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Disruption of Mks1 localization to the mother centriole causes cilia defects and developmental malformations in Meckel-Gruber syndrome.

Cui C, Chatterjee B, Francis D, Yu Q, SanAgustin JT, Francis R, Tansey T, Henry C, Wang B, Lemley B, Pazour GJ, Lo CW - Dis Model Mech (2010)

Bottom Line: Meckel-Gruber syndrome (MKS) is a recessive disorder resulting in multiple birth defects that are associated with mutations affecting ciliogenesis.By contrast, the Shh signaling domain was expanded in the anterior neural tube and anterior limb bud, consistent with reduced Gli3-repressor (Gli3R) function.On the basis of these results, we hypothesize a role for the B9 domain in mother centriole targeting, a possibility that warrants further future investigations.

View Article: PubMed Central - PubMed

Affiliation: University of Pittsburgh, Department of Developmental Biology, 8111 Rangos Research Center, 530 45th Street, Pittsburgh, PA 15201, USA.

ABSTRACT
Meckel-Gruber syndrome (MKS) is a recessive disorder resulting in multiple birth defects that are associated with mutations affecting ciliogenesis. We recovered a mouse mutant with a mutation in the Mks1 gene (Mks1(del64-323)) that caused a 260-amino-acid deletion spanning nine amino acids in the B9 domain, a protein motif with unknown function conserved in two other basal body proteins. We showed that, in wild-type cells, Mks1 was localized to the mother centriole from which the cilium was generated. However, in mutant Mks1(del64-323) cells, Mks1 was not localized to the centriole, even though it maintained a punctate distribution. Resembling MKS patients, Mks1 mutants had craniofacial defects, polydactyly, congenital heart defects, polycystic kidneys and randomized left-right patterning. These defects reflected disturbance of functions subserved by motile and non-motile cilia. In the kidney, glomerular and tubule cysts were observed along with short cilia, and cilia were reduced in number to a near-complete loss. Underlying the left-right patterning defects were fewer and shorter nodal cilia, and analysis with fluorescent beads showed no directional flow at the embryonic node. In the cochlea, the stereocilia were mal-patterned, with the kinocilia being abnormally positioned. Together, these defects suggested disruption of planar cell polarity, which is known to regulate node, kidney and cochlea development. In addition, we also showed that Shh signaling was disrupted. Thus, in the neural tube, the floor plate was not specified posteriorly even as expression of the Shh mediator Gli2 increased. By contrast, the Shh signaling domain was expanded in the anterior neural tube and anterior limb bud, consistent with reduced Gli3-repressor (Gli3R) function. The latter probably accounted for the preaxial digit duplication exhibited by the Mks1(del64-323) mutants. Overall, these findings indicate that centriole localization of Mks1 is required for ciliogenesis of motile and non-motile cilia, but not for centriole assembly. On the basis of these results, we hypothesize a role for the B9 domain in mother centriole targeting, a possibility that warrants further future investigations.

