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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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Morpholino knockdown of stil, aspm or wdr62 led to reduced clonal proliferation of retinal progenitors in vivo. Blocking apoptosis only partially rescued clonal potential. Cells from H2B-GFP-expressing wild-type (WT) or morphant donor embryos were transplanted into WT or morphant host embryos at approximately 3.5 hpf. Host embryo retinas were screened for GFP-expressing one to two cell clones at 24 hpf and those clones were analysed again at 48 hpf. Graphs (b–d) show the mean cells per clone at 48 hpf (derived from a single cell at 24 hpf). The average size of retinal clones derived from WT cells in WT hosts was 14.2 cells (n = 73). (a)(i) Two typical WT clones in a WT host retina at 48 hpf, each derived from a two-cell clone identified at 24 hpf. No significant difference in clone size was seen when cells from control embryos were transplanted into WT environments (not shown): CoMo: 13.9 cells (n = 7) versus WT: 14.2 cells (n = 73) (p > 0.05). (b) stil morphant cells had a markedly reduced clonal capacity in WT hosts: stil Mo 1.7 cells (n = 8) versus 14.2 cells for WT (n = 73) (p < 0.001). Partial rescue of clone size was achieved with injection of anti-p53 Mo to block apoptotic cell death: stil + p53 Mo donor cells in WT hosts: 5.1 cells (n = 25) versus 1.7 cells without p53 Mo (n = 8) (p > 0.05). However, clones remained significantly smaller than WT: 5.1 cells (n = 25) versus 14.2 cells (n = 73) (p < 0.001). A similar result was seen when WT or stil morphant cells were transplanted into stil morphant hosts. Within the morphant environment, stil morphant cells produced smaller retinal clones compared with WT cells: 5.2 cells (n = 9) versus 13.7 cells (n = 27) (p < 0.001). (a)(ii) A typical example of morphant cell clones within a morphant host environment at 48 hpf. GFP-expressing cells from a stil morphant donor were transplanted into a stil morphant host. Clone 1 contains seven cells with one cell (marked double asterisks (**)) presumed to be undergoing mitosis at the time of imaging. In addition, two small cells that appear to be shrinking (marked single asterisk (*)) were presumed to be undergoing apoptotic cell death. Clone 2 contains four cells. Partial rescue of clone size could be achieved by injection of anti-p53 Mo to block apoptotic cell death: stil + p53 Mo donor cells in stil hosts: 10.0 cells (n = 31) versus 5.2 cells (n = 9) (p < 0.05). However, clones remained significantly smaller than WT clones: 10.0 cells (n = 31) versus 13.7 cells (n = 27) (p < 0.001). (c) aspm morphant cells also produced smaller clones than WT cells; 6.9 cells (n = 3) versus 14.2 cells (n = 73) (p < 0.05). Partial rescue of clone size could be achieved by injection of anti-p53 Mo: aspm + p53 Mo donor cells in WT hosts: 7.4 cells (n = 20) versus 6.9 cells without p53 Mo (n = 3) (p > 0.05). However, clones remained significantly smaller than WT: 7.4 cells (n = 20) versus 14.2 cells (n = 73) (p < 0.01). Within the morphant environment, aspm morphant cells produced smaller retinal clones than WT cells: 8.1 cells (n = 15) versus 14.2 cells (n = 12) (p < 0.01). Partial rescue of clone size could be achieved with injection of anti-p53 Mo: aspm + p53 Mo donor cells in aspm hosts: 11.5 cells (n = 14) versus 8.1 cells (n = 15); p < 0.05. However, clone size still remained smaller than WT clones: 11.5 cells (n = 14) versus 14.2 cells (n = 12) (p > 0.05). (d) wdr62 morphant cells also produced smaller clones than WT; 1.5 cells (n = 12) versus 14.2 cells (n = 73) (p < 0.001). (a)(iii) A typical example