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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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MCPH gene depletion causes a block or delay at prometaphase. Mitotic retinal cells in stil mutant embryos appear to be delayed in prometaphase. A similar but less severe phenotype occurs in stil morphants and odf2 morphants. (a) Views of the retina at approximately 30 hpf demonstrate (i) the normal appearance of retinal progenitor cells in stilcz65+/? embryos (H2B-RFP marks nuclei red) and (iii) control embryos (H2B-GFP marks nuclei green). White arrows mark dividing cells (anaphase) at the apical membrane. (ii) By contrast, in stilcz65−/− embryos the retina appears disorganized with markedly more cells in mitosis. The appearance of these cells (white arrows) suggests they are in prometaphase. A similar but less severe phenotype was observed in (iv) stil morphants and (v) odf2 morphants. In both morphant conditions, numerous ‘prometaphase’-like cells were observed in the retina (white arrows), although in contrast to mutants these cells were localized near to the apical membrane. (b) Centrosomal abnormalities are present in stilcz65−/− embryo retinas, including reduced centrosome expression and loss of apical centrosome positioning. Normal apical centrosomes are seen in stilcz65+/? embryos (green; centrin-GFP). (i) As cells round up and enter mitosis in stilcz65+/? embryos two centrosomes can be seen. (ii) A dividing cell is shown in a stilcz65+/? embryo, with a single centrosome at each pole of the newly forming daughter cells. By contrast, in stilcz65−/− embryos mitotic cells lack one or both centrosomes. (iii) Many prometaphase-like cells appear to be associated with only a single centrosome (white arrows, and at high magnification in (v) or (iv) no centrosome. (c) Many mitotic cells in stilcz65−/− mutants and stil morphants remain arrested in mitosis throughout live 2–3 h movies. Here frames demonstrate cells arrested in mitosis (white arrows) in stilcz65−/− embryos (nuclei in red; marked by H2B-RFP; centrosomes in green; marked by centrin-GFP) over a period of at least 144 min. Over the same period, stil morphant cells (green; marked by H2B-GFP) are also seen arrested in mitosis (white arrows). (d) Throughout movies a marked reduction in the percentage of cells successfully completing division was noted in stil mutants and morphants, with many cells remaining delayed or stuck in mitosis. In control embryos, 100% of cells entering mitosis during the first 2 h of a 3-h movie successfully completed cell division before the end of the 3-h movie (n = 29). A similar outcome was observed in stilcz65+/? control embryos; 94% of cells successfully completed cell division with 6% of cells disappearing from view (n = 17). In stil morphants, only 24% of cells successfully completed division, with 11% disappearing from view and 65% remaining arrested or delayed in M-phase for 60 min or longer (n = 37). In stilcz65−/− mutants, the phenotype was even more severe, with 11% of mitotic cells disappearing, 80% remaining stuck or delayed in M-phase and only 9% successfully completing mitotic division (n = 54). Three separate 180-min movies were analysed for each condition. n = total number of mitotic cells analysed for each condition. (e) Successful mitotic divisions were slower in morphants and mutants versus control. Typical divisions are shown for control, stil morphant, odf2 morphant, stilcz65+/? and stilcz65−/− embryos. Black vertical arrows indicate the beginning of M-phase (0 min), when the dividing cell rounds up at prophase, and the end of M-phase, when two daughter cells have been formed and chromatin decondensation has commenced. In these examples, mitosis took approximately 30 min (control), 66 min (stil Mo), 54 min (odf2 Mo), 42 min (stilcz65+/?) and a minimum of 144 min (stilcz65−/−) (note that for the stilcz65−/− mutant the dividing cell had already entered M-phase before the movie commenced so the true length of time to complete division was longer than this minimum estimate). (f) The time for morphant and mutant retinal cells to successfully complete mitotic division was increased. Three separate movies were analysed for each condition. The mean time to complete mitotic cell division was increased in stil morphants (n = 23) versus control (n = 29) (51 versus 29 min; p < 0.001) and odf2 morphants (n = 55) (38.7 versus 29 min; p < 0.01). The mean time to complete mitotic cell division was also markedly increased in stilcz65−/− embryos (n = 5) versus stilcz65+/? (n = 15) (at least 66 versus 30 min; p < 0.001). Overall, mitotic cell division within the retina was most severely prolonged in stilcz65−/− mutants and moderately prolonged in both stil morphants and odf2 morphants. n = number of successful mitotic divisions analysed.
