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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

MCPH phenotypes in the zebrafish retina. Knockdown of MCPH genes causes a reduction in head size, retinal size and retinal cell number. (a) Reduction in head and eye size is demonstrated in whole-mount stilcz65−/− mutant embryos on day 5 of development. Healthy stilcz65+/? embryos are shown at the same developmental stage for comparison. While head and eye size were consistently reduced, overall body size/length of mutant embryos was variably affected. Here, one mutant (i) is smaller than the healthy embryo, whereas the other (ii) is similar in length and size to the healthy embryos. Note also the abnormally protruding lenses in the mutant embryo seen from (i), exposed owing to reduced retinal size. (b) DAPI-stained sections demonstrate reduced retinal area in stil morphants (stil Mo) at 72 hpf when compared with control morphants (CoMo). Similarly, the retinas of stilcz65−/− and stilhi1262Tg−/− mutant embryos at 72 hpf are markedly reduced in size. Labels in yellow: L, lens; R, retinal neuroepithelium; CMZ, ciliary marginal zone; ON, optic nerve; AM, apical membrane. (c) Retinal area is significantly reduced in stil morphant and mutant embryos: stil Mo 0.019 mm2 (n = 23) versus CoMo 0.024 mm2 (n = 34), p < 0.001; stilcz65−/− 0.019 mm2 (n = 23) versus stilcz65+/? 0.027 mm2 (n = 72) p < 0.001; stilhi1262Tg−/− 0.020 mm2 (n = 52) versus stilhi1262+/? 0.028 (n = 48), p < 0.001 (values are for mean area at 72 hpf). (d) Retinal cell number is reduced in stil morphants and mutants: stil Mo 471 cells (n = 23) versus CoMo 735 cells (n = 7), p < 0.001; stilcz65−/− 454 cells (n = 114) versus stilcz65+/? 780 cells (n = 72), p < 0.001; stilhi1262Tg−/− 468 cells (n = 52) versus stilhi1262+/? 744 cells (n = 48), p < 0.001 (values are for mean number of cells in central retinal sections at 72 hpf). (e) Retinal area increases as development progresses in stil, aspm, wdr62 and odf2 morphant embryos but remains reduced compared with control at all time-points examined (24, 48, 56 hpf at 72 hpf). (f) Retinal cell increases as development progresses in stil, aspm, wdr62 and odf2 morphant embryos but remains reduced compared with control at all time-points examined (24, 48, 56 hpf at 72 hpf), n = number of eyes analysed.
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RSOB130065F2: MCPH phenotypes in the zebrafish retina. Knockdown of MCPH genes causes a reduction in head size, retinal size and retinal cell number. (a) Reduction in head and eye size is demonstrated in whole-mount stilcz65−/− mutant embryos on day 5 of development. Healthy stilcz65+/? embryos are shown at the same developmental stage for comparison. While head and eye size were consistently reduced, overall body size/length of mutant embryos was variably affected. Here, one mutant (i) is smaller than the healthy embryo, whereas the other (ii) is similar in length and size to the healthy embryos. Note also the abnormally protruding lenses in the mutant embryo seen from (i), exposed owing to reduced retinal size. (b) DAPI-stained sections demonstrate reduced retinal area in stil morphants (stil Mo) at 72 hpf when compared with control morphants (CoMo). Similarly, the retinas of stilcz65−/− and stilhi1262Tg−/− mutant embryos at 72 hpf are markedly reduced in size. Labels in yellow: L, lens; R, retinal neuroepithelium; CMZ, ciliary marginal zone; ON, optic nerve; AM, apical membrane. (c) Retinal area is significantly reduced in stil morphant and mutant embryos: stil Mo 0.019 mm2 (n = 23) versus CoMo 0.024 mm2 (n = 34), p < 0.001; stilcz65−/− 0.019 mm2 (n = 23) versus stilcz65+/? 0.027 mm2 (n = 72) p < 0.001; stilhi1262Tg−/− 0.020 mm2 (n = 52) versus stilhi1262+/? 0.028 (n = 48), p < 0.001 (values are for mean area at 72 hpf). (d) Retinal cell number is reduced in stil morphants and mutants: stil Mo 471 cells (n = 23) versus CoMo 735 cells (n = 7), p < 0.001; stilcz65−/− 454 cells (n = 114) versus stilcz65+/? 780 cells (n = 72), p < 0.001; stilhi1262Tg−/− 468 cells (n = 52) versus stilhi1262+/? 744 cells (n = 48), p < 0.001 (values are for mean number of cells in central retinal sections at 72 hpf). (e) Retinal area increases as development progresses in stil, aspm, wdr62 and odf2 morphant embryos but remains reduced compared with control at all time-points examined (24, 48, 56 hpf at 72 hpf). (f) Retinal cell increases as development progresses in stil, aspm, wdr62 and odf2 morphant embryos but remains reduced compared with control at all time-points examined (24, 48, 56 hpf at 72 hpf), n = number of eyes analysed.

