Human Molecular Genetics

Human Molecular Genetics Advance Access originally published online on July 21, 2004
Human Molecular Genetics 2004 13(18):2133-2141; doi:10.1093/hmg/ddh219

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Human Molecular Genetics, Vol. 13, No. 18 © Oxford University Press 2004; all rights reserved

Dysfunction of axonemal dynein heavy chain Mdnah5 inhibits ependymal flow and reveals a novel mechanism for hydrocephalus formation

Inés Ibañez-Tallon¹, Axel Pagenstecher², Manfred Fliegauf³, Heike Olbrich³, Andreas Kispert⁴, Uwe-Peter Ketelsen³, Alison North⁵, Nathaniel Heintz¹ and Heymut Omran^3,*

¹Laboratory of Molecular Biology, Howard Hughes Medical Institute, Rockefeller University, New York, 10021 NY, USA, ²Department of Neuropathology, Albert-Ludwigs-University, 79106 Freiburg, Germany, ³Department of Pediatric Neurology and Muscle Disorders, Albert-Ludwigs-University, 79106 Freiburg, Germany, ⁴Institut für Molekularbiologie, Medizinische Hochschule Hannover, 30625 Hannover, Germany and ⁵Bio-Imaging Resource Center, Rockefeller University, 1230 York Avenue, New York, NY 10021, USA

Received June 1, 2004; Accepted July 9, 2004

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
Motility of unicellular organisms occurred early in evolution with the emergence of cilia and flagella. In vertebrates, motile cilia are required for numerous functions such as clearance of the airways and determination of left–right body asymmetry. Ependymal cells lining the brain ventricles also carry motile cilia, but their biological function has remained obscure. Here, we show that ependymal cilia generate a laminar flow of cerebrospinal fluid through the cerebral aqueduct, which we term as ‘ependymal flow’. The axonemal dynein heavy chain gene Mdnah5 is specifically expressed in ependymal cells, and is essential for ultrastructural and functional integrity of ependymal cilia. In Mdnah5-mutant mice, lack of ependymal flow causes closure of the aqueduct and subsequent formation of triventricular hydrocephalus during early postnatal brain development. The higher incidence of aqueduct stenosis and hydrocephalus formation in patients with ciliary defects proves the relevance of this novel mechanism in humans.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
Cilia are hair-like organelles extending from the cell membrane with either motile or sensory functions. In the unicellular alga Chlamydomonas rheinhardtii and in sperm, flagella resembling motile cilia, propel cells through a fluid. In vertebrates, multiple motile cilia or motile monocilia are located on various epithelial cells and move extracellular fluid. These include the respiratory epithelium of the airways, the embryonic node and the ependyma of the brain ventricles (1). Axonemal dyneins are the molecular motors that generate the movement of motile cilia and flagella by ATPase dependent reactions. Dynein heavy chain proteins assemble with intermediate and light chains into multiprotein complexes to form outer and inner dynein arms (ODA and IDA, respectively) which are attached to the axonemal microtubules (2). The very large dynein heavy chains form the globular heads and the stem of the complexes, and contain the ATPase and microtubule motor domains (3).