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RSOB130065F4: MCPH gene depletion causes a block or delay at prometaphase. Mitotic retinal cells in stil mutant embryos appear to be delayed in prometaphase. A similar but less severe phenotype occurs in stil morphants and odf2 morphants. (a) Views of the retina at approximately 30 hpf demonstrate (i) the normal appearance of retinal progenitor cells in stilcz65+/? embryos (H2B-RFP marks nuclei red) and (iii) control embryos (H2B-GFP marks nuclei green). White arrows mark dividing cells (anaphase) at the apical membrane. (ii) By contrast, in stilcz65−/− embryos the retina appears disorganized with markedly more cells in mitosis. The appearance of these cells (white arrows) suggests they are in prometaphase. A similar but less severe phenotype was observed in (iv) stil morphants and (v) odf2 morphants. In both morphant conditions, numerous ‘prometaphase’-like cells were observed in the retina (white arrows), although in contrast to mutants these cells were localized near to the apical membrane. (b) Centrosomal abnormalities are present in stilcz65−/− embryo retinas, including reduced centrosome expression and loss of apical centrosome positioning. Normal apical centrosomes are seen in stilcz65+/? embryos (green; centrin-GFP). (i) As cells round up and enter mitosis in stilcz65+/? embryos two centrosomes can be seen. (ii) A dividing cell is shown in a stilcz65+/? embryo, with a single centrosome at each pole of the newly forming daughter cells. By contrast, in stilcz65−/− embryos mitotic cells lack one or both centrosomes. (iii) Many prometaphase-like cells appear to be associated with only a single centrosome (white arrows, and at high magnification in (v) or (iv) no centrosome. (c) Many mitotic cells in stilcz65−/− mutants and stil morphants remain arrested in mitosis throughout live 2–3 h movies. Here frames demonstrate cells arrested in mitosis (white arrows) in stilcz65−/− embryos (nuclei in red; marked by H2B-RFP; centrosomes in green; marked by centrin-GFP) over a period of at least 144 min. Over the same period, stil morphant cells (green; marked by H2B-GFP) are also seen arrested in mitosis (white arrows). (d) Throughout movies a marked reduction in the percentage of cells successfully completing division was noted in stil mutants and morphants, with many cells remaining delayed or stuck in mitosis. In control embryos, 100% of cells entering mitosis during the first 2 h of a 3-h movie successfully completed cell division before the end of the 3-h movie (n = 29). A similar outcome was observed in stilcz65+/? control embryos; 94% of cells successfully completed cell division with 6% of cells disappearing from view (n = 17). In stil morphants, only 24% of cells successfully completed division, with 11% disappearing from view and 65% remaining arrested or delayed in M-phase for 60 min or longer (n = 37). In stilcz65−/− mutants, the phenotype was even more severe, with 11% of mitotic cells disappearing, 80% remaining stuck or delayed in M-phase and only 9% successfully completing mitotic division (n = 54). Three separate 180-min movies were analysed for each condition. n = total number of mitotic cells analysed for each condition. (e) Successful mitotic divisions were slower in morphants and mutants versus control. Typical divisions are shown for control, stil morphant, odf2 morphant, stilcz65+/? and stilcz65−/− embryos. Black vertical arrows indicate the beginning of M-phase (0 min), when the dividing cell rounds up at prophase, and the end of M-phase, when two daughter cells have been formed and chromatin decondensation has commenced. In these examples, mitosis took approximately 30 min (control), 66 min (stil Mo), 54 min (odf2 Mo), 42 min (stilcz65+/?) and a minimum of 144 min (stilcz65−/−) (note that for the stilcz65−/− mutant the dividing cell had already entered M-phase before the movie commenced so the true length of time to complete division was longer than this minimum estimate). (f) The time for morphant and mutant retinal cells to successfully complete mitotic division was increased. Three separate movies were analysed for each condition. The mean time to complete mitotic cell division was increased in stil morphants (n = 23) versus control (n = 29) (51 versus 29 min; p < 0.001) and odf2 morphants (n = 55) (38.7 versus 29 min; p < 0.01). The mean time to complete mitotic cell division was also markedly increased in stilcz65−/− embryos (n = 5) versus stilcz65+/? (n = 15) (at least 66 versus 30 min; p < 0.001). Overall, mitotic cell division within the retina was most severely prolonged in stilcz65−/− mutants and moderately prolonged in both stil morphants and odf2 morphants. n = number of successful mitotic divisions analysed.