of morphant cell clones within a WT host environment at 48 hpf. GFP-expressing wdr62 morphant cells were transplanted into WT host embryos. Two clones are seen, derived from two single cells identified at 24 hpf. One clone (white arrow) contains two cells. The second clone consists of three small cells (marked single asterisk (*)), all presumed to be undergoing apoptotic cell death. Partial rescue of clone size was achieved by injection of anti-p53 Mo: wdr62 + p53 Mo donor cells in WT hosts: 4.3 cells (n = 40) versus 1.4 cells without p53 Mo (n = 12) (p < 0.05). However, clones remained significantly smaller than WT clones: 4.3 cells (n = 40) versus 14.2 cells (n = 73) (p < 0.001). Within the morphant environment, wdr62 morphant cells produced smaller clones than WT cells: 4.3 cells (n = 26) versus 13.7 cells (n = 18) (p < 0.001). No significant rescue was achieved by injection of anti-p53 Mo to block apoptotic cell death: wdr62 + p53 Mo donor cells in wdr62 hosts: 4.9 cells (n = 28) versus 4.3 cells (n = 26) (p > 0.05). Clone size remained significantly smaller than WT clones: 4.9 cells (n = 28) versus 13.7 cells (n = 18) (p < 0.001). n = number of surviving clones examined at 48 hpf.
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RSOB130065F5: Morpholino knockdown of stil, aspm or wdr62 led to reduced clonal proliferation of retinal progenitors in vivo. Blocking apoptosis only partially rescued clonal potential. Cells from H2B-GFP-expressing wild-type (WT) or morphant donor embryos were transplanted into WT or morphant host embryos at approximately 3.5 hpf. Host embryo retinas were screened for GFP-expressing one to two cell clones at 24 hpf and those clones were analysed again at 48 hpf. Graphs (b–d) show the mean cells per clone at 48 hpf (derived from a single cell at 24 hpf). The average size of retinal clones derived from WT cells in WT hosts was 14.2 cells (n = 73). (a)(i) Two typical WT clones in a WT host retina at 48 hpf, each derived from a two-cell clone identified at 24 hpf. No significant difference in clone size was seen when cells from control embryos were transplanted into WT environments (not shown): CoMo: 13.9 cells (n = 7) versus WT: 14.2 cells (n = 73) (p > 0.05). (b) stil morphant cells had a markedly reduced clonal capacity in WT hosts: stil Mo 1.7 cells (n = 8) versus 14.2 cells for WT (n = 73) (p < 0.001). Partial rescue of clone size was achieved with injection of anti-p53 Mo to block apoptotic cell death: stil + p53 Mo donor cells in WT hosts: 5.1 cells (n = 25) versus 1.7 cells without p53 Mo (n = 8) (p > 0.05). However, clones remained significantly smaller than WT: 5.1 cells (n = 25) versus 14.2 cells (n = 73) (p < 0.001). A similar result was seen when WT or stil morphant cells were transplanted into stil morphant hosts. Within the morphant environment, stil morphant cells produced smaller retinal clones compared with WT cells: 5.2 cells (n = 9) versus 13.7 cells (n = 27) (p < 0.001). (a)(ii) A typical example of morphant cell clones within a morphant host environment at 48 hpf. GFP-expressing cells from a stil morphant donor were transplanted into a stil morphant host. Clone 1 contains seven cells with one cell (marked double asterisks (**)) presumed to be undergoing mitosis at the time of imaging. In addition, two small cells that appear to be shrinking (marked single asterisk (*)) were presumed to be undergoing apoptotic cell death. Clone 2 contains four cells. Partial rescue of clone size could be achieved by injection of anti-p53 Mo to block apoptotic cell death: stil + p53 Mo donor cells in stil hosts: 10.0 cells (n = 31) versus 5.2 cells (n = 9) (p < 0.05). However, clones remained significantly