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Deficient adaptation to centrosome duplication defects in neural progenitors causes microcephaly and subcortical heterotopias
José González-Martínez, … , Sagrario Ortega, Marcos Malumbres
José González-Martínez, … , Sagrario Ortega, Marcos Malumbres
Published July 8, 2021
Citation Information: JCI Insight. 2021;6(16):e146364. https://doi.org/10.1172/jci.insight.146364.
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Research Article Cell biology Development

Deficient adaptation to centrosome duplication defects in neural progenitors causes microcephaly and subcortical heterotopias

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Abstract

Congenital microcephaly (MCPH) is a neurodevelopmental disease associated with mutations in genes encoding proteins involved in centrosomal and chromosomal dynamics during mitosis. Detailed MCPH pathogenesis at the cellular level is still elusive, given the diversity of MCPH genes and lack of comparative in vivo studies. By generating a series of CRISPR/Cas9-mediated genetic KOs, we report here that — whereas defects in spindle pole proteins (ASPM, MCPH5) result in mild MCPH during development — lack of centrosome (CDK5RAP2, MCPH3) or centriole (CEP135, MCPH8) regulators induces delayed chromosome segregation and chromosomal instability in neural progenitors (NPs). Our mouse model of MCPH8 suggests that loss of CEP135 results in centriole duplication defects, TP53 activation, and cell death of NPs. Trp53 ablation in a Cep135-deficient background prevents cell death but not MCPH, and it leads to subcortical heterotopias, a malformation seen in MCPH8 patients. These results suggest that MCPH in some MCPH patients can arise from the lack of adaptation to centriole defects in NPs and may lead to architectural defects if chromosomally unstable cells are not eliminated during brain development.

Authors

José González-Martínez, Andrzej W. Cwetsch, Diego Martínez-Alonso, Luis R. López-Sainz, Jorge Almagro, Anna Melati, Jesús Gómez, Manuel Pérez-Martínez, Diego Megías, Jasminka Boskovic, Javier Gilabert-Juan, Osvaldo Graña-Castro, Alessandra Pierani, Axel Behrens, Sagrario Ortega, Marcos Malumbres

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Figure 3

Self-renewal ability and centrosome dynamics in Cep135-mutant neural progenitors.

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Self-renewal ability and centrosome dynamics in Cep135-mutant neural pro...
(A) Confocal imaging of neurospheres derived from E14.5 embryonic cortices stained with the indicated antibodies, and percentage of cyclin A+ cells with 1 or 2 centrosomes. (B) Self-renewal ability of neural progenitors as determined by limiting dilution assays. (C) Confocal imaging of cryosections of the rostral, medial, and caudal aspects of E14.5 embryonic brains with the indicated markers. Scale bar: 1 mm (left) and 100 μm (insets). Quantification of the thickness (left), SOX2+ cells (middle), and TBR2+ cells (right) in rostral (R), medial (M), and caudal (C) regions. (D) Immunofluorescence with the indicated antibodies in developing E14.5 neocortices. Control samples show typical bipolar spindles, whereas monopolar spindles (white arrowheads in middle panels), acentrosomal spindles (yellow arrowheads), and asymmetric centrosomes (arrowheads in bottom panels) are observed in Cep135-mutant samples. Scale bars: 25 μm. The histogram shows the quantification of polarity in mitotic spindles (monopolar, bipolar, or acentrosomal) in ventricular or abventricular mitoses of E14.5 neocortices. (E) Immunostaining with the indicated antibodies of anaphases in the ventricular surface of E14.5 Cep135-mutant and control embryos. Higher-magnification images of mitotic centrosomes are displayed in the bottom panels. Scale bar: 10 μm. (F) Mean fluorescence intensity (MFI; arbitrary units) of γ-tubulin at the centrosome from NPs of the indicated genotypes. ****P < 0.0001 (unpaired t test with Welsh correction). (G) Transmission electron micrographs showing representative pictures of centrioles contained in the first layer of APs of the ventricular surface. Scale bars: 200 nm. Scale bars: 10 μm (A, D, and E). Arrowheads indicate Cyclin A+ cells (A), TP53+ cells (B), or apoptotic cells (C). In A–D, data are mean ± SEM. **P < 0.01; ***P < 0.001; ****P<0.0001; 1-way ANOVA with Tukey’s multiple-comparison test (A, C, and F), χ2 (B), Student’s t test (D).

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