the brain demonstrate marked dilatation of the lateral ventricles (arrows) in mutant animals compared with wild-type controls. Scale bar: 4 mm.

View larger version (60K): [in a new window]   Fig. 2. Analysis of hydrocephalus progression in Tg737orpk mutant mice using T2 RARE MRI. Compartments containing CSF appear white while brain matter is gray. (A,C) Dilatation is evident in the lateral ventricles (white arrowheads) of 1-day-old mutants as compared with wild types. (E) By contrast, there is no sign of expansion in the fourth ventricle or in the aqueduct (arrows) at this age. (B,D) By day 6, the lateral ventricles of the mutants are markedly enlarged (white arrowheads), without overt differences in the (F) fourth ventricle and aqueduct, but protuberance is seen on the skull above the cerebellum (gray arrowheads). (D) In the subarachnoid space, no difference is detected between wild-type and mutant animals (black arrowheads). Scale bar: 10 mm. (G) Quantitative measurement of the relative ventricular volume in mutant and wild-type controls at each age (n=4; *P<0.05).

View larger version (64K): [in a new window]   Fig. 3. Altered cilia morphology on cells of the ventricular system in Tg737orpk mutant mice. Photomicrographs of brain sections from wild-type and mutant animals, showing immunolocalization of acetylated--tubulin (green) and polaris (red). White and yellow arrowheads indicate cilia. (A) Ependymal cilia in wild-type mice are in well-organized groups, with equal length, whereas cilia on the Tg737orpk mutant ependyma are fewer in number, shorter and anisometric. Polaris predominantly localizes to the basal body in the wild-type ependyma, but is found to accumulate at the cilia tip in the mutants. (B) Grouped and primary cilia are present on the CP of wild-type mice and polaris is concentrated at the basal bodies. Polaris accumulates at the tip of the grouped and primary cilia in Tg737orpk mice. Cilia often exhibit a large bulb-like structure in which polaris is concentrated. (C) Scanning electron microscopy of ependymal cilia of normal and Tg737orpk mutant mice. (D) Cilia on the CP of normal and Tg737orpk mutants. In mutants, the cilia are morphologically abnormal with a thickened axoneme. Scale bars: in A, 20 µm; in B, 10 µm; in C, 15 µm; in D 2.5 µm.

View larger version (74K): [in a new window]   Fig. 4. Defects in cilia beat of the Tg737orpk mutant result in impaired fluid flow over the ependymal cells. Red and yellow arrowheads label the ependyma and ependymal apical cilia, respectively. (A,B) DIC (A) and fluorescence (B) images of wild-type and mutant ependyma. Fluorescence images were overlaid with the movement of the fluorescently labeled beads, as recorded by motion tracking (yellow lines, see Movie 1 in the supplementary material). Movement of the beads propelled by wild-type cilia beating was rapid and directional, whereas movement of the beads in the mutant samples was random. Scale bar: 20 µm. (C) Graph showing quantitative analysis of the flow generated by the cilia in the left (LV) and fourth (4V) ventricles from mutant and wild-type samples (n=6; *P<0.005).

View larger version (151K): [in a new window]   Fig. 5. Analysis of cilia in the ventricular system in 1-day-old mice. Brain sections of a 1-day-old wild-type mouse containing the (A) lateral and (B) third ventricles, (C) the aqueduct and (D) the fourth ventricle were analyzed for the presence of cilia (anti-acetylated-tubulin, green; polaris, red) on the ependyma (white arrowheads) and the choroid plexus epithelia (white arrow). No multi-ciliated cells were evident on the ependyma of the (A) lateral, (B) third or (D) fourth ventricles at this age. Ependymal cells possess primary cilium, as shown by the SEM and immunofluorescence (inset in A,E; yellow arrowheads). (C) By contrast, the ependymal lining of the aqueduct was multi-ciliated (white arrowhead). Inset shows that multiple cilia are also present in the mutant aqueduct. (F) Multiple cilia cover cells in the aqueduct (yellow arrowheads). (G) Grouped and single cilia on the choroid plexus. Scale bars: in A-D, 200 µm; in E-G, 10 µm.

View larger version (131K): [in a new window]   Fig. 6. The initiation of hydrocephalus precedes aqueduct stenosis in Tg737orpk mutant mice. Movement of DiI (red) was tracked through brain sections of 2- and 6-day-old wild-type and Tg737orpk mutant mice, 10 minutes post-injection. (A,B) Horizontal view of brains showing the lateral ventricles (black arrows), third ventricle (black arrowheads) and fourth ventricle (white arrowheads). (C-H) Fluorescence images of brain sections through the indicated regions from (C,E,G) 2-day-old and (D,F,H) 6-day-old control and mutant mice. DiI is detectable in the fourth ventricle of 2-day-old mutants (A,G, right panels), but is not seen in 6-day-old mutants (B,H, right panels), indicating that CSF movement was obstructed in these mutants. Scale bar: 200 µm.

View larger version (120K): [in a new window]   Fig. 7. Tg737orpk mutant mice demonstrate no overt loss of epithelial polarity in the choroid plexus. Arrowheads indicate the apical surface of the choroid plexus. (A) Expression of -catenin (red) in sections of wild-type and mutant mice. (B) ZO-1 (red) is localized to the tight junctional complexes near the apical surface of wild-type and mutant choroid epithelia. (C) Analysis of transport proteins Na+/K+ATPase (green) and the anion exchanger type 2 (AE2, red) shows normal localization at the apical and basolateral membranes, respectively. Scale bar: 20 µm.

View larger version (68K): [in a new window]   Fig. 8. Altered localization of proteins in the cilial axoneme of Tg737orpk mutants. On the wild-type choroid plexus, polycystin 1 (red) was localized predominantly at the base of the cilia (acetylated--tubulin, green), whereas, in the mutants, polycystin 1 accumulated in the bulb-like structures at the cilia tip. Scale bar: 20 µm.

View larger version (11K): [in a new window]   Fig. 9. Choroid plexus physiology is altered in the Tg737orpk mutants. Graphs indicating (A) the chloride concentration in the CSF of wild-type and mutant mice, and (B) the intracellular cyclic AMP level ([cAMP]i) in the CP epithelium from wild-type and mutant animals (n=6 and n=7, respectively; A, *P<0.05; B, *P<0.005).