The ODA of the biflagellate alga Chlamydomonas comprises three (-, ß- and -) heavy chains. Mutations of the -heavy chain gene result in slow-swimming algae with ultrastructural axonemal defects in the ODA (4). The human and murine orthologs of Chlamydomonas -heavy chain are DNAH5 and Mdnah5, respectively. Recessive mutations of DNAH5 and Mdnah5 cause primary ciliary dyskinesia (PCD) (5,6). Symptoms of PCD include chronic respiratory infections and randomization of body situs (7). Respiratory cilia in affected patients and in mutant mice are immotile and completely lack ODAs. As a result of cilia immotility, affected individuals show impaired mucociliary clearance, which accounts for the observed chronic respiratory infections. Similarly, dysfunction of cilia at the embryonic node most likely results in random left/right axis determination and situs inversus in half of the affected patients and mutant mice (5,6). Consistently, Mdnah5 is specifically expressed in the respiratory epithelium and in the ventral surface of the node (5,8). In addition, Mdnah5-deficient mice develop severe hydrocephalus at early postnatal ages (6). Mutant mice with deficiencies in other axonemal proteins (Spag6) or in components involved in ciliogenesis (polaris, polymerase and Hfh-4) have also been reported to develop hydrocephalus (9–12). Moreover, WIC-Hyd rats and SUMS/NP mice are common hydrocephalic animal models with cilia-related but as yet unidentified genetic defects (13,14). These findings clearly indicate a link between hydrocephalus formation and cilia dysfunction. Interestingly, hydrocephalus has also been reported in patients with PCD, which suggests that cilia dysfunction might also contribute to human hydrocephalus formation (15–21). However, the precise mechanism that links cilia dysfunction to hydrocephalus formation has not been elucidated so far. Therefore, we investigated the role of axonemal dynein heavy chain Mdnah5 in ependymal cilia function and cerebrospinal fluid flow. We show that Mdnah5 is specifically expressed in ependymal cells lining the brain ventricles and the aqueduct. Mdnah5 deficiency results in cell-specific ultrastructural ODA defects in ependymal cilia. We demonstrate that ependymal cilia motility produces a directional ‘ependymal flow’ of the cerebrospinal fluid through the brain ventricles and the aqueduct. Furthermore, the absence of this ependymal flow is associated with the closure of the cerebral aqueduct during early postnatal brain development in Mdnah5-deficient mice, which subsequently leads to the formation of hydrocephalus. Thus, we show for the first time that a direct functional connection exists between the laminar flow produced by ependymal cilia and the correct development of the ventricular system during early postnatal brain development.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
Mdnah5 is expressed in ciliated ependymal cells lining the brain ventricles
Axonemal dynein heavy chains are highly conserved in evolution, and have retained their role as molecular motors of motile cilia and flagella (22). We have previously shown that Mdnah5 is expressed in motile respiratory cilia. We assumed that Mdnah5 is also an axonemal component of motile ependymal cilia. To confirm this, we carried out in situ hybridization experiments on brain sections from adult wild-type mice (Fig. 1). Mdnah5 is specifically expressed in ependymal cells lining the brain ventricles and the aqueduct. Expression of Mdnah5 was not seen in other cell types of the brain, which indicates an exclusive role in ciliated ependymal cells.

View larger version (141K): [in this window] [in a new window]   Figure 1. Mdnah5 expression is restricted to ependymal cells. Section in situ hybridization analysis of Mdnah5 expression in adult wild-type mice brains show specific staining in (A) lateral ventricles, (B) in the third ventricle and (C) the aqueduct of Sylvius. (D, E) Higher magnifications of details from (B) and (C), respectively. Please note the cilia on the ependymal cell surface.

 
Partial deficiency of ODA in ependymal cilia of Mdnah5-deficient mice
Mutations in the orthologous dynein heavy chain genes -HC, DNAH5 and Mdnah5 have been shown to cause a loss of ODA in axonemes from Chlamydomonas and respiratory cilia from humans and mice, respectively. To test whether deficiency of Mdnah5 also affects the ultrastructure of ependymal cilia, we performed transmission electron microscopy on brain samples from Mdnah5-deficient mice and control littermates (Fig. 2). To circumvent that secondary changes caused by increased intraventricular pressure lead to artificial results, we exclusively examined cilia derived from the fourth ventricle (Fig. 4). In both Mdnah5-deficient mice, we found a variable degree of ODA deficiency ranging from 0 to 5 ODA per cilium. This observation was supported by a large number of examined cilia. To calculate the mean number of ODA per cilium, we used only transverse sections (originating from both animals), which show all doublets in high quality. An average of 2.3 ODA (n=29) per transverse section was found in Mdnah5-deficient mice in contrast to eight in wild-type mice. Thus, the number of ODA in ependymal cilia of Mdnah5-deficient mice is markedly reduced.

View larger version (42K): [in this window] [in a new window]   Figure 2. Transverse sections of ependymal cilia from Mdnah5-deficient mice show variable ODA defects. Transmission electron microscopy of ependymal cilia of the fourth brain ventricle show that (A) eight ODAs (arrows) are visible in the control, whereas different transverse sections of ependymal cilia from the same Mdnah5-deficient mouse show (B) total and (C) partial absence of ODAs. Arrows indicate the location of the remaining ODAs.

