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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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Related in: MedlinePlus

Conditional inactivation of Pkd1 in the brain results in hydrocephalus.Knock-in Pkd1Myc/Myc mice were bred with a Nestin-Cre line to generate Pkd1Myc/+;Nestin-Cre mice. This line was next crossed with heterozygous mutant Pkd1+/ΔCMyc mice, to generate brain conditional mutant mice Pkd1Myc/ΔMyc;Nestin-Cre and Pkd1Myc/ΔMyc (control) littermates. (A, B) Brains from P7 mice were isolated and different regions were separated (OB: Olfactory bulb, CB: Cerebellum, STR: Striatum, Putamen, HI: Hippocampus, CX: Cortex, ME: Meninges). (A) By western blot analysis using anti-Myc antibodies, a reduction in the expression of full-length Myc-tagged PC-1 in conditional knock-out mice Pkd1Myc/ΔCMyc;Nestin-Cre could be seen compared to Pkd1Myc/ΔMyc control lines not expressing the Nestin-Cre transgene. GAPDH staining was used as a loading control. (B) The excision of Pkd1 exons 45/46 by Cre recombinase was analyzed by PCR. In samples from the brain of control Pkd1Myc/ΔCMyc mice, the knock-in Pkd1Myc (519 bp) and knock-out Pkd1ΔCMyc (862 bp) alleles were detected. In conditional knock-out brain of Pkd1Myc/ΔCMyc;Nestin-Cre mice, the appearance of an additional PCR product of 748 bp was observed identifying the excised allele. The excision occurred in all investigated brain regions and, as expected, to a much lesser extent in the meninges. (C–H) Coronal sections of Pkd1Flox/ΔC;Nestin-Cre brains (D, F, H) and the control Pkd1Flox/+;Nestin-Cre brains (C, E and G) at P8 stained with Hematoxylin-Eosin show hydrocephalus with dilatation of the third ventricle (arrow). (I, J) Coronal sections of Pkd1Flox/ΔC;Nestin-Cre brains (J) and the control Pkd1Flox/+;Nestin-Cre brains (I) at P10 show dilatation of the lateral ventricles (arrow). Scale bar C–J 1 mm.
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pone-0007137-g006: Conditional inactivation of Pkd1 in the brain results in hydrocephalus.Knock-in Pkd1Myc/Myc mice were bred with a Nestin-Cre line to generate Pkd1Myc/+;Nestin-Cre mice. This line was next crossed with heterozygous mutant Pkd1+/ΔCMyc mice, to generate brain conditional mutant mice Pkd1Myc/ΔMyc;Nestin-Cre and Pkd1Myc/ΔMyc (control) littermates. (A, B) Brains from P7 mice were isolated and different regions were separated (OB: Olfactory bulb, CB: Cerebellum, STR: Striatum, Putamen, HI: Hippocampus, CX: Cortex, ME: Meninges). (A) By western blot analysis using anti-Myc antibodies, a reduction in the expression of full-length Myc-tagged PC-1 in conditional knock-out mice Pkd1Myc/ΔCMyc;Nestin-Cre could be seen compared to Pkd1Myc/ΔMyc control lines not expressing the Nestin-Cre transgene. GAPDH staining was used as a loading control. (B) The excision of Pkd1 exons 45/46 by Cre recombinase was analyzed by PCR. In samples from the brain of control Pkd1Myc/ΔCMyc mice, the knock-in Pkd1Myc (519 bp) and knock-out Pkd1ΔCMyc (862 bp) alleles were detected. In conditional knock-out brain of Pkd1Myc/ΔCMyc;Nestin-Cre mice, the appearance of an additional PCR product of 748 bp was observed identifying the excised allele. The excision occurred in all investigated brain regions and, as expected, to a much lesser extent in the meninges. (C–H) Coronal sections of Pkd1Flox/ΔC;Nestin-Cre brains (D, F, H) and the control Pkd1Flox/+;Nestin-Cre brains (C, E and G) at P8 stained with Hematoxylin-Eosin show hydrocephalus with dilatation of the third ventricle (arrow). (I, J) Coronal sections of Pkd1Flox/ΔC;Nestin-Cre brains (J) and the control Pkd1Flox/+;Nestin-Cre brains (I) at P10 show dilatation of the lateral ventricles (arrow). Scale bar C–J 1 mm.