smaller than WT clones: 10.0 cells (n = 31) versus 13.7 cells (n = 27) (p < 0.001). (c) aspm morphant cells also produced smaller clones than WT cells; 6.9 cells (n = 3) versus 14.2 cells (n = 73) (p < 0.05). Partial rescue of clone size could be achieved by injection of anti-p53 Mo: aspm + p53 Mo donor cells in WT hosts: 7.4 cells (n = 20) versus 6.9 cells without p53 Mo (n = 3) (p > 0.05). However, clones remained significantly smaller than WT: 7.4 cells (n = 20) versus 14.2 cells (n = 73) (p < 0.01). Within the morphant environment, aspm morphant cells produced smaller retinal clones than WT cells: 8.1 cells (n = 15) versus 14.2 cells (n = 12) (p < 0.01). Partial rescue of clone size could be achieved with injection of anti-p53 Mo: aspm + p53 Mo donor cells in aspm hosts: 11.5 cells (n = 14) versus 8.1 cells (n = 15); p < 0.05. However, clone size still remained smaller than WT clones: 11.5 cells (n = 14) versus 14.2 cells (n = 12) (p > 0.05). (d) wdr62 morphant cells also produced smaller clones than WT; 1.5 cells (n = 12) versus 14.2 cells (n = 73) (p < 0.001). (a)(iii) A typical example of morphant cell clones within a WT host environment at 48 hpf. GFP-expressing wdr62 morphant cells were transplanted into WT host embryos. Two clones are seen, derived from two single cells identified at 24 hpf. One clone (white arrow) contains two cells. The second clone consists of three small cells (marked single asterisk (*)), all presumed to be undergoing apoptotic cell death. Partial rescue of clone size was achieved by injection of anti-p53 Mo: wdr62 + p53 Mo donor cells in WT hosts: 4.3 cells (n = 40) versus 1.4 cells without p53 Mo (n = 12) (p < 0.05). However, clones remained significantly smaller than WT clones: 4.3 cells (n = 40) versus 14.2 cells (n = 73) (p < 0.001). Within the morphant environment, wdr62 morphant cells produced smaller clones than WT cells: 4.3 cells (n = 26) versus 13.7 cells (n = 18) (p < 0.001). No significant rescue was achieved by injection of anti-p53 Mo to block apoptotic cell death: wdr62 + p53 Mo donor cells in wdr62 hosts: 4.9 cells (n = 28) versus 4.3 cells (n = 26) (p > 0.05). Clone size remained significantly smaller than WT clones: 4.9 cells (n = 28) versus 13.7 cells (n = 18) (p < 0.001). n = number of surviving clones examined at 48 hpf.

Mentions: To look at the proliferative potential of retinal progenitors affected by MCPH gene knockdown in more detail, we performed in vivo clonal analysis. Cells with GFP-marked nuclei from wild-type or morphant embryo donors were transplanted into wild-type or morphant host embryos early in development (approx. 3.5 hpf). Retinas of host embryos were then screened for GFP-expressing cells by fluorescence microscopy at 24 hpf. At this time-point, we identified clones of one or two cells to be tracked. We then found these clones again in vivo at 48 hpf and counted the cells. The average size of a wild-type retinal clone in a wild-type host at 48 hpf (figure 5a(i)) was 14.2 cells, and clones derived from control-morpholino-injected embryos gave an average clone size of 13.9 cells, demonstrating that the injection procedure had no significant effect on proliferation. When morphant cells were then transplanted into morphant hosts, clone size was considerably reduced. stil morphant cells (in stil morphant environments) (figure 5a(ii)) produced clones of mean size 5.2 cells, aspm morphant cells (in aspm morphant host environments) produced clones of mean 8.1 cells (figure 5c) and wdr62 morphant cells (in wdr62 morphant host environments) produced clones of mean 4.3 cells (figure 5d). Thus, individual morphant progenitors show a dramatic decrease in proliferation.Figure 5.