Mentions: To investigate the mitotic phenotype in more detail, in vivo time-lapse imaging of the developing retina was performed in stil cspcz65−/− mutants, stil morphants and odf2 morphants at approximately 30 hpf. Embryos with nuclei fluorescently marked were examined at 6-min intervals during 3-h movies. In stil mutants, the retina was strikingly disorganized, with large numbers of ‘rounded-up’ mitotic cells scattered throughout the retina (figure 4a(ii)). These cells appeared to contain scattered condensed chromosomes that were not forming a metaphase plate or entering anaphase. This was in contrast to the organized appearance of the unaffected cspcz65+/? retina (figure 4a(i)), in which mitotic cells (white arrows) were observed transiently and only at the apical membrane, often in metaphase or anaphase stages.Figure 4.


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)

MCPH gene depletion causes a block or delay at prometaphase. Mitotic retinal cells in stil mutant embryos appear to be delayed in prometaphase. A similar but less severe phenotype occurs in stil morphants and odf2 morphants. (a) Views of the retina at approximately 30 hpf demonstrate (i) the normal appearance of retinal progenitor cells in stilcz65+/? embryos (H2B-RFP marks nuclei red) and (iii) control embryos (H2B-GFP marks nuclei green). White arrows mark dividing cells (anaphase) at the apical membrane. (ii) By contrast, in stilcz65−/− embryos the retina appears disorganized with markedly more cells in mitosis. The appearance of these cells (white arrows) suggests they are in prometaphase. A similar but less severe phenotype was observed in (iv) stil morphants and (v) odf2 morphants. In both morphant conditions, numerous ‘prometaphase’-like cells were observed in the retina (white arrows), although in contrast to mutants these cells were localized near to the apical membrane. (b) Centrosomal abnormalities are present in stilcz65−/− embryo retinas, including reduced centrosome expression and loss of apical centrosome positioning. Normal apical centrosomes are seen in stilcz65+/? embryos (green; centrin-GFP). (i) As cells round up and enter mitosis in stilcz65+/? embryos two centrosomes can be seen. (ii) A dividing cell is shown in a stilcz65+/? embryo, with a single centrosome at each pole of the newly forming daughter cells. By contrast, in stilcz65−/− embryos mitotic cells lack one or both centrosomes. (iii) Many prometaphase-like cells appear to be associated with only a single centrosome (white arrows, and at high magnification in (v) or (iv) no centrosome. (c) Many mitotic cells in stilcz65−/− mutants and stil morphants remain arrested in mitosis throughout live 2–3 h movies. Here frames demonstrate cells arrested in mitosis (white arrows) in stilcz65−/− embryos (nuclei in red; marked by H2B-RFP; centrosomes in green; marked by centrin-GFP) over a period of at least 144 min. Over the same period, stil morphant cells (green; marked by H2B-GFP) are also seen arrested in mitosis (white arrows). (d) Throughout movies a marked reduction in the percentage of cells successfully completing division was noted in stil mutants and morphants, with many cells remaining delayed or stuck in mitosis. In control embryos, 100% of cells entering mitosis during the first 2 h of a 3-h movie successfully completed cell division before the end of the 3-h movie (n = 29). A similar outcome was observed in stilcz65+/? control embryos; 94% of cells successfully completed cell division with 6% of cells disappearing from view (n = 17). In stil morphants, only 24% of cells successfully completed division, with 11% disappearing from view and 65% remaining arrested or delayed in M-phase for 60 min or longer (n = 37). In stilcz65−/− mutants, the