Mentions: To follow the development of hydrocephalus at later time points we crossed our Pkd1Flox mice with a line carrying Cre recombinase in a restricted expression pattern to overcome the embryonic lethality of the Pkd1ΔC/ΔC mice, but with a broad expression pattern in the brain to allow targeting of all the regions of the CNS. We used the Nestin-Cre mouse line previously employed to inactivate Stumpy, another ciliary protein reported to cause hydrocephalus when inactivated [33], [42]. First of all, we analyzed the expression levels of Polycystin-1 in the different brain regions at P7 and found that the protein is expressed equally and abundantly in all regions (Figure 6A and not shown). Next, we compared the expression levels of the protein in Pkd1Myc/ΔCMyc in the presence or absence of Cre recombinase, and found that inactivation of the Pkd1 gene and reduction of the PC-1 protein occurring at equal rates in all of the brain regions examined (Figures 6A and B). Most importantly, analysis of brain sections derived from Nestin-Cre conditional mouse models revealed the presence of hydrocephalus with dilation of the third ventricle at day P8 (Figure 6C–H, n = 2), which was subsequently followed by the dilation of the lateral ventricles, showing a mild but typical triventricular phenotype by day P10 (Figure 6I–J, n = 3). As previously reported, the Nestin-Cre transgene is also active in the kidneys [33]. In agreement with this, the Pkd1Flox/ΔC;Nestin-Cre mouse developed renal cystogenesis and died of kidney failure around P12 (not shown). This prevented us from determining if a more severe phenotype would develop at later time points. In all of these experiments the Nestin-Cre transgene was employed as a heterozygous allele, since homozygosity of a similar transgene was previously reported to cause hydrocephalus itself [43]. In addition, Pkd1Flox/+;Nestin-Cre brains were used as negative controls (Figures 6C, E, G and I) and none developed any sign of hydrocephalus, excluding the possibility that the transgene alone induced this phenotype in the heterozygous state. The ventricular dilations observed are very similar to the phenotype present in the embryo and were again found with 100% penetrance (n = 5).


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)