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Related in: MedlinePlus

Recovery and analysis of an in-frame deletion mutation of Mks1. (A) Schematic of the mouse Mks1 gene, containing 18 exons, with the deleted genomic region highlighted, which corresponds to 5226 bases spanning exons 3–10. The sequencing trace file (not shown) from sequencing the genomic DNA (left trace file) showed that the sequence from intron 2 is contiguous with intron 10, whereas sequencing of the cDNA (right trace file) showed that the sequence from exon 2 is contiguous with exon 11 (mRNA). (B) Diagram showing the structure of wild-type and mutant Mks1 protein, with the region deleted delineated in yellow highlight. The in-frame deletion generates a truncated Mks1 protein with amino acid residues 64–323 deleted. (C) Western immunoblotting shows specificity of the anti-Mks1 antibody. Cell lysates from stably transfected HEK293 cells expressing N-terminal FLAG-tagged wild-type (Wt) or mutant (Mt) Mks1 protein were analyzed by western immunoblotting using two-color westerns with an anti-FLAG (green) and anti-Mks1 (red) antibodies. Cells expressing the FLAG-tagged wild-type Mks1 protein exhibited a 67-kD band (upper arrow) that was detected by both the anti-FLAG (green) and -Mks1 (red) antibodies (see merged panel), whereas a slightly smaller 64-kD band (lower arrow), the size expected for endogenous wild-type Mks1, was detected by anti-Mks1 antibody in nontransfected and transfected cells. In cells transfected with the mutant FLAG-tagged Mks1 construct, in addition to the expected 64-kD band, a lower 34-kD band was detected by both the anti-Mks1 and -FLAG antibodies (arrowhead), the size expected for the truncated mutant Mks1 protein. (D,E) Immunostaining of wild-type (D) and Mks1 mutant (E) MEFs with anti-γ-tubulin (green) and -Mks1 (red) antibodies showed that the mutant Mks1 protein is abnormally localized. In wild-type MEFs, γ-tubulin is localized in two punctate dots consistent with centrosome localization, with one showing colocalization with Mks1 (see arrowhead in D). However, in the mutant MEFs, Mks1 staining (red), although still localized in a punctate dot, is not colocalized with the γ-tubulin (green)-containing centrosomes (E, arrowhead). The inset in D of a wild-type MEF shows that Mks1 (red) is colocalized with γ-tubulin (green) and is in the centriole associated with the primary cilium, which is delineated with anti-acetylated-tubulin antibody staining (blue). By contrast, in the mutant MEFs, Mks1 (red) is not colocalized with γ-tubulin (green), either when the cilium is absent (left inset in E), or in rare instances where a cilium (blue)-like projection is observed (right inset in E). Panels D and E are at the same scale; scale bar in D: 5 μm. (F,G) IMCD kidney cells transfected with plasmids encoding GFP-tagged wild-type Mks1 (F) and mutant Mks1 (G) proteins. GFP-tagged wild-type Mks1 protein showed localization at the base of the cilium (F), but this was not observed with GFP-tagged mutant Mks1 protein (G). The cilia (arrowheads) were delineated with anti-IFT88 (G) or -IFT27 (F) antibodies. Panels F and G are at the same magnification; the scale bar in panel F: 5 μm.
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f4-0040043: Recovery and analysis of an in-frame deletion mutation of Mks1. (A) Schematic of the mouse Mks1 gene, containing 18 exons, with the deleted genomic region highlighted, which corresponds to 5226 bases spanning exons 3–10. The sequencing trace file (not shown) from sequencing the genomic DNA (left trace file) showed that the sequence from intron 2 is contiguous with intron 10, whereas sequencing of the cDNA (right trace file) showed that the sequence from exon 2 is contiguous with exon 11 (mRNA). (B) Diagram showing the structure of wild-type and mutant Mks1 protein, with the region deleted delineated in yellow highlight. The in-frame deletion generates a truncated Mks1 protein with amino acid residues 64–323 deleted. (C) Western immunoblotting shows specificity of the anti-Mks1 antibody. Cell lysates from stably transfected HEK293 cells expressing N-terminal FLAG-tagged wild-type (Wt) or mutant (Mt) Mks1 protein were analyzed by western immunoblotting using two-color westerns with an anti-FLAG (green) and anti-Mks1 (red) antibodies. Cells expressing the FLAG-tagged wild-type Mks1 protein exhibited a 67-kD band (upper arrow) that was detected by both the anti-FLAG (green) and -Mks1 (red) antibodies (see merged panel), whereas a slightly smaller 64-kD band (lower arrow), the size expected for endogenous wild-type Mks1, was detected by anti-Mks1 antibody in nontransfected and transfected cells. In cells transfected with the mutant FLAG-tagged Mks1 construct, in addition to the expected 64-kD band, a lower 34-kD band was detected by both the anti-Mks1 and -FLAG antibodies (arrowhead), the size expected for the truncated mutant Mks1 protein. (D,E) Immunostaining of wild-type (D) and Mks1 mutant (E) MEFs with anti-γ-tubulin (green) and -Mks1 (red) antibodies showed that the mutant Mks1 protein is abnormally localized. In wild-type MEFs, γ-tubulin is localized in two punctate dots consistent with centrosome localization, with one showing colocalization with Mks1 (see arrowhead in D). However, in the mutant MEFs, Mks1 staining (red), although still localized in a punctate dot, is not colocalized with the γ-tubulin (green)-containing centrosomes (E, arrowhead). The inset in D of a wild-type MEF shows that Mks1 (red) is colocalized with γ-tubulin (green) and is in the centriole associated with the primary cilium, which is delineated with anti-acetylated-tubulin antibody staining (blue). By contrast, in the mutant MEFs, Mks1 (red) is not colocalized with γ-tubulin (green), either when the cilium is absent (left inset in E), or in rare instances where a cilium (blue)-like projection is observed (right inset in E). Panels D and E are at the same scale; scale bar in D: 5 μm. (F,G) IMCD kidney cells transfected with plasmids encoding GFP-tagged wild-type Mks1 (F) and mutant Mks1 (G) proteins. GFP-tagged wild-type Mks1 protein showed localization at the base of the cilium (F), but this was not observed with GFP-tagged mutant Mks1 protein (G). The cilia (arrowheads) were delineated with anti-IFT88 (G) or -IFT27 (F) antibodies. Panels F and G are at the same magnification; the scale bar in panel F: 5 μm.