 

View larger version (112K): [in this window] [in a new window]   Figure 4. Mdnah5-deficient mice develop triventricular hydrocephalus owing to closure of the ventricular system at different sites along the cerebral aqueduct. (A, B) Sagittal brain sections of P0.5 control and Mdnah5-deficient mice show patent aqueducts connecting the third and fourth ventricles. (C) Normal brain anatomy of a control mouse (P12). (D) Hydrocephalic brain of an Mdnah5-deficient mouse (P12) with enlargement of both lateral ventricles and the third ventricle. The size of the fourth ventricle is normal. (E) Higher magnification of normal aqueduct anatomy (C). (F and G) Consecutive serial sagittal brain sections demonstrate aqueduct discontinuity in another Mdnah5-deficient mouse brain (P12). The proximal (F) and the distal part of the aqueduct (G) are closed and lack connection, because the central part of the aqueduct is missing. (H–K) Consecutive sagittal brain sections of an additional Mdnah5-deficient mouse also demonstrate aqueduct discontinuity and show abnormal narrowing of the aqueduct. III, IV, Lv, third, fourth, lateral ventricles; aq, respectively aqueduct.

 
Ependymal cilia produce a directed fluid flow, which is absent in Mdnah5-deficient mice
We next assessed, whether the observed ultrastructural alterations affect the motility of the ependymal cilia. We carried out high-speed video recordings using differential interference contrast (DIC) and fluorescence microscopy on brain samples in the presence of a liquid suspension of fluorescent latex beads. In wild-type mice brains (Fig. 3A), the fast and synchronized movement of ependymal cilia produced a laminar fluid flow above the surface of the ependymal cells. The fluid flow caused a continuous movement of fluorescent beads (Supplementary Material). This result indicates that the ependymal cilia lining the brain ventricles produce a constant directed flow of cerebrospinal fluid. We term this movement as ‘ependymal flow’ in analogy to the ‘nodal flow’ of the extraembryonic perinodal fluid generated by nodal cilia.

View larger version (134K): [in this window] [in a new window]   Figure 3. Ependymal flow produced by ciliary activity in wild-type mouse brain ventricles is absent in Mdnah5-deficient mice. (A) Movement of fluorescent particles visualizes the directed laminar ‘ependymal flow’ (red lines/arrows) in the wild-type control. Yellow arrows indicate a fluorescent particle along its trace (right panel). Particles move with an average flow velocity of 21.84 µm/s. (B) Mdnah5-deficient mice lack ependymal flow. The residual particle movements are indistinguishable from (C) background. A total of 200 frames were recorded over a period of 11.72 s (17 frames/s). Four individual images at different times; t0, t1: 0.77 s, t2: 2.99 s and t3: 6.98 s are shown on the right.

 
In contrast, the beat frequency of ependymal cilia from Mdnah5-deficient mice was severely reduced, irregular and unsynchronized compared with control littermates (Supplementary Material), and therefore unable to move fluorescent beads in a continuous and directed manner (Fig. 3B). Movement of fluorescent beads was random and indistinguishable from background movement (Fig. 3C). We concluded, that the ependymal flow is absent in Mdnah5-deficient mice because of the dysmotility of ependymal cilia in these animals.

