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Microcephaly models in the developing zebrafish retinal neuroepithelium point to an underlying defect in metaphase progression.

Novorol C, Burkhardt J, Wood KJ, Iqbal A, Roque C, Coutts N, Almeida AD, He J, Wilkinson CJ, Harris WA - Open Biol (2013)

Bottom Line: Autosomal recessive primary microcephaly (MCPH) is a congenital disorder characterized by significantly reduced brain size and mental retardation.Mutant or morpholino-mediated knockdown of three known MCPH genes (stil, aspm and wdr62) and a fourth centrosomal gene, odf2, which is linked to several MCPH proteins, results in a marked reduction in head and eye size.There was also increased apoptosis in all the MCPH models but this appears to be secondary to the mitotic defect as we frequently saw mitotically arrested cells disappear, and knocking down p53 apoptosis did not rescue the mitotic phenotype, either in whole retinas or clones.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Development and Neuroscience, Cambridge University, Cambridge CB2 3DY, UK.

ABSTRACT
Autosomal recessive primary microcephaly (MCPH) is a congenital disorder characterized by significantly reduced brain size and mental retardation. Nine genes are currently known to be associated with the condition, all of which encode centrosomal or spindle pole proteins. MCPH is associated with a reduction in proliferation of neural progenitors during fetal development. The cellular mechanisms underlying the proliferation defect, however, are not fully understood. The zebrafish retinal neuroepithelium provides an ideal system to investigate this question. Mutant or morpholino-mediated knockdown of three known MCPH genes (stil, aspm and wdr62) and a fourth centrosomal gene, odf2, which is linked to several MCPH proteins, results in a marked reduction in head and eye size. Imaging studies reveal a dramatic rise in the fraction of proliferating cells in mitosis in all cases, and time-lapse microscopy points to a failure of progression through prometaphase. There was also increased apoptosis in all the MCPH models but this appears to be secondary to the mitotic defect as we frequently saw mitotically arrested cells disappear, and knocking down p53 apoptosis did not rescue the mitotic phenotype, either in whole retinas or clones.

