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A novel mouse model reveals that polycystin-1 deficiency in ependyma and choroid plexus results in dysfunctional cilia and hydrocephalus.

Wodarczyk C, Rowe I, Chiaravalli M, Pema M, Qian F, Boletta A - PLoS ONE (2009)

Bottom Line: Here, we show that our approach was successful in generating a fully functional and easily detectable endogenous PC-1.Both choroid plexus and ependymal cilia were morphologically normal in these mice, suggesting a role for PC-1 in ciliary function or signalling in this compartment, rather than in ciliogenesis.We propose that the role of PC-1 in the brain cilia might be to prevent hydrocephalus, a previously unrecognized role for this receptor and one that might have important implications for other genetic or sporadic diseases.

View Article: PubMed Central - PubMed

Affiliation: Dulbecco Telethon Institute (DTI) at Dibit, San Raffaele Scientific Institute, Milan, Italy.

ABSTRACT
Polycystin-1 (PC-1), the product of the PKD1 gene, mutated in the majority of cases of Autosomal Dominant Polycystic Kidney Disease (ADPKD), is a very large (approximately 520 kDa) plasma membrane receptor localized in several subcellular compartments including cell-cell/matrix junctions as well as cilia. While heterologous over-expression systems have allowed identification of several of the potential biological roles of this receptor, its precise function remains largely elusive. Studying PC-1 in vivo has been a challenging task due to its complexity and low expression levels. To overcome these limitations and facilitate the study of endogenous PC-1, we have inserted HA- or Myc-tag sequences into the Pkd1 locus by homologous recombination. Here, we show that our approach was successful in generating a fully functional and easily detectable endogenous PC-1. Characterization of PC-1 distribution in vivo showed that it is expressed ubiquitously and is developmentally-regulated in most tissues. Furthermore, our novel tool allowed us to investigate the role of PC-1 in brain, where the protein is abundantly expressed. Subcellular localization of PC-1 revealed strong and specific staining in ciliated ependymal and choroid plexus cells. Consistent with this distribution, we observed hydrocephalus formation both in the ubiquitous knock-out embryos and in newborn mice with conditional inactivation of the Pkd1 gene in the brain. Both choroid plexus and ependymal cilia were morphologically normal in these mice, suggesting a role for PC-1 in ciliary function or signalling in this compartment, rather than in ciliogenesis. We propose that the role of PC-1 in the brain cilia might be to prevent hydrocephalus, a previously unrecognized role for this receptor and one that might have important implications for other genetic or sporadic diseases.

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Cilia morphology and apico-basal polarity in the brain from knock-out embryos at E16.5.(A to H) Centrosomes and cilia in the choroid plexus from E16.5 brain were stained with anti-pericentrin (red) and anti-acetylated alpha-tubulin antibodies (green), respectively. Multicilia, monocilia, and bundles from lateral ventricles in knock-out (B, D, and F, respectively) and in wild-type (A, C, E) do not show differences in shape or number. Cilia from choroid plexus of the third ventricle of wild-type (G) and knock-out (H) animals do not show any differences. Staining of β-catenin of the choroid plexus in the third ventricle (I and J) and Aquaporin-1 in the choroid plexus of the lateral ventricle (K, L) did not reveal major differences. Transverse sections of the cilium of an ependymal cell from the third ventricle of wild-type (M) or mutant (N) animals carried out by electronic microscopy revealed the normal structure of the cilium. Scale bar A to F 5 µm; G to L 10 µm; M and N 100 nm.
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pone-0007137-g007: Cilia morphology and apico-basal polarity in the brain from knock-out embryos at E16.5.(A to H) Centrosomes and cilia in the choroid plexus from E16.5 brain were stained with anti-pericentrin (red) and anti-acetylated alpha-tubulin antibodies (green), respectively. Multicilia, monocilia, and bundles from lateral ventricles in knock-out (B, D, and F, respectively) and in wild-type (A, C, E) do not show differences in shape or number. Cilia from choroid plexus of the third ventricle of wild-type (G) and knock-out (H) animals do not show any differences. Staining of β-catenin of the choroid plexus in the third ventricle (I and J) and Aquaporin-1 in the choroid plexus of the lateral ventricle (K, L) did not reveal major differences. Transverse sections of the cilium of an ependymal cell from the third ventricle of wild-type (M) or mutant (N) animals carried out by electronic microscopy revealed the normal structure of the cilium. Scale bar A to F 5 µm; G to L 10 µm; M and N 100 nm.