Mentions: To map the mutation, we intercrossed the mutant that was generated in a C57BL6 background with C3H mice to generate C57BL6:C3H hybrid offspring. The mutant embryos and fetuses obtained from these intercrosses were analyzed by genome scanning using C57BL6/C3H polymorphic DNA markers, and linkage data obtained from ten mutants localized the mutation to mouse chromosome 11. Analysis of an additional 95 mutants using six additional microsatellite DNA markers and 16 SNPs narrowed the interval to a 9.8-Mb region positioned between SNP rs13481117 and rs27099917. This map interval contained 143 genes, including Mks1. Reverse transcriptase PCR (RT-PCR) analysis of transcripts derived from genes in the interval in the mutant embryos revealed a deletion of 780 nucleotides in transcripts of Mks1 that resulted in an in-frame deletion of amino acid residues 64–323 (Fig. 4). In agreement with this finding, sequencing of the genomic DNA from the mutant revealed a corresponding deletion of 5226 bases that spanned intron 2 to intron 10 of Mks1 (Fig. 4). Subsequent genotyping analysis showed that all offspring exhibiting the mutant phenotype were homozygous for the Mks1del64-323 mutation, whereas all viable adult animals were wild-type or heterozygous (Mks1del64-323/+).


Disruption of Mks1 localization to the mother centriole causes cilia defects and developmental malformations in Meckel-Gruber syndrome.

Cui C, Chatterjee B, Francis D, Yu Q, SanAgustin JT, Francis R, Tansey T, Henry C, Wang B, Lemley B, Pazour GJ, Lo CW - Dis Model Mech (2010)