Mentions: To investigate the phenotype associated with stil knockdown, we were able to take advantage of two previously characterized loss-of-function mutants, cspcz65−/− and stilhi1262Tg−/− (see electronic supplementary material, figure S2A). We also designed antisense morpholinos against all four genes of interest (see electronic supplementary material, figure S2B–E). Both mutants and all four morphants exhibited a consistent MCPH-like phenotype involving marked reduction in head size (figure 2a and not shown) and eye size (see electronic supplementary material, figure S2A–E), which became increasingly obvious as the development progressed from 24 hpf through to 72 hpf (figure 2d–e). Other abnormalities noted in some but not all mutant and morphant embryos included dorsal or ventral tail curvature, cardiac oedema and a reduction in overall size of the embryo (figure 2a and not shown). Examination of DAPI-stained histological sections at 24, 48, 56 and 72 hpf revealed a significant reduction in retinal size and cell number in all mutant and morphant conditions when compared with control embryos. As well as reduced retinal size, morphant and mutant embryos typically lacked the normal retinal lamination patterns apparent in wild-type embryos at 56–72 hpf suggesting possible delayed development (figure 2a). We also noted in severely disorganized retinas of mutant embryos that there were patchy areas of increased fluorescence suggestive of cell debris.Figure 2.


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 phenotypes in the zebrafish retina. Knockdown of MCPH genes causes a reduction in head size, retinal size and retinal cell number. (a) Reduction in head and eye size is demonstrated in whole-mount stilcz65−/− mutant embryos on day 5 of development. Healthy stilcz65+/? embryos are shown at the same developmental stage for comparison. While head and eye size were consistently reduced, overall body size/length of mutant embryos was variably affected. Here, one mutant (i) is smaller than the healthy embryo, whereas the other (ii) is similar in length and size to the healthy embryos. Note also the abnormally protruding lenses in the mutant embryo seen from (i), exposed owing to reduced retinal size. (b) DAPI-stained sections demonstrate reduced retinal area in stil morphants (stil Mo) at 72 hpf when compared with control morphants (CoMo). Similarly, the retinas of stilcz65−/− and stilhi1262Tg−/− mutant embryos at 72 hpf are markedly reduced in size. Labels in yellow: L, lens; R, retinal neuroepithelium; CMZ, ciliary marginal zone; ON, optic nerve; AM, apical membrane. (c) Retinal area is significantly reduced in stil morphant and mutant embryos: stil Mo 0.019 mm2 (n = 23) versus CoMo 0.024 mm2 (n = 34), p < 0.001; stilcz65−/− 0.019 mm2 (n = 23) versus stilcz65+/? 0.027 mm2 (n = 72) p < 0.001; stilhi1262Tg−/− 0.020 mm2 (n = 52) versus stilhi1262+/? 0.028 (n = 48), p < 0.001 (values are for mean area at 72 hpf). (d) Retinal cell number is reduced in stil morphants and mutants: stil Mo 471 cells (n = 23) versus CoMo 735 cells (n = 7), p < 0.001; stilcz65−/− 454 cells (n = 114) versus stilcz65+/? 780 cells (n = 72), p < 0.001; stilhi1262Tg−/− 468 cells (n = 52) versus stilhi1262+/? 744 cells (n = 48), p < 0.001 (values are for mean number of cells in central retinal sections at 72 hpf). (e) Retinal area increases as development progresses in stil, aspm, wdr62 and odf2 morphant embryos but remains reduced compared with control at all time-points examined (24, 48, 56 hpf at 72 hpf). (f) Retinal cell increases as development progresses in stil, aspm, wdr62 and odf2 morphant embryos but remains reduced compared with control at all time-points examined (24, 48, 56 hpf at 72 hpf), n = number of eyes analysed.
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Related In: Results  -  Collection