Mdnah5-deficient mice develop hydrocephalus owing to closure of the cerebral aqueduct
Our ultrastructural and functional data showed that cilia motility and ependymal flow are severely impaired in Mdnah5-mutant mice. As previously reported (6), hydrocephalus occurs in all mice homozygous for Mdnah5 insertional mutation and becomes pathologically evident by enlarged head sizes at early postnatal age. The condition is progressive and affected mice grow to markedly smaller size than wild-type littermates and die before breeding age. To determine how the absence of ependymal flow affects the development of the ventricular system and when the hydrocephalus forms, we examined the brain morphology of Mdnah5-mutant mice and control littermates at different embryonic and early postnatal ages in serial sagittal and coronal semi-thin sections. At embryonic day 16.5 (E16.5) (n=2), postnatal day 0.5 (P0.5) (n=2) and P2 (n=2) brains from Mdnah5-deficient mice were indistinguishable from controls and did not show any indication of hydrocephalus or aqueduct aberrations (Fig. 4A and B). These findings demonstrate that Mdnah5 deficiency does not cause congenital brain malformation. However, in Mdnah5-deficient mice brains (n=12) at early postnatal ages (days 6, 12 and 19) we noticed formation of triventricular hydrocephalus with massive dilatation of the lateral ventricles and enlargement of the third ventricle (Fig. 4C and D). Further observations include thinning of the cortex and compression and distortion of the diencephalon and striatum. The fourth ventricle was never enlarged, consistent with an obstruction of the cerebrospinal fluid flow at the site of the cerebral aqueduct (aqueduct of Sylvius). We observed obstructions of the ventricular system at different sites along the aqueduct in all hydrocephalic Mdnah5-deficient mice studied. Some animals displayed discontinuity of the central part of the aqueduct (Fig. 4E–G). Others showed aqueductal stenosis or abnormal stenotic narrowing of the third ventricle at the junction of the aqueduct.

Development of transient dilatation of brain ventricles or triventricular hydrocephalus in PCD patients
The specific finding of triventricular hydrocephalus formation due to aqueduct closure in Mdnah5-deficient mice prompted us to review medical records of PCD patients with DNAH5 mutations (5). Although hydrocephalus was not documented, a transient dilatation of lateral brain ventricles in the neonatal period was reported in one patient (F753). We also reviewed all available clinical data from 80 patients participating in a German genetic PCD study. Two of these PCD patients had a triventricular hydrocephalus with closure of the aqueduct, which is referred to as ‘aqueduct stenosis’ by clinical neurologists. In both patients, raised intraventricular pressure had to be relieved by neurosurgical therapy. The incidence of congenital hydrocephalus is usually estimated to be three per 1000 (23). However, aqueduct stenosis is only responsible for a small proportion of congenital hydrocephalus cases. Incidence of aqueduct stenosis ranges from 2 to 16% (24,25). Therefore, a careful estimate for the incidence of congenital hydrocephalus caused by aqueduct stenosis is 3 per 10 000. Thus, the incidence of hydrocephalus caused by aqueduct stenosis in PCD patients (1 : 40) is significantly increased when compared with the general incidence of this disorder (3 : 10 000).

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
A role for ependymal cilia dysfunction in the etiology of hydrocephalus has been suggested on the basis of the occurrence of hydrocephalus in animal models with cilia defects (9–14). Likewise, humans affected by PCD are prone to a higher incidence of hydrocephalus. However, the specific mechanism linking cilia dysfunction with the development of hydrocephalus has remained unclear. Here, we report that axonemal dynein Mdnah5-mutant mice have dysfunctional ependymal cilia, ultrastructural ODA deficiencies and altered motility. Our studies demonstrate that normal ependymal cilia generate a directional flow of cerebrospinal fluid, which we term as ‘ependymal flow’. This ependymal flow is absent in Mdnah5-deficient mice as a consequence of cilia dysmotility. Our anatomical examinations indicate that Mdnah5 deficiency causes secondary closure of the aqueduct during early postnatal brain development and not congenital hydrocephalus, because anatomical alterations were first observed in early postnatal Mdnah5-deficient animals. Obstruction of the cerebrospinal fluid system at the site of the aqueduct explains the consecutive widening of the three proximal brain ventricles and the development of triventricular hydrocephalus in Mdnah5-deficient animals. We observed that closure of the aqueduct occurred, when the cerebral aqueduct lies within a very constricted flexure at the pontine level and becomes the narrowest and longest part for the passage of cerebrospinal fluid connecting the third and the fourth ventricles. This invagination, constriction and elongation take place only during late embryonic–early postnatal brain development (Fig. 5). On the basis of our functional data, we conclude that a steady ependymal flow generated by motile ependymal cilia lining the brain ventricles and the aqueduct is essential to maintain the structural integrity of the cerebral aqueduct during that critical time of brain development. On the contrary, if ependymal flow within the aqueduct is insufficient, as in the case of Mdnah5-deficient mice, secondary aqueduct closure and consecutive widening of the three brain ventricles located proximally from the occlusion (triventricular hydrocephalus) develop. Thus, we provide the first evidence that cilia movement is required for the propulsion of cerebrospinal fluid to avoid stenosis of the cerebral aqueduct during early postnatal development. Our data are supported by observations in the spontaneous WIC-Hyd rat mutant, a well-characterized animal model for PCD with unknown genetic defect (26). Similar to the results presented here, impaired ependymal ciliary motility has been demonstrated in hydrocephalic WIC-Hyd rats (27). However, the exact mechanism of hydrocephalus formation in these rats remained unknown, because obstructive lesions responsible for the origin of the hydrocephalus were not identified. Therefore, the WIC-Hyd model has been considered to be a non-obstructive hydrocephalus model. In this report, serial sections of the aqueductal region enabled us to detect aqueduct closure in Mdnah5-deficient mice. In light of our results, animal models with cilia-related defects should be specifically investigated for the presence of aqueduct alterations. Hydrocephalus development in mutant mice with deficiencies in the axonemal protein Spag (at 6–8 weeks after birth) or in polymerase involved in ciliogenesis (soon after birth) is also compatible with a secondary aqueduct closure in these animal models (9,11).