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)

Morpholino knockdown of stil, aspm or wdr62 led to reduced clonal proliferation of retinal progenitors in vivo. Blocking apoptosis only partially rescued clonal potential. Cells from H2B-GFP-expressing wild-type (WT) or morphant donor embryos were transplanted into WT or morphant host embryos at approximately 3.5 hpf. Host embryo retinas were screened for GFP-expressing one to two cell clones at 24 hpf and those clones were analysed again at 48 hpf. Graphs (b–d) show the mean cells per clone at 48 hpf (derived from a single cell at 24 hpf). The average size of retinal clones derived from WT cells in WT hosts was 14.2 cells (n = 73). (a)(i) Two typical WT clones in a WT host retina at 48 hpf, each derived from a two-cell clone identified at 24 hpf. No significant difference in clone size was seen when cells from control embryos were transplanted into WT environments (not shown): CoMo: 13.9 cells (n = 7) versus WT: 14.2 cells (n = 73) (p > 0.05). (b) stil morphant cells had a markedly reduced clonal capacity in WT hosts: stil Mo 1.7 cells (n = 8) versus 14.2 cells for WT (n = 73) (p < 0.001). Partial rescue of clone size was achieved with injection of anti-p53 Mo to block apoptotic cell death: stil + p53 Mo donor cells in WT hosts: 5.1 cells (n = 25) versus 1.7 cells without p53 Mo (n = 8) (p > 0.05). However, clones remained significantly smaller than WT: 5.1 cells (n = 25) versus 14.2 cells (n = 73) (p < 0.001). A similar result was seen when WT or stil morphant cells were transplanted into stil morphant hosts. Within the morphant environment, stil morphant cells produced smaller retinal clones compared with WT cells: 5.2 cells (n = 9) versus 13.7 cells (n = 27) (p < 0.001). (a)(ii) A typical example of morphant cell clones within a morphant host environment at 48 hpf. GFP-expressing cells from a stil morphant donor were transplanted into a stil morphant host. Clone 1 contains seven cells with one cell (marked double asterisks (**)) presumed to be undergoing mitosis at the time of imaging. In addition, two small cells that appear to be shrinking (marked single asterisk (*)) were presumed to be undergoing apoptotic cell death. Clone 2 contains four cells. Partial rescue of clone size could be achieved by injection of anti-p53 Mo to block apoptotic cell death: stil + p53 Mo donor cells in stil hosts: 10.0 cells (n = 31) versus 5.2 cells (n = 9) (p < 0.05). However, clones remained significantly smaller than WT clones: 10.0 cells (n = 31) versus 13.7 cells (n = 27) (p < 0.001). (c) aspm morphant cells also produced smaller clones than WT cells; 6.9 cells (n = 3) versus 14.2 cells (n = 73) (p < 0.05). Partial rescue of clone size could be achieved by injection of anti-p53 Mo: aspm + p53 Mo donor cells in WT hosts: 7.4 cells (n = 20) versus 6.9 cells without p53 Mo (n = 3) (p > 0.05). However, clones remained significantly smaller than WT: 7.4 cells (n = 20) versus 14.2 cells (n = 73) (p < 0.01). Within the morphant environment, aspm morphant cells produced smaller retinal clones than WT cells: 8.1 cells (n = 15) versus 14.2 cells (n = 12) (p < 0.01). Partial rescue of clone size could be achieved with injection of anti-p53 Mo: aspm + p53 Mo donor cells in aspm hosts: 11.5 cells (n = 14) versus 8.1 cells (n = 15); p < 0.05. However, clone size still remained smaller than WT clones: 11.5 cells (n = 14) versus 14.2 cells (n = 12) (p > 0.05). (d) wdr62 morphant cells also produced smaller clones than WT; 1.5 cells (n = 12) versus 14.2 cells (n = 73) (p < 0.001). (a)(iii) A typical example of morphant cell clones within a WT host environment at 48 hpf. GFP-expressing wdr62 