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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

Mks1 mutants exhibit kidney cysts and defects in ciliogenesis. (A,B) Hematoxylin and eosin (H&E) staining of paraffin sections from E18.5 wild-type (ctrl; A) and Mks1 mutant (m/m; B) animals showed the presence of prominent glomerular and tubule cysts (‘c’) in the mutant animal kidney cortex. Arrows mark glomerular tufts. (C,D) At E18.5, immunostaining with antibody to IFT88 (green) to delineate the cilia (arrows in C,D) showed that cilia were shorter in epithelial cells of mutant glomerular capsule (D) as compared to that of control (C). γ-tubulin (red) was used to delineate the basal body. (E,F) Cilia were less abundant in E17.5 mutant (F) collecting ducts as compared to control (E). The collecting duct was stained with DBA (green) and cilia were stained with anti-acetylated-tubulin antibody (red). Note the presence of cilia (arrow) in the lumen of the control (E) but not the mutant (F) ducts. Images were maximum projections of confocal z-stacks. (G–J) Sections of the kidney from control wild-type (G,I) and mutant (H,J) embryos were immunostained with antibodies to γ-tubulin (red) and IFT88 (green). Arrows denote the opposing apical surfaces of the unilaminar kidney tubule epithelia where the centrioles are localized in the control and mutant kidney tubules. The same regions denoted by arrows in G and H are digitally enlarged in I and J. The enlarged panels show that cilia are abundant in the control sample (I), but very few cilia are observed in the mutant kidney tubule (J). (K,L) Cilia (arrow) were shorter in mutant MEFs (L) as compared with control wild-type MEFs (K). The tip and basal body were stained with IFT140 (green) and the ciliary axoneme was stained with anti-acetylated-tubulin antibody (red). (M) Quantitation of cilia observed in the MEF cultures and kidney sections showed significant reductions in the percentage of ciliated cells in the Mks1 mutant kidney (P<0.0001) and mutant MEFs (P<0.0001). Scale bars: 100 μm (A,B); 10 μm (C–H); 5 μm (I,K,L).
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f2-0040043: Mks1 mutants exhibit kidney cysts and defects in ciliogenesis. (A,B) Hematoxylin and eosin (H&E) staining of paraffin sections from E18.5 wild-type (ctrl; A) and Mks1 mutant (m/m; B) animals showed the presence of prominent glomerular and tubule cysts (‘c’) in the mutant animal kidney cortex. Arrows mark glomerular tufts. (C,D) At E18.5, immunostaining with antibody to IFT88 (green) to delineate the cilia (arrows in C,D) showed that cilia were shorter in epithelial cells of mutant glomerular capsule (D) as compared to that of control (C). γ-tubulin (red) was used to delineate the basal body. (E,F) Cilia were less abundant in E17.5 mutant (F) collecting ducts as compared to control (E). The collecting duct was stained with DBA (green) and cilia were stained with anti-acetylated-tubulin antibody (red). Note the presence of cilia (arrow) in the lumen of the control (E) but not the mutant (F) ducts. Images were maximum projections of confocal z-stacks. (G–J) Sections of the kidney from control wild-type (G,I) and mutant (H,J) embryos were immunostained with antibodies to γ-tubulin (red) and IFT88 (green). Arrows denote the opposing apical surfaces of the unilaminar kidney tubule epithelia where the centrioles are localized in the control and mutant kidney tubules. The same regions denoted by arrows in G and H are digitally enlarged in I and J. The enlarged panels show that cilia are abundant in the control sample (I), but very few cilia are observed in the mutant kidney tubule (J). (K,L) Cilia (arrow) were shorter in mutant MEFs (L) as compared with control wild-type MEFs (K). The tip and basal body were stained with IFT140 (green) and the ciliary axoneme was stained with anti-acetylated-tubulin antibody (red). (M) Quantitation of cilia observed in the MEF cultures and kidney sections showed significant reductions in the percentage of ciliated cells in the Mks1 mutant kidney (P<0.0001) and mutant MEFs (P<0.0001). Scale bars: 100 μm (A,B); 10 μm (C–H); 5 μm (I,K,L).

Mentions: A mouse mutant was recovered that exhibited a wide range of anomalies, including: cleft lip (Fig. 1C); pointy snout (Fig. 1D); eye defects ranging from deeply recessed eyes (enopthalmia), small eyes (microphthalmia) to no eye (anophthalmia) (Fig. 1A,D); polydactyly (Fig. 1A); and congenital heart defects such as transposition of the great arteries (Fig. 1B,E). These anomalies were accompanied by heterotaxy – the randomized left-right positioning of the heart and other visceral organs in the body (Fig. 1B). Analyses of 23 mutants at embryonic day 10.5 (E10.5)-E11.0 showed that 11 had reversed heart looping, and approximately half of the mutants examined at later stages showed dextrocardia, some with a right-sided aortic arch (Fig. 1B,E). We also observed other visceral organ situs defects, including randomized left-right positioning of the stomach (data not shown), abnormal liver situs with some exhibiting symmetric midline liver (Fig. 1F), and polysplenia or asplenia (Fig. 1I and data not shown). In addition, enlargement of the kidneys was observed that, in some cases, was associated with duplex kidneys (Fig. 1J). Histological sections revealed the presence of glomerular cysts and cysts in the kidney tubules (Fig. 2A–D). The latter seem to be derived from the proximal tubules, because only a small number of cysts were positive for the collecting duct and ureteric bud lectin marker Dolichos biflorus agglutinin (DBA) (Fig. 2E,F and data not shown). The appearance of the liver also suggested possible cystic changes (Fig. 1G).