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Related in: MedlinePlus

Apoptosis is associated with the MCPH phenotype. (a,b) High levels of apoptotic cell death occur within the retina of developing stil mutant embryos. (a) Little or no apoptotic cell death, as marked by antiactivated caspase-3 antibody (green) was seen in stilcz65+/? retinas at 72 hpf (also shown with DAPI-counterstain in blue). By contrast, high levels of apoptotic cell death were seen throughout the retina of stilcz65−/− embryos. A similar pattern was seen in stilhi1262Tg−/− embryos, with little apoptotic death in stilhi1262+/? retinas, but high levels of apoptosis in the stilhi1262Tg−/− mutant. (b). In stilhi1262Tg−/− mutants at 72 hpf, 8.7% of retinal cells were observed to be undergoing apoptosis (n = 52) versus 0.2% in stilcz65+/? (n = 24) (p < 0.001). In stilhi1262Tg−/− embryos, 12.4% of retinal cells were apoptotic (n = 16) versus 0.2% in stilhi1262+/? (n = 22) (p < 0.001). (c–g) Blocking apoptosis in stil mutant embryos partially rescued the retinal phenotype but did not rescue the mitotic phenotype. (c–d) anti-p53 Mo injection led to a reduction in apoptosis (green; antiactivated caspase-3) in stilcz65−/− mutants at 72 hpf, from 9.8% of cells (n = 39) to 4.3% (n = 15), p < 0.001. By comparison, 0.47% of retinal cells underwent apoptosis in stilcz65+/? embryos (n = 38) with no significant difference with anti-p53 Mo (1.0%; n = 2), p > 0.05. This anti-p53 Mo-related reduction in apoptosis led to partial rescue of retinal size (c,e), with an increase in mean cells per retinal section to 568 (n = 15) from 429 (n = 39), p < 0.01. A smaller, non-significant increase in retinal cells was seen in stilcz65+/? embryos; 944 (n = 2) versus 895 (n = 38), p > 0.05). (f) While anti-p53 Mo reduces apoptosis and led to an increase in retinal size and cell number, there was no significant effect on the mitotic phenotype, as demonstrated by anti-PH3 immunostaining (red). (g) anti-p53 Mo injection had no significant effect on MI (stilcz65−/− with p53 Mo; 21.2% (n = 12) versus 23.5% (n = 7) for stilcz65−/− without p53 Mo, p > 0.5. As a control, anti-p53 Mo was also injected into stilcz65+/? embryos with no significant effect on percentage of mitotic cells (stilcz65+/? with p53 Mo 1.7% (n = 13) versus 0.8% (n = 3) in stilcz65+/? embryos without p53 Mo; p < 0.05. n = number of eyes analysed.
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RSOB130065F6: Apoptosis is associated with the MCPH phenotype. (a,b) High levels of apoptotic cell death occur within the retina of developing stil mutant embryos. (a) Little or no apoptotic cell death, as marked by antiactivated caspase-3 antibody (green) was seen in stilcz65+/? retinas at 72 hpf (also shown with DAPI-counterstain in blue). By contrast, high levels of apoptotic cell death were seen throughout the retina of stilcz65−/− embryos. A similar pattern was seen in stilhi1262Tg−/− embryos, with little apoptotic death in stilhi1262+/? retinas, but high levels of apoptosis in the stilhi1262Tg−/− mutant. (b). In stilhi1262Tg−/− mutants at 72 hpf, 8.7% of retinal cells were observed to be undergoing apoptosis (n = 52) versus 0.2% in stilcz65+/? (n = 24) (p < 0.001). In stilhi1262Tg−/− embryos, 12.4% of retinal cells were apoptotic (n = 16) versus 0.2% in stilhi1262+/? (n = 22) (p < 0.001). (c–g) Blocking apoptosis in stil mutant embryos partially rescued the retinal phenotype but did not rescue the mitotic phenotype. (c–d) anti-p53 Mo injection led to a reduction in apoptosis (green; antiactivated caspase-3) in stilcz65−/− mutants at 72 hpf, from 9.8% of cells (n = 39) to 4.3% (n = 15), p < 0.001. By comparison, 0.47% of retinal cells underwent apoptosis in stilcz65+/? embryos (n = 38) with no significant difference with anti-p53 Mo (1.0%; n = 2), p > 0.05. This anti-p53 Mo-related reduction in apoptosis led to partial rescue of retinal size (c,e), with an increase in mean cells per retinal section to 568 (n = 15) from 429 (n = 39), p < 0.01. A smaller, non-significant increase in retinal cells was seen in stilcz65+/? embryos; 944 (n = 2) versus 895 (n = 38), p > 0.05). (f) While anti-p53 Mo reduces apoptosis and led to an increase in retinal size and cell number, there was no significant effect on the mitotic phenotype, as demonstrated by anti-PH3 immunostaining (red). (g) anti-p53 Mo injection had no significant effect on MI (stilcz65−/− with p53 Mo; 21.2% (n = 12) versus 23.5% (n = 7) for stilcz65−/− without p53 Mo, p > 0.5. As a control, anti-p53 Mo was also injected into stilcz65+/? embryos with no significant effect on percentage of mitotic cells (stilcz65+/? with p53 Mo 1.7% (n = 13) versus 0.8% (n = 3) in stilcz65+/? embryos without p53 Mo; p < 0.05. n = number of eyes analysed.

Mentions: Increased levels of apoptosis have previously been observed in cspcz65−/− embryos by whole-mount TUNEL staining [34]. In addition, we observed significant levels of hyperfluorescent cellular debris in DAPI-stained sections of cspcz65−/− mutants (figure 2a) as well as in vivo time-lapse movies (not shown). This led us to investigate whether apoptosis was a consistent feature of the zebrafish retinal MCPH phenotype by performing immunostaining on fixed retinal sections using anti-activated caspase-3. This confirmed high rates of apoptosis in the retina of both the cspcz65−/− mutant and the stilhi1262−/− mutant at 72 hpf (figure 6a). Whereas levels of apoptosis were very low in unaffected embryos (0.2%), 8.7% of cells stained positive for activated caspase-3 in the retinas of cspcz65−/− mutant embryos and 12.5% in the retina of stilhi1262−/− mutants (figure 6b). We were interested in elucidating to what degree this apoptotic cell death might be contributing to the observed reduction in retinal cell number. To explore this, we made use of the anti-p53 Mo. While p53 expression cannot be considered synonymous with cell death, we found that blocking p53 activity in cspcz65−/− mutants led to a 56% reduction in apoptotic death, from 9.8% of cells to 4.3% of cells (figure 6c,d) and to a 32% increase in mean retinal cell number (figure 6e). This suggests that apoptotic cell death directly leads to some of the reduction in retinal cell number observed in stil mutant embryos. This is consistent with our data from p53-Mo-injected morphant MCPH cells that were transplanted into morphant retinas. In these cases, the average clone size also increased by rather similar amounts (figure 5b–d). These results indicate that both an increase in apoptosis and a reduction in proliferative potential combine to cause the observed reduction in retinal clone size.Figure 6.


