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Microcephaly disease gene Wdr62 regulates mitotic progression of embryonic neural stem cells and brain size.

Chen JF, Zhang Y, Wilde J, Hansen KC, Lai F, Niswander L - Nat Commun (2014)

Bottom Line: Mutations in WDR62 are associated with microcephaly and other cortical abnormalities in humans.Wdr62 depleted cells show spindle instability, spindle assembly checkpoint (SAC) activation, mitotic arrest and cell death.Mechanistically, Wdr62 associates and genetically interacts with Aurora A to regulate spindle formation, mitotic progression and brain size.

View Article: PubMed Central - PubMed

Affiliation: 1] Howard Hughes Medical Institute, Department of Pediatrics, University of Colorado Anschutz Medical Campus, Children's Hospital Colorado, Aurora, Colorado 80045, USA [2] Department of Genetics, Department of Biochemistry & Molecular Biology, University of Georgia, Athens, Georgia 30602, USA.

ABSTRACT
Human genetic studies have established a link between a class of centrosome proteins and microcephaly. Current studies of microcephaly focus on defective centrosome/spindle orientation. Mutations in WDR62 are associated with microcephaly and other cortical abnormalities in humans. Here we create a mouse model of Wdr62 deficiency and find that the mice exhibit reduced brain size due to decreased neural progenitor cells (NPCs). Wdr62 depleted cells show spindle instability, spindle assembly checkpoint (SAC) activation, mitotic arrest and cell death. Mechanistically, Wdr62 associates and genetically interacts with Aurora A to regulate spindle formation, mitotic progression and brain size. Our results suggest that Wdr62 interacts with Aurora A to control mitotic progression, and loss of these interactions leads to mitotic delay and cell death of NPCs, which could be a potential cause of human microcephaly.

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Wdr62 regulates spindle assembly and its depletion activates the spindle checkpoint. (a,b) Wild type and mutant MEFs at metaphase were co-stained for CREST (green), Mad2 (red in a), and BubR1 (red in b). Inserts show single focal planes of boxed regions. White arrowhead in a points to unaligned chromosome. Bar: 2 um. (c) Quantification of percentage of Mad2 or BubR1 positive metaphase cells out of total metaphase cells in a, b, respectively. Data represent s.e.m. of three independent experiments, counting at least 50 metaphase cells per experiment (p< 0.05, student’s t-test). (d) Confocal microscope images of mutant cells at anaphase stained with BubR1. Hoechst stains nuclei (blue). White arrowheads mark unaligned or lagging chromosomes. Bar: 2.5 um, 5 um. (e) Quantification of percentage of mitotic cells with unaligned or lagging chromosomes. Data represent s.e.m. of three independent experiments, counting at least 50 metaphase or anaphase cells per experiment (**p< 0.01, *p<0.05, student’s t-test). (f) Confocal microscope images of WT and mutant MEFs in metaphase stained with antibodies against α-tubulin (red) and γ-tubulin (green). Hoechst stains nuclei (blue). Bar: 2 um. (g) Confocal microscope images of MEFs incubated at 4°C for 40 min and co-stained with antibodies against α-tubulin (red) and CREST (green). Hoechst stains nuclei (blue). Inserts show single focal planes of boxed regions. White arrowhead points to unaligned chromosome. Bar: 2 um. (h) The relative intensity of α-tubulin (red) within metaphase spindle was measured using quantitative fluorescence imaging. Data represent s.e.m. of >25 cells for each genotype (p = 0.002, student’s t-test).
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Figure 5: Wdr62 regulates spindle assembly and its depletion activates the spindle checkpoint. (a,b) Wild type and mutant MEFs at metaphase were co-stained for CREST (green), Mad2 (red in a), and BubR1 (red in b). Inserts show single focal planes of boxed regions. White arrowhead in a points to unaligned chromosome. Bar: 2 um. (c) Quantification of percentage of Mad2 or BubR1 positive metaphase cells out of total metaphase cells in a, b, respectively. Data represent s.e.m. of three independent experiments, counting at least 50 metaphase cells per experiment (p< 0.05, student’s t-test). (d) Confocal microscope images of mutant cells at anaphase stained with BubR1. Hoechst stains nuclei (blue). White arrowheads mark unaligned or lagging chromosomes. Bar: 2.5 um, 5 um. (e) Quantification of percentage of mitotic cells with unaligned or lagging chromosomes. Data represent s.e.m. of three independent experiments, counting at least 50 metaphase or anaphase cells per experiment (**p< 0.01, *p<0.05, student’s t-test). (f) Confocal microscope images of WT and mutant MEFs in metaphase stained with antibodies against α-tubulin (red) and γ-tubulin (green). Hoechst stains nuclei (blue). Bar: 2 um. (g) Confocal microscope images of MEFs incubated at 4°C for 40 min and co-stained with antibodies against α-tubulin (red) and CREST (green). Hoechst stains nuclei (blue). Inserts show single focal planes of boxed regions. White arrowhead points to unaligned chromosome. Bar: 2 um. (h) The relative intensity of α-tubulin (red) within metaphase spindle was measured using quantitative fluorescence imaging. Data represent s.e.m. of >25 cells for each genotype (p = 0.002, student’s t-test).