phenotype was even more severe, with 11% of mitotic cells disappearing, 80% remaining stuck or delayed in M-phase and only 9% successfully completing mitotic division (n = 54). Three separate 180-min movies were analysed for each condition. n = total number of mitotic cells analysed for each condition. (e) Successful mitotic divisions were slower in morphants and mutants versus control. Typical divisions are shown for control, stil morphant, odf2 morphant, stilcz65+/? and stilcz65−/− embryos. Black vertical arrows indicate the beginning of M-phase (0 min), when the dividing cell rounds up at prophase, and the end of M-phase, when two daughter cells have been formed and chromatin decondensation has commenced. In these examples, mitosis took approximately 30 min (control), 66 min (stil Mo), 54 min (odf2 Mo), 42 min (stilcz65+/?) and a minimum of 144 min (stilcz65−/−) (note that for the stilcz65−/− mutant the dividing cell had already entered M-phase before the movie commenced so the true length of time to complete division was longer than this minimum estimate). (f) The time for morphant and mutant retinal cells to successfully complete mitotic division was increased. Three separate movies were analysed for each condition. The mean time to complete mitotic cell division was increased in stil morphants (n = 23) versus control (n = 29) (51 versus 29 min; p < 0.001) and odf2 morphants (n = 55) (38.7 versus 29 min; p < 0.01). The mean time to complete mitotic cell division was also markedly increased in stilcz65−/− embryos (n = 5) versus stilcz65+/? (n = 15) (at least 66 versus 30 min; p < 0.001). Overall, mitotic cell division within the retina was most severely prolonged in stilcz65−/− mutants and moderately prolonged in both stil morphants and odf2 morphants. n = number of successful mitotic divisions analysed.
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RSOB130065F4: MCPH gene depletion causes a block or delay at prometaphase. Mitotic retinal cells in stil mutant embryos appear to be delayed in prometaphase. A similar but less severe phenotype occurs in stil morphants and odf2 morphants. (a) Views of the retina at approximately 30 hpf demonstrate (i) the normal appearance of retinal progenitor cells in stilcz65+/? embryos (H2B-RFP marks nuclei red) and (iii) control embryos (H2B-GFP marks nuclei green). White arrows mark dividing cells (anaphase) at the apical membrane. (ii) By contrast, in stilcz65−/− embryos the retina appears disorganized with markedly more cells in mitosis. The appearance of these cells (white arrows) suggests they are in prometaphase. A similar but less severe phenotype was observed in (iv) stil morphants and (v) odf2 morphants. In both morphant conditions, numerous ‘prometaphase’-like cells were observed in the retina (white arrows), although in contrast to mutants these cells were localized near to the apical membrane. (b) Centrosomal abnormalities are present in stilcz65−/− embryo retinas, including reduced centrosome expression and loss of apical centrosome positioning. Normal apical centrosomes are seen in stilcz65+/? embryos (green; centrin-GFP). (i) As cells round up and enter mitosis in stilcz65+/? embryos two centrosomes can be seen. (ii) A dividing cell is shown in a stilcz65+/? embryo, with a single centrosome at each pole of the newly forming daughter cells. By contrast, in stilcz65−/− embryos mitotic cells lack one or both centrosomes. (iii) Many prometaphase-like cells appear to be associated with only a single centrosome (white arrows, and at high magnification in (v) or (iv) no centrosome. (c) Many mitotic cells in stilcz65−/− mutants and stil morphants remain arrested in mitosis throughout live 2–3 h movies. Here frames demonstrate cells