Conditional inactivation of Pkd1 in the brain results in hydrocephalus.Knock-in Pkd1Myc/Myc mice were bred with a Nestin-Cre line to generate Pkd1Myc/+;Nestin-Cre mice. This line was next crossed with heterozygous mutant Pkd1+/ΔCMyc mice, to generate brain conditional mutant mice Pkd1Myc/ΔMyc;Nestin-Cre and Pkd1Myc/ΔMyc (control) littermates. (A, B) Brains from P7 mice were isolated and different regions were separated (OB: Olfactory bulb, CB: Cerebellum, STR: Striatum, Putamen, HI: Hippocampus, CX: Cortex, ME: Meninges). (A) By western blot analysis using anti-Myc antibodies, a reduction in the expression of full-length Myc-tagged PC-1 in conditional knock-out mice Pkd1Myc/ΔCMyc;Nestin-Cre could be seen compared to Pkd1Myc/ΔMyc control lines not expressing the Nestin-Cre transgene. GAPDH staining was used as a loading control. (B) The excision of Pkd1 exons 45/46 by Cre recombinase was analyzed by PCR. In samples from the brain of control Pkd1Myc/ΔCMyc mice, the knock-in Pkd1Myc (519 bp) and knock-out Pkd1ΔCMyc (862 bp) alleles were detected. In conditional knock-out brain of Pkd1Myc/ΔCMyc;Nestin-Cre mice, the appearance of an additional PCR product of 748 bp was observed identifying the excised allele. The excision occurred in all investigated brain regions and, as expected, to a much lesser extent in the meninges. (C–H) Coronal sections of Pkd1Flox/ΔC;Nestin-Cre brains (D, F, H) and the control Pkd1Flox/+;Nestin-Cre brains (C, E and G) at P8 stained with Hematoxylin-Eosin show hydrocephalus with dilatation of the third ventricle (arrow). (I, J) Coronal sections of Pkd1Flox/ΔC;Nestin-Cre brains (J) and the control Pkd1Flox/+;Nestin-Cre brains (I) at P10 show dilatation of the lateral ventricles (arrow). Scale bar C–J 1 mm.
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pone-0007137-g006: Conditional inactivation of Pkd1 in the brain results in hydrocephalus.Knock-in Pkd1Myc/Myc mice were bred with a Nestin-Cre line to generate Pkd1Myc/+;Nestin-Cre mice. This line was next crossed with heterozygous mutant Pkd1+/ΔCMyc mice, to generate brain conditional mutant mice Pkd1Myc/ΔMyc;Nestin-Cre and Pkd1Myc/ΔMyc (control) littermates. (A, B) Brains from P7 mice were isolated and different regions were separated (OB: Olfactory bulb, CB: Cerebellum, STR: Striatum, Putamen, HI: Hippocampus, CX: Cortex, ME: Meninges). (A) By western blot analysis using anti-Myc antibodies, a reduction in the expression of full-length Myc-tagged PC-1 in conditional knock-out mice Pkd1Myc/ΔCMyc;Nestin-Cre could be seen compared to Pkd1Myc/ΔMyc control lines not expressing the Nestin-Cre transgene. GAPDH staining was used as a loading control. (B) The excision of Pkd1 exons 45/46 by Cre recombinase was analyzed by PCR. In samples from the brain of control Pkd1Myc/ΔCMyc mice, the knock-in Pkd1Myc (519 bp) and knock-out Pkd1ΔCMyc (862 bp) alleles were detected. In conditional knock-out brain of Pkd1Myc/ΔCMyc;Nestin-Cre mice, the appearance of an additional PCR product of 748 bp was observed identifying the excised allele. The excision occurred in all investigated brain regions and, as expected, to a much lesser extent in the meninges. (C–H) Coronal sections of Pkd1Flox/ΔC;Nestin-Cre brains (D, F, H) and the control Pkd1Flox/+;Nestin-Cre brains (C, E and G) at P8 stained with Hematoxylin-Eosin show hydrocephalus with dilatation of the third ventricle (arrow). (I, J) Coronal sections of Pkd1Flox/ΔC;Nestin-Cre brains (J) and the control Pkd1Flox/+;Nestin-Cre brains (I) at P10 show dilatation of the lateral ventricles (arrow). Scale bar C–J 1 mm.
Mentions: To follow the development of hydrocephalus at later time points we crossed our Pkd1Flox mice with a line carrying Cre recombinase in a restricted expression pattern to overcome the embryonic lethality of the Pkd1ΔC/ΔC mice, but with a broad expression pattern in the brain to allow targeting of all the regions of the CNS. We used the Nestin-Cre mouse line previously employed to inactivate Stumpy, another ciliary protein reported to cause hydrocephalus when inactivated [33], [42]. First of all, we analyzed the expression levels of Polycystin-1 in the different brain regions at P7 and found that the protein is expressed equally and abundantly in all regions (Figure 6A and not shown). Next, we compared the expression levels of the protein in Pkd1Myc/ΔCMyc in the presence or absence of Cre recombinase, and found that inactivation of the Pkd1 gene and reduction of the PC-1 protein occurring at equal rates in all of the brain regions examined (Figures 6A and B). Most importantly, analysis of brain sections derived from Nestin-Cre conditional mouse models revealed the presence of hydrocephalus with dilation of the third ventricle at day P8 (Figure 6C–H, n = 2), which was subsequently followed by the dilation of the lateral ventricles, showing a mild but typical triventricular phenotype by day P10 (Figure 6I–J, n = 3). As previously reported, the Nestin-Cre transgene is also active in the kidneys [33]. In agreement with this, the Pkd1Flox/ΔC;Nestin-Cre mouse developed renal cystogenesis and died of kidney failure around P12 (not shown). This prevented us from determining if a more severe phenotype would develop at later time points. In all of these experiments the Nestin-Cre transgene was employed as a heterozygous allele, since homozygosity of a similar transgene was previously reported to cause hydrocephalus itself [43]. In addition, Pkd1Flox/+;Nestin-Cre brains were used as negative controls (Figures 6C, E, G and I) and none developed any sign of hydrocephalus, excluding the possibility that the transgene alone induced this phenotype in the heterozygous state. The ventricular dilations observed are very similar to the phenotype present in the embryo and were again found with 100% penetrance (n = 5).

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