Mentions: The mitotic arrest and cell death phenotypes in Wdr62 mutant NPCs (Fig. 3) and MEFs (Fig. 4) suggest the activation of the spindle assembly checkpoint (SAC) 17,20. We examined the expression of the kinetochore-associated checkpoint protein Mad2, which monitors the microtubule attachment on kinetochores 21,22. In wild type metaphase cells, Mad2 has left the kinetochores, however this checkpoint protein remained present on the kinetochores of mutant cells in metaphase (Fig. 5a,c), including on unaligned chromosomes (white arrowheads and white box 1 in Fig. 5a). We next monitored tension across sister kinetochores using the checkpoint protein BubR1 21,22. Indeed, the numbers of metaphase cells with BubR1 expression were significantly increased in mutant MEFs compared to control cells (Fig. 5b,c). Furthermore, there was a > 4-fold increase in cells with unaligned or lagging chromosomes upon depletion of Wdr62 (Fig. 5d,e). Consistent with the association of Wdr62 with centrosomes and spindle microtubules (Supplementary Fig. 3), these studies indicate that loss of Wdr62 functions compromises microtubule-kinetochore attachment and the tension across sister kinetochores, leading to activation of the spindle checkpoint.


Microcephaly disease gene Wdr62 regulates mitotic progression of embryonic neural stem cells and brain size.

Chen JF, Zhang Y, Wilde J, Hansen KC, Lai F, Niswander L - Nat Commun (2014)