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RSOB130065F2: MCPH phenotypes in the zebrafish retina. Knockdown of MCPH genes causes a reduction in head size, retinal size and retinal cell number. (a) Reduction in head and eye size is demonstrated in whole-mount stilcz65−/− mutant embryos on day 5 of development. Healthy stilcz65+/? embryos are shown at the same developmental stage for comparison. While head and eye size were consistently reduced, overall body size/length of mutant embryos was variably affected. Here, one mutant (i) is smaller than the healthy embryo, whereas the other (ii) is similar in length and size to the healthy embryos. Note also the abnormally protruding lenses in the mutant embryo seen from (i), exposed owing to reduced retinal size. (b) DAPI-stained sections demonstrate reduced retinal area in stil morphants (stil Mo) at 72 hpf when compared with control morphants (CoMo). Similarly, the retinas of stilcz65−/− and stilhi1262Tg−/− mutant embryos at 72 hpf are markedly reduced in size. Labels in yellow: L, lens; R, retinal neuroepithelium; CMZ, ciliary marginal zone; ON, optic nerve; AM, apical membrane. (c) Retinal area is significantly reduced in stil morphant and mutant embryos: stil Mo 0.019 mm2 (n = 23) versus CoMo 0.024 mm2 (n = 34), p < 0.001; stilcz65−/− 0.019 mm2 (n = 23) versus stilcz65+/? 0.027 mm2 (n = 72) p < 0.001; stilhi1262Tg−/− 0.020 mm2 (n = 52) versus stilhi1262+/? 0.028 (n = 48), p < 0.001 (values are for mean area at 72 hpf). (d) Retinal cell number is reduced in stil morphants and mutants: stil Mo 471 cells (n = 23) versus CoMo 735 cells (n = 7), p < 0.001; stilcz65−/− 454 cells (n = 114) versus stilcz65+/? 780 cells (n = 72), p < 0.001; stilhi1262Tg−/− 468 cells (n = 52) versus stilhi1262+/? 744 cells (n = 48), p < 0.001 (values are for mean number of cells in central retinal sections at 72 hpf). (e) Retinal area increases as development progresses in stil, aspm, wdr62 and odf2 morphant embryos but remains reduced compared with control at all time-points examined (24, 48, 56 hpf at 72 hpf). (f) Retinal cell increases as development progresses in stil, aspm, wdr62 and odf2 morphant embryos but remains reduced compared with control at all time-points examined (24, 48, 56 hpf at 72 hpf), n = number of eyes analysed.
Mentions: To investigate the phenotype associated with stil knockdown, we were able to take advantage of two previously characterized loss-of-function mutants, cspcz65−/− and stilhi1262Tg−/− (see electronic supplementary material, figure S2A). We also designed antisense morpholinos against all four genes of interest (see electronic supplementary material, figure S2B–E). Both mutants and all four morphants exhibited a consistent MCPH-like phenotype involving marked reduction in head size (figure 2a and not shown) and eye size (see electronic supplementary material, figure S2A–E), which became increasingly obvious as the development progressed from 24 hpf through to 72 hpf (figure 2d–e). Other abnormalities noted in some but not all mutant and morphant embryos included dorsal or ventral tail curvature, cardiac oedema and a reduction in overall size of the embryo (figure 2a and not shown). Examination of DAPI-stained histological sections at 24, 48, 56 and 72 hpf revealed a significant reduction in retinal size and cell number in all mutant and morphant conditions when compared with control embryos. As well as reduced retinal size, morphant and mutant embryos typically lacked the normal retinal lamination patterns apparent in wild-type embryos at 56–72 hpf suggesting possible delayed development (figure 2a). We also noted in severely disorganized retinas of mutant embryos that there were patchy areas of increased fluorescence suggestive of cell debris.Figure 2.

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