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)

Mks1 mutants exhibit kidney cysts and defects in ciliogenesis. (A,B) Hematoxylin and eosin (H&E) staining of paraffin sections from E18.5 wild-type (ctrl; A) and Mks1 mutant (m/m; B) animals showed the presence of prominent glomerular and tubule cysts (‘c’) in the mutant animal kidney cortex. Arrows mark glomerular tufts. (C,D) At E18.5, immunostaining with antibody to IFT88 (green) to delineate the cilia (arrows in C,D) showed that cilia were shorter in epithelial cells of mutant glomerular capsule (D) as compared to that of control (C). γ-tubulin (red) was used to delineate the basal body. (E,F) Cilia were less abundant in E17.5 mutant (F) collecting ducts as compared to control (E). The collecting duct was stained with DBA (green) and cilia were stained with anti-acetylated-tubulin antibody (red). Note the presence of cilia (arrow) in the lumen of the control (E) but not the mutant (F) ducts. Images were maximum projections of confocal z-stacks. (G–J) Sections of the kidney from control wild-type (G,I) and mutant (H,J) embryos were immunostained with antibodies to γ-tubulin (red) and IFT88 (green). Arrows denote the opposing apical surfaces of the unilaminar kidney tubule epithelia where the centrioles are localized in the control and mutant kidney tubules. The same regions denoted by arrows in G and H are digitally enlarged in I and J. The enlarged panels show that cilia are abundant in the control sample (I), but very few cilia are observed in the mutant kidney tubule (J). (K,L) Cilia (arrow) were shorter in mutant MEFs (L) as compared with control wild-type MEFs (K). The tip and basal body were stained with IFT140 (green) and the ciliary axoneme was stained with anti-acetylated-tubulin antibody (red). (M) Quantitation of cilia observed in the MEF cultures and kidney sections showed significant reductions in the percentage of ciliated cells in the Mks1 mutant kidney (P<0.0001) and mutant MEFs (P<0.0001). Scale bars: 100 μm (A,B); 10 μm (C–H); 5 μm (I,K,L).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2-0040043: Mks1 mutants exhibit kidney cysts and defects in ciliogenesis. (A,B) Hematoxylin and eosin (H&E) staining of paraffin sections from E18.5 wild-type (ctrl; A) and Mks1 mutant (m/m; B) animals showed the presence of prominent glomerular and tubule cysts (‘c’) in the mutant animal kidney cortex. Arrows mark glomerular tufts. (C,D) At E18.5, immunostaining with antibody to IFT88 (green) to delineate the cilia (arrows in C,D) showed that cilia were shorter in epithelial cells of mutant glomerular capsule (D) as compared to that of control (C). γ-tubulin (red) was used to delineate the basal body. (E,F) Cilia were less abundant in E17.5 mutant (F) collecting ducts as compared to control (E). The collecting duct was stained with DBA (green) and cilia were stained with anti-acetylated-tubulin antibody (red). Note the presence of cilia (arrow) in the lumen of the control (E) but not the mutant (F) ducts. Images were maximum projections of confocal z-stacks. (G–J) Sections of the kidney from control wild-type (G,I) and mutant (H,J) embryos were immunostained with antibodies to γ-tubulin (red) and IFT88 (green). Arrows denote the opposing apical surfaces of the unilaminar kidney tubule epithelia where the centrioles are localized in the control and mutant kidney tubules. The same regions denoted by arrows in G and H are digitally enlarged in I and J. The enlarged panels show that cilia are abundant in the control sample (I), but very few cilia are observed in the mutant kidney tubule (J). (K,L) Cilia (arrow) were shorter in mutant MEFs (L) as compared with control wild-type MEFs (K). The tip and basal body were stained with IFT140 (green) and the ciliary axoneme was stained with anti-acetylated-tubulin antibody (red). (M) Quantitation of cilia observed in the MEF cultures and kidney sections showed significant reductions in the percentage of ciliated cells in the Mks1 mutant kidney (P<0.0001) and mutant MEFs (P<0.0001). Scale bars: 100 μm (A,B); 10 μm (C–H); 5 μm (I,K,L).
Mentions: A mouse mutant was recovered that exhibited a wide range of anomalies, including: cleft lip (Fig. 1C); pointy snout (Fig. 1D); eye defects ranging from deeply recessed eyes (enopthalmia), small eyes (microphthalmia) to no eye (anophthalmia) (Fig. 1A,D); polydactyly (Fig. 1A); and congenital heart defects such as transposition of the great arteries (Fig. 1B,E). These anomalies were accompanied by heterotaxy – the randomized left-right positioning of the heart and other visceral organs in the body (Fig. 1B). Analyses of 23 mutants at embryonic day 10.5 (E10.5)-E11.0 showed that 11 had reversed heart looping, and approximately half of the mutants examined at later stages showed dextrocardia, some with a right-sided aortic arch (Fig. 1B,E). We also observed other visceral organ situs defects, including randomized left-right positioning of the stomach (data not shown), abnormal liver situs with some exhibiting symmetric midline liver (Fig. 1F), and polysplenia or asplenia (Fig. 1I and data not shown). In addition, enlargement of the kidneys was observed that, in some cases, was associated with duplex kidneys (Fig. 1J). Histological sections revealed the presence of glomerular cysts and cysts in the kidney tubules (Fig. 2A–D). The latter seem to be derived from the proximal tubules, because only a small number of cysts were positive for the collecting duct and ureteric bud lectin marker Dolichos biflorus agglutinin (DBA) (Fig. 2E,F and data not shown). The appearance of the liver also suggested possible cystic changes (Fig. 1G).

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