Recovery and analysis of an in-frame deletion mutation of Mks1. (A) Schematic of the mouse Mks1 gene, containing 18 exons, with the deleted genomic region highlighted, which corresponds to 5226 bases spanning exons 3–10. The sequencing trace file (not shown) from sequencing the genomic DNA (left trace file) showed that the sequence from intron 2 is contiguous with intron 10, whereas sequencing of the cDNA (right trace file) showed that the sequence from exon 2 is contiguous with exon 11 (mRNA). (B) Diagram showing the structure of wild-type and mutant Mks1 protein, with the region deleted delineated in yellow highlight. The in-frame deletion generates a truncated Mks1 protein with amino acid residues 64–323 deleted. (C) Western immunoblotting shows specificity of the anti-Mks1 antibody. Cell lysates from stably transfected HEK293 cells expressing N-terminal FLAG-tagged wild-type (Wt) or mutant (Mt) Mks1 protein were analyzed by western immunoblotting using two-color westerns with an anti-FLAG (green) and anti-Mks1 (red) antibodies. Cells expressing the FLAG-tagged wild-type Mks1 protein exhibited a 67-kD band (upper arrow) that was detected by both the anti-FLAG (green) and -Mks1 (red) antibodies (see merged panel), whereas a slightly smaller 64-kD band (lower arrow), the size expected for endogenous wild-type Mks1, was detected by anti-Mks1 antibody in nontransfected and transfected cells. In cells transfected with the mutant FLAG-tagged Mks1 construct, in addition to the expected 64-kD band, a lower 34-kD band was detected by both the anti-Mks1 and -FLAG antibodies (arrowhead), the size expected for the truncated mutant Mks1 protein. (D,E) Immunostaining of wild-type (D) and Mks1 mutant (E) MEFs with anti-γ-tubulin (green) and -Mks1 (red) antibodies showed that the mutant Mks1 protein is abnormally localized. In wild-type MEFs, γ-tubulin is localized in two punctate dots consistent with centrosome localization, with one showing colocalization with Mks1 (see arrowhead in D). However, in the mutant MEFs, Mks1 staining (red), although still localized in a punctate dot, is not colocalized with the γ-tubulin (green)-containing centrosomes (E, arrowhead). The inset in D of a wild-type MEF shows that Mks1 (red) is colocalized with γ-tubulin (green) and is in the centriole associated with the primary cilium, which is delineated with anti-acetylated-tubulin antibody staining (blue). By contrast, in the mutant MEFs, Mks1 (red) is not colocalized with γ-tubulin (green), either when the cilium is absent (left inset in E), or in rare instances where a cilium (blue)-like projection is observed (right inset in E). Panels D and E are at the same scale; scale bar in D: 5 μm. (F,G) IMCD kidney cells transfected with plasmids encoding GFP-tagged wild-type Mks1 (F) and mutant Mks1 (G) proteins. GFP-tagged wild-type Mks1 protein showed localization at the base of the cilium (F), but this was not observed with GFP-tagged mutant Mks1 protein (G). The cilia (arrowheads) were delineated with anti-IFT88 (G) or -IFT27 (F) antibodies. Panels F and G are at the same magnification; the scale bar in panel F: 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f4-0040043: Recovery and analysis of an in-frame deletion mutation of Mks1. (A) Schematic of the mouse Mks1 gene, containing 18 exons, with the deleted genomic region highlighted, which corresponds to 5226 bases spanning exons 3–10. The sequencing trace file (not shown) from sequencing the genomic DNA (left trace file) showed that the sequence from intron 2 is contiguous with intron 10, whereas sequencing of the cDNA (right trace file) showed that the sequence from exon 2 is contiguous with exon 11 (mRNA). (B) Diagram showing the structure of wild-type and mutant Mks1 protein, with the region deleted delineated in yellow highlight. The in-frame deletion generates a truncated Mks1 protein with amino acid residues 64–323 deleted. (C) Western immunoblotting shows specificity of the anti-Mks1 antibody. Cell lysates from stably transfected HEK293 cells expressing N-terminal FLAG-tagged wild-type (Wt) or mutant (Mt) Mks1 protein were analyzed by western immunoblotting using two-color westerns with an anti-FLAG (green) and anti-Mks1 (red) antibodies. Cells expressing the FLAG-tagged wild-type Mks1 protein exhibited a 67-kD band (upper arrow) that was detected by both the anti-FLAG (green) and -Mks1 (red) antibodies (see merged panel), whereas a slightly smaller 64-kD band (lower arrow), the size expected for endogenous wild-type Mks1, was detected by anti-Mks1 antibody in nontransfected and transfected cells. In cells transfected with the mutant FLAG-tagged Mks1 construct, in addition to the expected 64-kD band, a lower 34-kD band was detected by both the anti-Mks1 and -FLAG antibodies (arrowhead), the size expected for the truncated mutant Mks1 protein. (D,E) Immunostaining of wild-type (D) and Mks1 mutant (E) MEFs with anti-γ-tubulin (green) and -Mks1 (red) antibodies showed that the mutant Mks1 protein is abnormally localized. In wild-type MEFs, γ-tubulin is localized in two punctate dots consistent with centrosome localization, with one showing colocalization with Mks1 (see arrowhead in D). However, in the mutant MEFs, Mks1 staining (red), although still localized in a punctate dot, is not colocalized with the γ-tubulin (green)-containing centrosomes (E, arrowhead). The inset in D of a wild-type MEF shows that Mks1 (red) is colocalized with γ-tubulin (green) and is in the centriole associated with the primary cilium, which is delineated with anti-acetylated-tubulin antibody staining (blue). By contrast, in the mutant MEFs, Mks1 (red) is not colocalized with γ-tubulin (green), either when the cilium is absent (left inset in E), or in rare instances where a cilium (blue)-like projection is observed (right inset in E). Panels D and E are at the same scale; scale bar in D: 5 μm. (F,G) IMCD kidney cells transfected with plasmids encoding GFP-tagged wild-type Mks1 (F) and mutant Mks1 (G) proteins. GFP-tagged wild-type Mks1 protein showed localization at the base of the cilium (F), but this was not observed with GFP-tagged mutant Mks1 protein (G). The cilia (arrowheads) were delineated with anti-IFT88 (G) or -IFT27 (F) antibodies. Panels F and G are at the same magnification; the scale bar in panel F: 5 μm.
Mentions: To map the mutation, we intercrossed the mutant that was generated in a C57BL6 background with C3H mice to generate C57BL6:C3H hybrid offspring. The mutant embryos and fetuses obtained from these intercrosses were analyzed by genome scanning using C57BL6/C3H polymorphic DNA markers, and linkage data obtained from ten mutants localized the mutation to mouse chromosome 11. Analysis of an additional 95 mutants using six additional microsatellite DNA markers and 16 SNPs narrowed the interval to a 9.8-Mb region positioned between SNP rs13481117 and rs27099917. This map interval contained 143 genes, including Mks1. Reverse transcriptase PCR (RT-PCR) analysis of transcripts derived from genes in the interval in the mutant embryos revealed a deletion of 780 nucleotides in transcripts of Mks1 that resulted in an in-frame deletion of amino acid residues 64–323 (Fig. 4). In agreement with this finding, sequencing of the genomic DNA from the mutant revealed a corresponding deletion of 5226 bases that spanned intron 2 to intron 10 of Mks1 (Fig. 4). Subsequent genotyping analysis showed that all offspring exhibiting the mutant phenotype were homozygous for the Mks1del64-323 mutation, whereas all viable adult animals were wild-type or heterozygous (Mks1del64-323/+).