View larger version (36K): [in this window] [in a new window]   Figure 5. Representative scheme of the ventricular system during mouse brain development [E10, E11.5, E14.5, P0.5 and adult (Ad)]. At E10 the brain has developed from the anterior end of the neural tube into three primary vesicles: telencephalic (yellow), mesencephalic (blue) and rhombencephalic (orange). Later the lateral (I and II), third and fourth ventricles develop. Cerebrospinal fluid, predominantly produced in the lateral ventricles is transported through the ventricular system and enters the subarachnoid space through foramina at the fourth ventricle, where it is finally re-absorbed. During late embryonic brain development the cerebral aqueduct connecting the third and fourth ventricle is formed and becomes the narrowest part of the cerebrospinal fluid system.

 
The specific finding of triventricular hydrocephalus formation due to aqueduct stenosis in Mdnah5-deficient mice prompted us to study the relevance of this mechanism in humans. We did not find evidence for hydrocephalus formation in PCD patients carrying DNAH5 mutations (5). However, we found triventricular hydrocephalus with aqueduct stenosis in two of 80 PCD patients, which clearly indicates a higher incidence of this rare disorder in PCD patients. This observation is in agreement with other case reports of hydrocephalus in PCD patients (15–21). We conclude that cilia dysmotility is not sufficient for hydrocephalus formation but increases the risk for hydrocephalus development in humans. The brain morphology of humans with a shorter and wider aqueduct compared with mice, most likely explains the reduced vulnerability for secondary aqueduct occlusion in PCD patients. Vice versa, human individuals with an in-born narrow aqueduct might only develop hydrocephalus, if an additional primary or secondary (i.e. caused by bacterial toxins in meningitis) ependymal cilia dysfunction is present. Hydrocephalus is a frequent neurological disorder commonly caused by obstruction or malabsorption of cerebrospinal fluid (23). We propose here a novel mechanism, termed ependymal flow, as a relevant factor contributing to obstructive triventricular hydrocephalus in human patients.

So far, cell-specific differences between motile cilia of different origin are poorly understood. Variability in the beating pattern, movement frequency and length between respiratory and ependymal cilia have been observed (28). As in other motile cilia, respiratory and ependymal cilia have two central microtubules and nine peripheral microtubule doublets with dynein arms (1). Identical axonemal ultrastructures regarding the IDAs and the ODAs have been reported for both cilia types. In this report, we show that ependymal cilia in Mdnah5- deficient mice have partial absence of ODAs and severely reduced motility. Interestingly, we previously showed that the same mutation causes complete absence of ODAs and immotility in respiratory cilia (6). The observed differences provide novel evidence that the molecular composition of ciliary axonemes varies between cell-specific cilia types. Identification of the differences in the molecular architecture of respiratory and ependymal cilia will help to gain insight into the mechanisms that generate and control the cilia beat.