arrested in mitosis (white arrows) in stilcz65−/− embryos (nuclei in red; marked by H2B-RFP; centrosomes in green; marked by centrin-GFP) over a period of at least 144 min. Over the same period, stil morphant cells (green; marked by H2B-GFP) are also seen arrested in mitosis (white arrows). (d) Throughout movies a marked reduction in the percentage of cells successfully completing division was noted in stil mutants and morphants, with many cells remaining delayed or stuck in mitosis. In control embryos, 100% of cells entering mitosis during the first 2 h of a 3-h movie successfully completed cell division before the end of the 3-h movie (n = 29). A similar outcome was observed in stilcz65+/? control embryos; 94% of cells successfully completed cell division with 6% of cells disappearing from view (n = 17). In stil morphants, only 24% of cells successfully completed division, with 11% disappearing from view and 65% remaining arrested or delayed in M-phase for 60 min or longer (n = 37). In stilcz65−/− mutants, the phenotype was even more severe, with 11% of mitotic cells disappearing, 80% remaining stuck or delayed in M-phase and only 9% successfully completing mitotic division (n = 54). Three separate 180-min movies were analysed for each condition. n = total number of mitotic cells analysed for each condition. (e) Successful mitotic divisions were slower in morphants and mutants versus control. Typical divisions are shown for control, stil morphant, odf2 morphant, stilcz65+/? and stilcz65−/− embryos. Black vertical arrows indicate the beginning of M-phase (0 min), when the dividing cell rounds up at prophase, and the end of M-phase, when two daughter cells have been formed and chromatin decondensation has commenced. In these examples, mitosis took approximately 30 min (control), 66 min (stil Mo), 54 min (odf2 Mo), 42 min (stilcz65+/?) and a minimum of 144 min (stilcz65−/−) (note that for the stilcz65−/− mutant the dividing cell had already entered M-phase before the movie commenced so the true length of time to complete division was longer than this minimum estimate). (f) The time for morphant and mutant retinal cells to successfully complete mitotic division was increased. Three separate movies were analysed for each condition. The mean time to complete mitotic cell division was increased in stil morphants (n = 23) versus control (n = 29) (51 versus 29 min; p < 0.001) and odf2 morphants (n = 55) (38.7 versus 29 min; p < 0.01). The mean time to complete mitotic cell division was also markedly increased in stilcz65−/− embryos (n = 5) versus stilcz65+/? (n = 15) (at least 66 versus 30 min; p < 0.001). Overall, mitotic cell division within the retina was most severely prolonged in stilcz65−/− mutants and moderately prolonged in both stil morphants and odf2 morphants. n = number of successful mitotic divisions analysed.
Mentions: To investigate the mitotic phenotype in more detail, in vivo time-lapse imaging of the developing retina was performed in stil cspcz65−/− mutants, stil morphants and odf2 morphants at approximately 30 hpf. Embryos with nuclei fluorescently marked were examined at 6-min intervals during 3-h movies. In stil mutants, the retina was strikingly disorganized, with large numbers of ‘rounded-up’ mitotic cells scattered throughout the retina (figure 4a(ii)). These cells appeared to contain scattered condensed chromosomes that were not forming a metaphase plate or entering anaphase. This was in contrast to the organized appearance of the unaffected cspcz65+/? retina (figure 4a(i)), in which mitotic cells (white arrows) were observed transiently and only at the apical membrane, often in metaphase or anaphase stages.Figure 4.

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