Bottom Line: Meckel-Gruber syndrome (MKS) is a recessive disorder resulting in multiple birth defects that are associated with mutations affecting ciliogenesis.By contrast, the Shh signaling domain was expanded in the anterior neural tube and anterior limb bud, consistent with reduced Gli3-repressor (Gli3R) function.On the basis of these results, we hypothesize a role for the B9 domain in mother centriole targeting, a possibility that warrants further future investigations.

View Article: PubMed Central - PubMed

Affiliation: University of Pittsburgh, Department of Developmental Biology, 8111 Rangos Research Center, 530 45th Street, Pittsburgh, PA 15201, USA.

ABSTRACT
Meckel-Gruber syndrome (MKS) is a recessive disorder resulting in multiple birth defects that are associated with mutations affecting ciliogenesis. We recovered a mouse mutant with a mutation in the Mks1 gene (Mks1(del64-323)) that caused a 260-amino-acid deletion spanning nine amino acids in the B9 domain, a protein motif with unknown function conserved in two other basal body proteins. We showed that, in wild-type cells, Mks1 was localized to the mother centriole from which the cilium was generated. However, in mutant Mks1(del64-323) cells, Mks1 was not localized to the centriole, even though it maintained a punctate distribution. Resembling MKS patients, Mks1 mutants had craniofacial defects, polydactyly, congenital heart defects, polycystic kidneys and randomized left-right patterning. These defects reflected disturbance of functions subserved by motile and non-motile cilia. In the kidney, glomerular and tubule cysts were observed along with short cilia, and cilia were reduced in number to a near-complete loss. Underlying the left-right patterning defects were fewer and shorter nodal cilia, and analysis with fluorescent beads showed no directional flow at the embryonic node. In the cochlea, the stereocilia were mal-patterned, with the kinocilia being abnormally positioned. Together, these defects suggested disruption of planar cell polarity, which is known to regulate node, kidney and cochlea development. In addition, we also showed that Shh signaling was disrupted. Thus, in the neural tube, the floor plate was not specified posteriorly even as expression of the Shh mediator Gli2 increased. By contrast, the Shh signaling domain was expanded in the anterior neural tube and anterior limb bud, consistent with reduced Gli3-repressor (Gli3R) function. The latter probably accounted for the preaxial digit duplication exhibited by the Mks1(del64-323) mutants. Overall, these findings indicate that centriole localization of Mks1 is required for ciliogenesis of motile and non-motile cilia, but not for centriole assembly. On the basis of these results, we hypothesize a role for the B9 domain in mother centriole targeting, a possibility that warrants further future investigations.

Show MeSH
Related in: MedlinePlus