To avoid associated morbidity, prevention of hydrocephalus is an important goal of health care. Recent interventional studies in cultured rat brainstem slices have shown that application of serotonin can significantly increase the beat frequency of ependymal cilia (29). This observation suggests strategies to control cilia movement in vivo by administration of neurotransmitters and/or interference of signaling pathways. Our findings will aid the understanding of hydrocephalus formation and might help to identify new therapeutic options for the prevention or treatment of this common neurological disorder using ependymal cilia as a novel target.

	MATERIALS AND METHODS

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In situ hybridization experiments on brain sections from adult wild-type mice
In situ hybridization experiments on brain sections from adult wild-type mice using a 1.1 kb digoxigenin labeled antisense riboprobe transcribed from an Mdnah5-cDNA clone (AA880561) were performed as described following a published modification (30,31). Color reactions were extended up to 4 days to visualize the weak expression in the brain ventricles. Specimens were photographed using a Leica DC200 digital camera on a Leica M420 photomicroscope or under Nomarski optics using a Fujix digital camera HC300Z on a Zeiss Axioplan.

Ultrastructural analysis of ependymal cilia
Ultrastructural analyses of ependymal cilia were performed by transmission electron microscopy (Zeiss EM 900) on brain samples from two Mdnah5-deficient mice and two control littermates according to the standard procedures (32). We examined cilia of the fourth ventricle to circumvent artificial secondary changes caused by increased intraventricular pressure.

Videomicroscopic analysis of ependymal cilia function
Ependymal cilia function was analyzed by video microscopy using a Princeton Instruments Pentamax intensified CCD camera mounted on a Zeiss Axiovert 200 microscope with a 100x1.3 NA objective and DIC imaging. Studies were performed on fresh brain samples obtained from nine Mdnah5-deficient animals and seven age-matched control littermates at ages ranging from P 7 to P 14. Immediately after euthanasia by decapitation, brains were removed from the skull and kept in sterile L-15 oocyte medium (specialty media) at room temperature. Under a dissecting microscope, brains were cut sagittally into two using a sharp blade, and tissue brushings of the brain ventricles were performed using a small brush with plastic bristles. Tissue brushing suspensions were collected in eppendorf tubes, kept in L-15 medium at room temperature and processed immediately. Before videomicroscopy analysis, 0.1 ml of tissue brushing suspensions, either alone or mixed with 10 µl of a 0.1% suspension of green fluorescent latex beads (0.046 µm, Sigma) were transferred to the central chamber of a 35 mm glass-bottom microwell dish for inverted microscopes (Plastek cultureware). For each mouse studied, the average processing time from euthanasia to videomicroscopy analysis was of 10 min. In each case the samples were examined at the microscope for 10–30 min at room temperature. During that time no change of flow characteristics was noted. We monitored the spontaneous particle movement of the fluorescent beads suspension as background control. For documentation of ependymal flow directionality and ciliary beating pattern 200 frames were recorded over a period of 7.14 s (28 frames/s). Recordings were analyzed by using MetaMorph software (Universal Imaging). A similar method was described for measurement of the nodal flow (33).

Brain pathology of Mdnah5-deficient and control mice
We determined the origin of hydrocephalus in Mdnah5-deficient and control mice in serial sagittal and coronal semi-thin sections. Brains of mice were analyzed at various ages ranging from E16.5 to P12. The sections were stained using routine protocols.

Incidence of hydrocephalus in PCD patients
Medical data available from PCD patients enrolled in a German genetic PCD study were evaluated for evidence of intrauterine dilatation of brain ventricles or hydrocephalus formation.

	SUPPLEMENTARY MATERIAL

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Supplementary Material is available at HMG online.

	ACKNOWLEDGEMENTS

 
We would like to thank D. Elreda for assistance with cilia motility recordings, D. Morales for help with mouse embryonal studies and M. Petry for technical assistance. We are grateful for the help of the German patient support group ‘Primaere Ciliaere Dyskinesie und Kartagener Syndrom e.V.’ I.I.-T. and N.H. received support from the Howard Hughes Medical Institute. A.K., A.P. and H.O. were supported by the German Research Foundation (DFG Ki728/1, DFG Pa602/3 and DFG Om 6/2).

	FOOTNOTES

 
^* To whom correspondence should be addressed. Tel: +49 7612704301; Fax: +49 7612704344; Email: omran{at}kikli.ukl.uni-freiburg.de

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TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 

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