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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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Scanning EM images of embryonic node cilia. Whereas the control (ctrl) embryo exhibited cells with cuboidal shaped (A), the mutant (m/m) node exhibited flat epithelial cell morphology (B). Cilia in the control node projected posteriorly (arrows in C) from the posterior hemisphere of nodal cells but, in Mks1-mutant embryos, node cells exhibited mostly amorphous protrusions (circle in D) and, when cilia were observed, they were shorter (10–30% of length in control node) and were randomly oriented (arrowheads in D–F). Arrows in C–F point to the roots of cilia, with the arrow aligned with the direction of cilium-projection. The two-ended arrow in panel A indicates the orientation of images in all panels, with posterior end of the embryonic node pointing down and anterior end pointing up. Scale bars: 20 μm (A,B); 2 μm (C–F).
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f5-0040043: Scanning EM images of embryonic node cilia. Whereas the control (ctrl) embryo exhibited cells with cuboidal shaped (A), the mutant (m/m) node exhibited flat epithelial cell morphology (B). Cilia in the control node projected posteriorly (arrows in C) from the posterior hemisphere of nodal cells but, in Mks1-mutant embryos, node cells exhibited mostly amorphous protrusions (circle in D) and, when cilia were observed, they were shorter (10–30% of length in control node) and were randomly oriented (arrowheads in D–F). Arrows in C–F point to the roots of cilia, with the arrow aligned with the direction of cilium-projection. The two-ended arrow in panel A indicates the orientation of images in all panels, with posterior end of the embryonic node pointing down and anterior end pointing up. Scale bars: 20 μm (A,B); 2 μm (C–F).

Mentions: Scanning EM analysis of E7.5–E8.0 embryos showed cuboidal epithelial cell morphology in the embryonic node of wild-type or heterozygous mutant embryos (Fig. 5A), whereas, in the homozygous mutant embryos (n=4), the node had a flattened epithelial morphology (Fig. 5B). In wild-type or heterozygous embryos, most cells in the embryonic node exhibited a single cilium that projected posteriorly. By contrast, in homozygous Mks1-mutant embryos, only a few randomly oriented abnormal short cilia-like projections were observed in a few cells in the node (arrows in Fig. 5D–F), but these were not immunostained by an anti-acetylated-tubulin antibody (data not shown). Videomicroscopy also showed no ciliary motion and fluorescent beads placed over the node showed no net nodal flow (see supplementary material Movies 1 and 2).


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)

Scanning EM images of embryonic node cilia. Whereas the control (ctrl) embryo exhibited cells with cuboidal shaped (A), the mutant (m/m) node exhibited flat epithelial cell morphology (B). Cilia in the control node projected posteriorly (arrows in C) from the posterior hemisphere of nodal cells but, in Mks1-mutant embryos, node cells exhibited mostly amorphous protrusions (circle in D) and, when cilia were observed, they were shorter (10–30% of length in control node) and were randomly oriented (arrowheads in D–F). Arrows in C–F point to the roots of cilia, with the arrow aligned with the direction of cilium-projection. The two-ended arrow in panel A indicates the orientation of images in all panels, with posterior end of the embryonic node pointing down and anterior end pointing up. Scale bars: 20 μm (A,B); 2 μm (C–F).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3008963&req=5

f5-0040043: Scanning EM images of embryonic node cilia. Whereas the control (ctrl) embryo exhibited cells with cuboidal shaped (A), the mutant (m/m) node exhibited flat epithelial cell morphology (B). Cilia in the control node projected posteriorly (arrows in C) from the posterior hemisphere of nodal cells but, in Mks1-mutant embryos, node cells exhibited mostly amorphous protrusions (circle in D) and, when cilia were observed, they were shorter (10–30% of length in control node) and were randomly oriented (arrowheads in D–F). Arrows in C–F point to the roots of cilia, with the arrow aligned with the direction of cilium-projection. The two-ended arrow in panel A indicates the orientation of images in all panels, with posterior end of the embryonic node pointing down and anterior end pointing up. Scale bars: 20 μm (A,B); 2 μm (C–F).
Mentions: Scanning EM analysis of E7.5–E8.0 embryos showed cuboidal epithelial cell morphology in the embryonic node of wild-type or heterozygous mutant embryos (Fig. 5A), whereas, in the homozygous mutant embryos (n=4), the node had a flattened epithelial morphology (Fig. 5B). In wild-type or heterozygous embryos, most cells in the embryonic node exhibited a single cilium that projected posteriorly. By contrast, in homozygous Mks1-mutant embryos, only a few randomly oriented abnormal short cilia-like projections were observed in a few cells in the node (arrows in Fig. 5D–F), but these were not immunostained by an anti-acetylated-tubulin antibody (data not shown). Videomicroscopy also showed no ciliary motion and fluorescent beads placed over the node showed no net nodal flow (see supplementary material Movies 1 and 2).

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