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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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Pkd1 knock-out embryos show mild hydrocephalus at E16.5.Upper view (A) and lateral view (B, C) of wild-type and knockout brains at E16.5 show macroscopic differences with dilated vessels (arrows) and olfactory bulbs (ob) pointing down. Coronal sections corresponding to 1 (D, E), 2 (F, G), 3 (H, I), and 4 (J, K) show that the third ventricle (arrow) from the mutant brains is strongly dilated compared to the wild-type. The lateral ventricle was only mildly dilated. (arrowhead). All the images are representative of an identical phenotype observed in 8 different mutant mice and never observed in 8 different wild-type controls. Scale bar A 1 mm; D to G 500 µm.
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pone-0007137-g005: Pkd1 knock-out embryos show mild hydrocephalus at E16.5.Upper view (A) and lateral view (B, C) of wild-type and knockout brains at E16.5 show macroscopic differences with dilated vessels (arrows) and olfactory bulbs (ob) pointing down. Coronal sections corresponding to 1 (D, E), 2 (F, G), 3 (H, I), and 4 (J, K) show that the third ventricle (arrow) from the mutant brains is strongly dilated compared to the wild-type. The lateral ventricle was only mildly dilated. (arrowhead). All the images are representative of an identical phenotype observed in 8 different mutant mice and never observed in 8 different wild-type controls. Scale bar A 1 mm; D to G 500 µm.

Mentions: Since we found strong ciliary staining both in ependymal and choroid plexus cilia, we wondered whether the absence of Polycystin-1 in these structures would cause an overt phenotype or if PC-1 function in this compartment is redundant and compensated for by other factors, given that a defect at this level was never reported before in Pkd1 mutant mice. To answer this question we analyzed the brains of Pkd1ΔC/ΔC mice compared to the wild-type littermates at day E16.5. As shown in Figure 5, the brains of wild-type and Pkd1ΔC/ΔC mice appear macroscopically different, with the two separated frontal lobes (Figure 5A) and several dilated vessels (Figure 5A, arrows; n = 8). Lateral views show that the brains of Pkd1ΔC/ΔC mice appeared more curved, with the olfactory bulbs (ob) pointing down due to the dome-shape of the brain (Figures 5B and C, ob; n = 8). Morphological analysis of sections showed a mild dilatation of the lateral ventricles and a more prominent dilatation of the third ventricle, with a considerable defect in choroid plexus (CP) morphology (Figure 5D–K, n = 5). Interestingly, the aqueduct of Sylvius does not show obvious signs of stenosis, excluding the possibility of ventricle dilatation as a secondary cause of aqueduct stenosis (not shown). The images shown in figure 5A–C are representative of an identical phenotype observed in eight distinct mutant mice (100% penetrance) and completely absent from eight distinct littermate wild type controls withdrawn at the same time under identical conditions, thus excluding the possibility that the defects observed might be secondary to problems in dissection. Likewise, the images in figure 5D–K are representative of five distinct mutant and wild-type littermate controls subject to cross-sections showing an identical phenotype.


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)

Pkd1 knock-out embryos show mild hydrocephalus at E16.5.Upper view (A) and lateral view (B, C) of wild-type and knockout brains at E16.5 show macroscopic differences with dilated vessels (arrows) and olfactory bulbs (ob) pointing down. Coronal sections corresponding to 1 (D, E), 2 (F, G), 3 (H, I), and 4 (J, K) show that the third ventricle (arrow) from the mutant brains is strongly dilated compared to the wild-type. The lateral ventricle was only mildly dilated. (arrowhead). All the images are representative of an identical phenotype observed in 8 different mutant mice and never observed in 8 different wild-type controls. Scale bar A 1 mm; D to G 500 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2743994&req=5

pone-0007137-g005: Pkd1 knock-out embryos show mild hydrocephalus at E16.5.Upper view (A) and lateral view (B, C) of wild-type and knockout brains at E16.5 show macroscopic differences with dilated vessels (arrows) and olfactory bulbs (ob) pointing down. Coronal sections corresponding to 1 (D, E), 2 (F, G), 3 (H, I), and 4 (J, K) show that the third ventricle (arrow) from the mutant brains is strongly dilated compared to the wild-type. The lateral ventricle was only mildly dilated. (arrowhead). All the images are representative of an identical phenotype observed in 8 different mutant mice and never observed in 8 different wild-type controls. Scale bar A 1 mm; D to G 500 µm.
Mentions: Since we found strong ciliary staining both in ependymal and choroid plexus cilia, we wondered whether the absence of Polycystin-1 in these structures would cause an overt phenotype or if PC-1 function in this compartment is redundant and compensated for by other factors, given that a defect at this level was never reported before in Pkd1 mutant mice. To answer this question we analyzed the brains of Pkd1ΔC/ΔC mice compared to the wild-type littermates at day E16.5. As shown in Figure 5, the brains of wild-type and Pkd1ΔC/ΔC mice appear macroscopically different, with the two separated frontal lobes (Figure 5A) and several dilated vessels (Figure 5A, arrows; n = 8). Lateral views show that the brains of Pkd1ΔC/ΔC mice appeared more curved, with the olfactory bulbs (ob) pointing down due to the dome-shape of the brain (Figures 5B and C, ob; n = 8). Morphological analysis of sections showed a mild dilatation of the lateral ventricles and a more prominent dilatation of the third ventricle, with a considerable defect in choroid plexus (CP) morphology (Figure 5D–K, n = 5). Interestingly, the aqueduct of Sylvius does not show obvious signs of stenosis, excluding the possibility of ventricle dilatation as a secondary cause of aqueduct stenosis (not shown). The images shown in figure 5A–C are representative of an identical phenotype observed in eight distinct mutant mice (100% penetrance) and completely absent from eight distinct littermate wild type controls withdrawn at the same time under identical conditions, thus excluding the possibility that the defects observed might be secondary to problems in dissection. Likewise, the images in figure 5D–K are representative of five distinct mutant and wild-type littermate controls subject to cross-sections showing an identical phenotype.

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