morphant cells were transplanted into WT host embryos. Two clones are seen, derived from two single cells identified at 24 hpf. One clone (white arrow) contains two cells. The second clone consists of three small cells (marked single asterisk (*)), all presumed to be undergoing apoptotic cell death. Partial rescue of clone size was achieved by injection of anti-p53 Mo: wdr62 + p53 Mo donor cells in WT hosts: 4.3 cells (n = 40) versus 1.4 cells without p53 Mo (n = 12) (p < 0.05). However, clones remained significantly smaller than WT clones: 4.3 cells (n = 40) versus 14.2 cells (n = 73) (p < 0.001). Within the morphant environment, wdr62 morphant cells produced smaller clones than WT cells: 4.3 cells (n = 26) versus 13.7 cells (n = 18) (p < 0.001). No significant rescue was achieved by injection of anti-p53 Mo to block apoptotic cell death: wdr62 + p53 Mo donor cells in wdr62 hosts: 4.9 cells (n = 28) versus 4.3 cells (n = 26) (p > 0.05). Clone size remained significantly smaller than WT clones: 4.9 cells (n = 28) versus 13.7 cells (n = 18) (p < 0.001). n = number of surviving clones examined at 48 hpf.
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RSOB130065F5: Morpholino knockdown of stil, aspm or wdr62 led to reduced clonal proliferation of retinal progenitors in vivo. Blocking apoptosis only partially rescued clonal potential. Cells from H2B-GFP-expressing wild-type (WT) or morphant donor embryos were transplanted into WT or morphant host embryos at approximately 3.5 hpf. Host embryo retinas were screened for GFP-expressing one to two cell clones at 24 hpf and those clones were analysed again at 48 hpf. Graphs (b–d) show the mean cells per clone at 48 hpf (derived from a single cell at 24 hpf). The average size of retinal clones derived from WT cells in WT hosts was 14.2 cells (n = 73). (a)(i) Two typical WT clones in a WT host retina at 48 hpf, each derived from a two-cell clone identified at 24 hpf. No significant difference in clone size was seen when cells from control embryos were transplanted into WT environments (not shown): CoMo: 13.9 cells (n = 7) versus WT: 14.2 cells (n = 73) (p > 0.05). (b) stil morphant cells had a markedly reduced clonal capacity in WT hosts: stil Mo 1.7 cells (n = 8) versus 14.2 cells for WT (n = 73) (p < 0.001). Partial rescue of clone size was achieved with injection of anti-p53 Mo to block apoptotic cell death: stil + p53 Mo donor cells in WT hosts: 5.1 cells (n = 25) versus 1.7 cells without p53 Mo (n = 8) (p > 0.05). However, clones remained significantly smaller than WT: 5.1 cells (n = 25) versus 14.2 cells (n = 73) (p < 0.001). A similar result was seen when WT or stil morphant cells were transplanted into stil morphant hosts. Within the morphant environment, stil morphant cells produced smaller retinal clones compared with WT cells: 5.2 cells (n = 9) versus 13.7 cells (n = 27) (p < 0.001). (a)(ii) A typical example of morphant cell clones within a morphant host environment at 48 hpf. GFP-expressing cells from a stil morphant donor were transplanted into a stil morphant host. Clone 1 contains seven cells with one cell (marked double asterisks (**)) presumed to be undergoing mitosis at the time of imaging. In addition, two small cells that appear to be shrinking (marked single asterisk (*)) were presumed to be undergoing apoptotic cell death. Clone 2 contains four cells. Partial rescue of clone size could be achieved by injection of anti-p53 Mo to block apoptotic cell death: stil + p53 Mo donor cells in stil hosts: 10.0 cells (n = 31) versus 5.2 cells (n = 9) (p < 0.05). However, clones remained significantly smaller than WT clones: 10.0 cells (n = 31) versus 13.7 cells (n = 