Microcephaly models in the developing zebrafish retinal neuroepithelium point to an underlying defect in metaphase progression.

Novorol C, Burkhardt J, Wood KJ, Iqbal A, Roque C, Coutts N, Almeida AD, He J, Wilkinson CJ, Harris WA - Open Biol (2013)

Apoptosis is associated with the MCPH phenotype. (a,b) High levels of apoptotic cell death occur within the retina of developing stil mutant embryos. (a) Little or no apoptotic cell death, as marked by antiactivated caspase-3 antibody (green) was seen in stilcz65+/? retinas at 72 hpf (also shown with DAPI-counterstain in blue). By contrast, high levels of apoptotic cell death were seen throughout the retina of stilcz65−/− embryos. A similar pattern was seen in stilhi1262Tg−/− embryos, with little apoptotic death in stilhi1262+/? retinas, but high levels of apoptosis in the stilhi1262Tg−/− mutant. (b). In stilhi1262Tg−/− mutants at 72 hpf, 8.7% of retinal cells were observed to be undergoing apoptosis (n = 52) versus 0.2% in stilcz65+/? (n = 24) (p < 0.001). In stilhi1262Tg−/− embryos, 12.4% of retinal cells were apoptotic (n = 16) versus 0.2% in stilhi1262+/? (n = 22) (p < 0.001). (c–g) Blocking apoptosis in stil mutant embryos partially rescued the retinal phenotype but did not rescue the mitotic phenotype. (c–d) anti-p53 Mo injection led to a reduction in apoptosis (green; antiactivated caspase-3) in stilcz65−/− mutants at 72 hpf, from 9.8% of cells (n = 39) to 4.3% (n = 15), p < 0.001. By comparison, 0.47% of retinal cells underwent apoptosis in stilcz65+/? embryos (n = 38) with no significant difference with anti-p53 Mo (1.0%; n = 2), p > 0.05. This anti-p53 Mo-related reduction in apoptosis led to partial rescue of retinal size (c,e), with an increase in mean cells per retinal section to 568 (n = 15) from 429 (n = 39), p < 0.01. A smaller, non-significant increase in retinal cells was seen in stilcz65+/? embryos; 944 (n = 2) versus 895 (n = 38), p > 0.05). (f) While anti-p53 Mo reduces apoptosis and led to an increase in retinal size and cell number, there was no significant effect on the mitotic phenotype, as demonstrated by anti-PH3 immunostaining (red). (g) anti-p53 Mo injection had no significant effect on MI (stilcz65−/− with p53 Mo; 21.2% (n = 12) versus 23.5% (n = 7) for stilcz65−/− without p53 Mo, p > 0.5. As a control, anti-p53 Mo was also injected into stilcz65+/? embryos with no significant effect on percentage of mitotic cells (stilcz65+/? with p53 Mo 1.7% (n = 13) versus 0.8% (n = 3) in stilcz65+/? embryos without p53 Mo; p < 0.05. n = number of eyes analysed.
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RSOB130065F6: Apoptosis is associated with the MCPH phenotype. (a,b) High levels of apoptotic cell death occur within the retina of developing stil mutant embryos. (a) Little or no apoptotic cell death, as marked by antiactivated caspase-3 antibody (green) was seen in stilcz65+/? retinas at 72 hpf (also shown with DAPI-counterstain in blue). By contrast, high levels of apoptotic cell death were seen throughout the retina of stilcz65−/− embryos. A similar pattern was seen in stilhi1262Tg−/− embryos, with little apoptotic death in stilhi1262+/? retinas, but high levels of apoptosis in the stilhi1262Tg−/− mutant. (b). In stilhi1262Tg−/− mutants at 72 hpf, 8.7% of retinal cells were observed to be undergoing apoptosis (n = 52) versus 0.2% in stilcz65+/? (n = 24) (p < 0.001). In stilhi1262Tg−/− embryos, 12.4% of retinal cells were apoptotic (n = 16) versus 0.2% in stilhi1262+/? (n = 22) (p < 0.001). (c–g) Blocking apoptosis in stil mutant embryos partially rescued the retinal phenotype but did not rescue the mitotic phenotype. (c–d) anti-p53 Mo injection led to a reduction in apoptosis (green; antiactivated caspase-3) in stilcz65−/− mutants at 72 hpf, from 9.8% of cells (n = 39) to 4.3% (n = 15), p < 0.001. By comparison, 0.47% of retinal cells underwent apoptosis in stilcz65+/? embryos (n = 38) with no significant difference with anti-p53 Mo (1.0%; n = 2), p > 0.05. This anti-p53 Mo-related reduction in apoptosis led to partial rescue of retinal size (c,e), with an increase in mean cells per retinal section to 568 (n = 15) from 429 (n = 39), p < 0.01. A smaller, non-significant increase in retinal cells was seen in stilcz65+/? embryos; 944 (n = 2) versus 895 (n = 38), p > 0.05). (f) While anti-p53 Mo reduces apoptosis and led to an increase in retinal size and cell number, there was no significant effect on the mitotic phenotype, as demonstrated by anti-PH3 immunostaining (red). (g) anti-p53 Mo injection had no significant effect on MI (stilcz65−/− with p53 Mo; 21.2% (n = 12) versus 23.5% (n = 7) for stilcz65−/− without p53 Mo, p > 0.5. As a control, anti-p53 Mo was also injected into stilcz65+/? embryos with no significant effect on percentage of mitotic cells (stilcz65+/? with p53 Mo 1.7% (n = 13) versus 0.8% (n = 3) in stilcz65+/? embryos without p53 Mo; p < 0.05. n = number of eyes analysed.
Mentions: Increased levels of apoptosis have previously been observed in cspcz65−/− embryos by whole-mount TUNEL staining [34]. In addition, we observed significant levels of hyperfluorescent cellular debris in DAPI-stained sections of cspcz65−/− mutants (figure 2a) as well as in vivo time-lapse movies (not shown). This led us to investigate whether apoptosis was a consistent feature of the zebrafish retinal MCPH phenotype by performing immunostaining on fixed retinal sections using anti-activated caspase-3. This confirmed high rates of apoptosis in the retina of both the cspcz65−/− mutant and the stilhi1262−/− mutant at 72 hpf (figure 6a). Whereas levels of apoptosis were very low in unaffected embryos (0.2%), 8.7% of cells stained positive for activated caspase-3 in the retinas of cspcz65−/− mutant embryos and 12.5% in the retina of stilhi1262−/− mutants (figure 6b). We were interested in elucidating to what degree this apoptotic cell death might be contributing to the observed reduction in retinal cell number. To explore this, we made use of the anti-p53 Mo. While p53 expression cannot be considered synonymous with cell death, we found that blocking p53 activity in cspcz65−/− mutants led to a 56% reduction in apoptotic death, from 9.8% of cells to 4.3% of cells (figure 6c,d) and to a 32% increase in mean retinal cell number (figure 6e). This suggests that apoptotic cell death directly leads to some of the reduction in retinal cell number observed in stil mutant embryos. This is consistent with our data from p53-Mo-injected morphant MCPH cells that were transplanted into morphant retinas. In these cases, the average clone size also increased by rather similar amounts (figure 5b–d). These results indicate that both an increase in apoptosis and a reduction in proliferative potential combine to cause the observed reduction in retinal clone size.Figure 6.