Mentions: Hydrocephalus is a condition associated with dilatation of the brain ventricles caused by accumulation of the cerebrospinal fluid which is either: I) secreted in excess by the choroid plexus; II) not properly propelled through the aqueduct and ventricles due to a failure of the ependymal cilia beating or stenosis of the aqueduct; III) improper re-absorption by the archnoid villi [31], [34], [37]. In previous mouse models of hydrocephalus due to mutations of ciliary proteins a dual mechanism of hydrocephalus formation due to both lack of proper cilia beating and defective fluid secretion, possibly regulated by cilia themselves, was proposed [34], [37]. We therefore analysed the morphology of cilia in E16.5 brains and found no differences in the shape of different cilia types on the choroid plexus (Figures 7A–H), nor in their relative numbers (not shown). In addition, electron microscopy analysis showed the normal 9+2 structure of the cilia of ependymal cells of the third ventricle (Figures 7M–N). These data are in agreement with the previously proposed role of Polycystin-1 in ciliary function rather than in ciliogenesis [27], [28]. Since defects in the apicobasal polarity of epithelial cells has been reported in the renal cystic epithelium of ADPKD patients, we wondered whether major defects in the morphology of the choroid plexus epithelium might account for the phenotype observed. Staining with Aquaporin-1, a marker of apical membrane, or beta-catenin, a marker of cell-cell junctions, revealed that no major differences could be observed in the choroid plexus epithelia between Pkd1 wild-type and knock-out brains (Figures 7I–L).


A novel mouse model reveals that polycystin-1 deficiency in ependyma and choroid plexus results in dysfunctional cilia and hydrocephalus.

Wodarczyk C, Rowe I, Chiaravalli M, Pema M, Qian F, Boletta A - PLoS ONE (2009)

Cilia morphology and apico-basal polarity in the brain from knock-out embryos at E16.5.(A to H) Centrosomes and cilia in the choroid plexus from E16.5 brain were stained with anti-pericentrin (red) and anti-acetylated alpha-tubulin antibodies (green), respectively. Multicilia, monocilia, and bundles from lateral ventricles in knock-out (B, D, and F, respectively) and in wild-type (A, C, E) do not show differences in shape or number. Cilia from choroid plexus of the third ventricle of wild-type (G) and knock-out (H) animals do not show any differences. Staining of β-catenin of the choroid plexus in the third ventricle (I and J) and Aquaporin-1 in the choroid plexus of the lateral ventricle (K, L) did not reveal major differences. Transverse sections of the cilium of an ependymal cell from the third ventricle of wild-type (M) or mutant (N) animals carried out by electronic microscopy revealed the normal structure of the cilium. Scale bar A to F 5 µm; G to L 10 µm; M and N 100 nm.
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Related In: Results  -  Collection