Wdr62 regulates spindle assembly and its depletion activates the spindle checkpoint. (a,b) Wild type and mutant MEFs at metaphase were co-stained for CREST (green), Mad2 (red in a), and BubR1 (red in b). Inserts show single focal planes of boxed regions. White arrowhead in a points to unaligned chromosome. Bar: 2 um. (c) Quantification of percentage of Mad2 or BubR1 positive metaphase cells out of total metaphase cells in a, b, respectively. Data represent s.e.m. of three independent experiments, counting at least 50 metaphase cells per experiment (p< 0.05, student’s t-test). (d) Confocal microscope images of mutant cells at anaphase stained with BubR1. Hoechst stains nuclei (blue). White arrowheads mark unaligned or lagging chromosomes. Bar: 2.5 um, 5 um. (e) Quantification of percentage of mitotic cells with unaligned or lagging chromosomes. Data represent s.e.m. of three independent experiments, counting at least 50 metaphase or anaphase cells per experiment (**p< 0.01, *p<0.05, student’s t-test). (f) Confocal microscope images of WT and mutant MEFs in metaphase stained with antibodies against α-tubulin (red) and γ-tubulin (green). Hoechst stains nuclei (blue). Bar: 2 um. (g) Confocal microscope images of MEFs incubated at 4°C for 40 min and co-stained with antibodies against α-tubulin (red) and CREST (green). Hoechst stains nuclei (blue). Inserts show single focal planes of boxed regions. White arrowhead points to unaligned chromosome. Bar: 2 um. (h) The relative intensity of α-tubulin (red) within metaphase spindle was measured using quantitative fluorescence imaging. Data represent s.e.m. of >25 cells for each genotype (p = 0.002, student’s t-test).
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Figure 5: Wdr62 regulates spindle assembly and its depletion activates the spindle checkpoint. (a,b) Wild type and mutant MEFs at metaphase were co-stained for CREST (green), Mad2 (red in a), and BubR1 (red in b). Inserts show single focal planes of boxed regions. White arrowhead in a points to unaligned chromosome. Bar: 2 um. (c) Quantification of percentage of Mad2 or BubR1 positive metaphase cells out of total metaphase cells in a, b, respectively. Data represent s.e.m. of three independent experiments, counting at least 50 metaphase cells per experiment (p< 0.05, student’s t-test). (d) Confocal microscope images of mutant cells at anaphase stained with BubR1. Hoechst stains nuclei (blue). White arrowheads mark unaligned or lagging chromosomes. Bar: 2.5 um, 5 um. (e) Quantification of percentage of mitotic cells with unaligned or lagging chromosomes. Data represent s.e.m. of three independent experiments, counting at least 50 metaphase or anaphase cells per experiment (**p< 0.01, *p<0.05, student’s t-test). (f) Confocal microscope images of WT and mutant MEFs in metaphase stained with antibodies against α-tubulin (red) and γ-tubulin (green). Hoechst stains nuclei (blue). Bar: 2 um. (g) Confocal microscope images of MEFs incubated at 4°C for 40 min and co-stained with antibodies against α-tubulin (red) and CREST (green). Hoechst stains nuclei (blue). Inserts show single focal planes of boxed regions. White arrowhead points to unaligned chromosome. Bar: 2 um. (h) The relative intensity of α-tubulin (red) within metaphase spindle was measured using quantitative fluorescence imaging. Data represent s.e.m. of >25 cells for each genotype (p = 0.002, student’s t-test).
Mentions: The mitotic arrest and cell death phenotypes in Wdr62 mutant NPCs (Fig. 3) and MEFs (Fig. 4) suggest the activation of the spindle assembly checkpoint (SAC) 17,20. We examined the expression of the kinetochore-associated checkpoint protein Mad2, which monitors the microtubule attachment on kinetochores 21,22. In wild type metaphase cells, Mad2 has left the kinetochores, however this checkpoint protein remained present on the kinetochores of mutant cells in metaphase (Fig. 5a,c), including on unaligned chromosomes (white arrowheads and white box 1 in Fig. 5a). We next monitored tension across sister kinetochores using the checkpoint protein BubR1 21,22. Indeed, the numbers of metaphase cells with BubR1 expression were significantly increased in mutant MEFs compared to control cells (Fig. 5b,c). Furthermore, there was a > 4-fold increase in cells with unaligned or lagging chromosomes upon depletion of Wdr62 (Fig. 5d,e). Consistent with the association of Wdr62 with centrosomes and spindle microtubules (Supplementary Fig. 3), these studies indicate that loss of Wdr62 functions compromises microtubule-kinetochore attachment and the tension across sister kinetochores, leading to activation of the spindle checkpoint.

Bottom Line: Mutations in WDR62 are associated with microcephaly and other cortical abnormalities in humans.Wdr62 depleted cells show spindle instability, spindle assembly checkpoint (SAC) activation, mitotic arrest and cell death.Mechanistically, Wdr62 associates and genetically interacts with Aurora A to regulate spindle formation, mitotic progression and brain size.

View Article: PubMed Central - PubMed

Affiliation: 1] Howard Hughes Medical Institute, Department of Pediatrics, University of Colorado Anschutz Medical Campus, Children's Hospital Colorado, Aurora, Colorado 80045, USA [2] Department of Genetics, Department of Biochemistry & Molecular Biology, University of Georgia, Athens, Georgia 30602, USA.

ABSTRACT
Human genetic studies have established a link between a class of centrosome proteins and microcephaly. Current studies of microcephaly focus on defective centrosome/spindle orientation. Mutations in WDR62 are associated with microcephaly and other cortical abnormalities in humans. Here we create a mouse model of Wdr62 deficiency and find that the mice exhibit reduced brain size due to decreased neural progenitor cells (NPCs). Wdr62 depleted cells show spindle instability, spindle assembly checkpoint (SAC) activation, mitotic arrest and cell death. Mechanistically, Wdr62 associates and genetically interacts with Aurora A to regulate spindle formation, mitotic progression and brain size. Our results suggest that Wdr62 interacts with Aurora A to control mitotic progression, and loss of these interactions leads to mitotic delay and cell death of NPCs, which could be a potential cause of human microcephaly.

Show MeSH
Related in: MedlinePlus