Bottom Line: Autosomal recessive primary microcephaly (MCPH) is a congenital disorder characterized by significantly reduced brain size and mental retardation.Mutant or morpholino-mediated knockdown of three known MCPH genes (stil, aspm and wdr62) and a fourth centrosomal gene, odf2, which is linked to several MCPH proteins, results in a marked reduction in head and eye size.There was also increased apoptosis in all the MCPH models but this appears to be secondary to the mitotic defect as we frequently saw mitotically arrested cells disappear, and knocking down p53 apoptosis did not rescue the mitotic phenotype, either in whole retinas or clones.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Development and Neuroscience, Cambridge University, Cambridge CB2 3DY, UK.

ABSTRACT
Autosomal recessive primary microcephaly (MCPH) is a congenital disorder characterized by significantly reduced brain size and mental retardation. Nine genes are currently known to be associated with the condition, all of which encode centrosomal or spindle pole proteins. MCPH is associated with a reduction in proliferation of neural progenitors during fetal development. The cellular mechanisms underlying the proliferation defect, however, are not fully understood. The zebrafish retinal neuroepithelium provides an ideal system to investigate this question. Mutant or morpholino-mediated knockdown of three known MCPH genes (stil, aspm and wdr62) and a fourth centrosomal gene, odf2, which is linked to several MCPH proteins, results in a marked reduction in head and eye size. Imaging studies reveal a dramatic rise in the fraction of proliferating cells in mitosis in all cases, and time-lapse microscopy points to a failure of progression through prometaphase. There was also increased apoptosis in all the MCPH models but this appears to be secondary to the mitotic defect as we frequently saw mitotically arrested cells disappear, and knocking down p53 apoptosis did not rescue the mitotic phenotype, either in whole retinas or clones.

Show MeSH
Related in: MedlinePlus