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pone-0007137-g007: Cilia morphology and apico-basal polarity in the brain from knock-out embryos at E16.5.(A to H) Centrosomes and cilia in the choroid plexus from E16.5 brain were stained with anti-pericentrin (red) and anti-acetylated alpha-tubulin antibodies (green), respectively. Multicilia, monocilia, and bundles from lateral ventricles in knock-out (B, D, and F, respectively) and in wild-type (A, C, E) do not show differences in shape or number. Cilia from choroid plexus of the third ventricle of wild-type (G) and knock-out (H) animals do not show any differences. Staining of β-catenin of the choroid plexus in the third ventricle (I and J) and Aquaporin-1 in the choroid plexus of the lateral ventricle (K, L) did not reveal major differences. Transverse sections of the cilium of an ependymal cell from the third ventricle of wild-type (M) or mutant (N) animals carried out by electronic microscopy revealed the normal structure of the cilium. Scale bar A to F 5 µm; G to L 10 µm; M and N 100 nm.
Mentions: Hydrocephalus is a condition associated with dilatation of the brain ventricles caused by accumulation of the cerebrospinal fluid which is either: I) secreted in excess by the choroid plexus; II) not properly propelled through the aqueduct and ventricles due to a failure of the ependymal cilia beating or stenosis of the aqueduct; III) improper re-absorption by the archnoid villi [31], [34], [37]. In previous mouse models of hydrocephalus due to mutations of ciliary proteins a dual mechanism of hydrocephalus formation due to both lack of proper cilia beating and defective fluid secretion, possibly regulated by cilia themselves, was proposed [34], [37]. We therefore analysed the morphology of cilia in E16.5 brains and found no differences in the shape of different cilia types on the choroid plexus (Figures 7A–H), nor in their relative numbers (not shown). In addition, electron microscopy analysis showed the normal 9+2 structure of the cilia of ependymal cells of the third ventricle (Figures 7M–N). These data are in agreement with the previously proposed role of Polycystin-1 in ciliary function rather than in ciliogenesis [27], [28]. Since defects in the apicobasal polarity of epithelial cells has been reported in the renal cystic epithelium of ADPKD patients, we wondered whether major defects in the morphology of the choroid plexus epithelium might account for the phenotype observed. Staining with Aquaporin-1, a marker of apical membrane, or beta-catenin, a marker of cell-cell junctions, revealed that no major differences could be observed in the choroid plexus epithelia between Pkd1 wild-type and knock-out brains (Figures 7I–L).

Bottom Line: Here, we show that our approach was successful in generating a fully functional and easily detectable endogenous PC-1.Both choroid plexus and ependymal cilia were morphologically normal in these mice, suggesting a role for PC-1 in ciliary function or signalling in this compartment, rather than in ciliogenesis.We propose that the role of PC-1 in the brain cilia might be to prevent hydrocephalus, a previously unrecognized role for this receptor and one that might have important implications for other genetic or sporadic diseases.

View Article: PubMed Central - PubMed

Affiliation: Dulbecco Telethon Institute (DTI) at Dibit, San Raffaele Scientific Institute, Milan, Italy.

ABSTRACT
Polycystin-1 (PC-1), the product of the PKD1 gene, mutated in the majority of cases of Autosomal Dominant Polycystic Kidney Disease (ADPKD), is a very large (approximately 520 kDa) plasma membrane receptor localized in several subcellular compartments including cell-cell/matrix junctions as well as cilia. While heterologous over-expression systems have allowed identification of several of the potential biological roles of this receptor, its precise function remains largely elusive. Studying PC-1 in vivo has been a challenging task due to its complexity and low expression levels. To overcome these limitations and facilitate the study of endogenous PC-1, we have inserted HA- or Myc-tag sequences into the Pkd1 locus by homologous recombination. Here, we show that our approach was successful in generating a fully functional and easily detectable endogenous PC-1. Characterization of PC-1 distribution in vivo showed that it is expressed ubiquitously and is developmentally-regulated in most tissues. Furthermore, our novel tool allowed us to investigate the role of PC-1 in brain, where the protein is abundantly expressed. Subcellular localization of PC-1 revealed strong and specific staining in ciliated ependymal and choroid plexus cells. Consistent with this distribution, we observed hydrocephalus formation both in the ubiquitous knock-out embryos and in newborn mice with conditional inactivation of the Pkd1 gene in the brain. Both choroid plexus and ependymal cilia were morphologically normal in these mice, suggesting a role for PC-1 in ciliary function or signalling in this compartment, rather than in ciliogenesis. We propose that the role of PC-1 in the brain cilia might be to prevent hydrocephalus, a previously unrecognized role for this receptor and one that might have important implications for other genetic or sporadic diseases.

Show MeSH
Related in: MedlinePlus