27) (p < 0.001). (c) aspm morphant cells also produced smaller clones than WT cells; 6.9 cells (n = 3) versus 14.2 cells (n = 73) (p < 0.05). Partial rescue of clone size could be achieved by injection of anti-p53 Mo: aspm + p53 Mo donor cells in WT hosts: 7.4 cells (n = 20) versus 6.9 cells without p53 Mo (n = 3) (p > 0.05). However, clones remained significantly smaller than WT: 7.4 cells (n = 20) versus 14.2 cells (n = 73) (p < 0.01). Within the morphant environment, aspm morphant cells produced smaller retinal clones than WT cells: 8.1 cells (n = 15) versus 14.2 cells (n = 12) (p < 0.01). Partial rescue of clone size could be achieved with injection of anti-p53 Mo: aspm + p53 Mo donor cells in aspm hosts: 11.5 cells (n = 14) versus 8.1 cells (n = 15); p < 0.05. However, clone size still remained smaller than WT clones: 11.5 cells (n = 14) versus 14.2 cells (n = 12) (p > 0.05). (d) wdr62 morphant cells also produced smaller clones than WT; 1.5 cells (n = 12) versus 14.2 cells (n = 73) (p < 0.001). (a)(iii) A typical example of morphant cell clones within a WT host environment at 48 hpf. GFP-expressing wdr62 morphant cells were transplanted into WT host embryos. Two clones are seen, derived from two single cells identified at 24 hpf. One clone (white arrow) contains two cells. The second clone consists of three small cells (marked single asterisk (*)), all presumed to be undergoing apoptotic cell death. Partial rescue of clone size was achieved by injection of anti-p53 Mo: wdr62 + p53 Mo donor cells in WT hosts: 4.3 cells (n = 40) versus 1.4 cells without p53 Mo (n = 12) (p < 0.05). However, clones remained significantly smaller than WT clones: 4.3 cells (n = 40) versus 14.2 cells (n = 73) (p < 0.001). Within the morphant environment, wdr62 morphant cells produced smaller clones than WT cells: 4.3 cells (n = 26) versus 13.7 cells (n = 18) (p < 0.001). No significant rescue was achieved by injection of anti-p53 Mo to block apoptotic cell death: wdr62 + p53 Mo donor cells in wdr62 hosts: 4.9 cells (n = 28) versus 4.3 cells (n = 26) (p > 0.05). Clone size remained significantly smaller than WT clones: 4.9 cells (n = 28) versus 13.7 cells (n = 18) (p < 0.001). n = number of surviving clones examined at 48 hpf.
Mentions: To look at the proliferative potential of retinal progenitors affected by MCPH gene knockdown in more detail, we performed in vivo clonal analysis. Cells with GFP-marked nuclei from wild-type or morphant embryo donors were transplanted into wild-type or morphant host embryos early in development (approx. 3.5 hpf). Retinas of host embryos were then screened for GFP-expressing cells by fluorescence microscopy at 24 hpf. At this time-point, we identified clones of one or two cells to be tracked. We then found these clones again in vivo at 48 hpf and counted the cells. The average size of a wild-type retinal clone in a wild-type host at 48 hpf (figure 5a(i)) was 14.2 cells, and clones derived from control-morpholino-injected embryos gave an average clone size of 13.9 cells, demonstrating that the injection procedure had no significant effect on proliferation. When morphant cells were then transplanted into morphant hosts, clone size was considerably reduced. stil morphant cells (in stil morphant environments) (figure 5a(ii)) produced clones of mean size 5.2 cells, aspm morphant cells (in aspm morphant host environments) produced clones of mean 8.1 cells (figure 5c) and wdr62 morphant cells (in wdr62 morphant host environments) produced clones of mean 4.3 cells (figure 5d). Thus, individual morphant progenitors show a dramatic decrease in proliferation.Figure 5.

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