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. 2017 Mar 8:7:43708.
doi: 10.1038/srep43708.

Disruptions in asymmetric centrosome inheritance and WDR62-Aurora kinase B interactions in primary microcephaly

Paraskevi Sgourdou  1 , Ketu Mishra-Gorur  1 , Ichiko Saotome  1 , Octavian Henagariu  1 , Beyhan Tuysuz  2 , Cynthia Campos  1 , Keiko Ishigame  1 , Krinio Giannikou  1 , Jennifer L Quon  1 , Nenad Sestan  3 , Ahmet O Caglayan  1   4 , Murat Gunel  1   5 , Angeliki Louvi  1
Affiliations

Disruptions in asymmetric centrosome inheritance and WDR62-Aurora kinase B interactions in primary microcephaly

Paraskevi Sgourdou et al. Sci Rep. .

Abstract

Recessive mutations in WD repeat domain 62 (WDR62) cause microcephaly and a wide spectrum of severe brain malformations. Disruption of the mouse ortholog results in microcephaly underlain by reduced proliferation of neocortical progenitors during late neurogenesis, abnormalities in asymmetric centrosome inheritance leading to neuronal migration delays, and altered neuronal differentiation. Spindle pole localization of WDR62 and mitotic progression are defective in patient-derived fibroblasts, which, similar to mouse neocortical progenitors, transiently arrest at prometaphase. Expression of WDR62 is closely correlated with components of the chromosome passenger complex (CPC), a key regulator of mitosis. Wild type WDR62, but not disease-associated mutant forms, interacts with the CPC core enzyme Aurora kinase B and staining of CPC components at centromeres is altered in patient-derived fibroblasts. Our findings demonstrate critical and diverse functions of WDR62 in neocortical development and provide insight into the mechanisms by which its disruption leads to a plethora of structural abnormalities.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Microcephaly and abnormal brain cytoarchitecture in Wdr621-21/1-21 mice.
(A–L) Wdr621-21/1-21mice exhibit microcephaly. (A) Representative images of whole brains from WT and Wdr621-21/1-21 littermates at P3. (B) Brain weight is significantly decreased in Wdr621-21/1-21 pups compared with WT at P3 and P6. (C,D) Measurements of the anterior to posterior (A–P) white dotted brackets in (A) and medial to lateral (M-L, black dotted line in (A) axis length of WT and Wdr621-21/1-21 forebrains at P1, P3 and P6 show significant differences in (A–P) axis length (C) and a transient decrease in (M-L axis length at P3 (D) in Wdr621-21/1-21 forebrains compared with WT. Measurements were obtained from at least 3 and up to 9 pairs of brains per stage. (E–G) Total brain and cerebral cortical area are reduced in Wdr621-21/1-21mice. (E) Measurements were performed at six coronal levels along the A-P axis as indicated (n = 6 pairs, 6 sections per brain analyzed). (F,G) Significantly smaller brain (highlighted in F) and cerebral cortical (highlighted in G) areas in Wdr621-21/1-21 animals compared with WT. (H) Representative images of whole brains from WT and Wdr621-21/1-21 littermates at 9 weeks of age. (I–K) Reduced weight of whole brain (I) and cerebral cortex (J) and reduced length of A-P (K), but not M-L (see Figure S1), axis in Wdr621-21/1-21 mice compared with WT. (L) Decrease in total cell number in cerebral cortex and hippocampus of Wdr621-21/1-21 mice compared with WT as determined by the isotropic fractionator method (n = 4 pairs). (M–T) Wdr621-21/1-21mice exhibit abnormal cytoarchitecture in the cerebral cortex. Analyses of Nissl stained coronal brain sections at rostral (M–P) and caudal (Q–T) A-P levels at 5 weeks (M,N,Q,R), P7 (O,S) and P3 (P,T) reveals reduced thickness and abnormalities in neocortical cytoarchitecture in Wdr621-21/1-21brains compared with control (as indicated). At early postnatal stages, upper cortical layers appear condensed and deep layers are disorganized. These defects are more pronounced rostrally. In panels N–P and R–T, sections are shown in a medial (to the left) to lateral (to the right) orientation. Error bars represent s.e.m; ★p < 0.05, ★★p < 0.01, ★★★p < 0.005; NS: not significant (two-tailed Student’s t-test) Scale bar: (A,H): 1 mm; (M,Q): 0.5 mm; (P; applies to N–P and R–T): 0.1 mm.
Figure 2
Figure 2. Proliferation and cell cycle defects in Wdr621-21/1-21 neural progenitors.
(A–D) BrdU incorporation analyses. (A,C) Coronal sections of wild type and Wdr621-21/1-21 embryonic brains at E13.5 (A) and E17.5 (C) immunostained with an antibody specific to BrdU. Embryonic neocortices were analyzed following a 30-minute BrdU pulse. (B,D) Quantification of the number of BrdU+ cells at E11.5, E13.5, E15.5 (B) and E17.5 (D) reveals decreased proliferation in Wdr621-21/1-21 brains compared with wild type at late stages of neocortical development (n = 3 pairs, 3–4 sections analyzed per brain). (E,F) Detection of apoptotic cells using the TUNEL assay. Quantification of cells stained with TUNEL in coronal brain sections (E) reveals increased apoptosis (F) at E15.5 in Wdr621-21/1-21 neocortex compared with wild type (n = 3 pairs of brains, 3 sections analyzed per brain; boxes in E delineate the area of each section where cells were counted). (G–J) Analyses of cell cycle exit. Immunofluorescence staining for CldU and Ki67 at E14.5 (G) and E17.5 (I) following a 24-hour CldU pulse. (H,J) Quantification of CldU+ Ki67−/CldU+ cells indicates reduced cell cycle exit at late stages of neurogenesis (E17.5) in Wdr621-21/1-21 compared with wild type brains (n = 3–4 pairs of brains, 3–4 sections analyzed per brain). (K–P) Analyses of progenitor cell division. (K,L) Immunofluorescence staining for PH3 at E13.5 (K) and E17.5 (L). (M–P) Quantification of PH3+ cells reveals significantly more mitotic cells in the developing neocortex of Wdr621-21/1-21 embryos compared with wild type littermates at E13.5 (M). In contrast, at the end of neurogenesis, the number of mitotic cells is decreased in Wdr621-21/1-21 neocortex (N). There are no significant differences in the distribution of PH3+ cells at the ventricular surface (VS) or at a distance from it between wild type and Wdr621-21/1-21 embryonic neocortex (O). Mitotic index (PH3+ cells/total number of cells) in the VS is increased in Wdr621-21/1-21 neocortex compared with wild type at E13.5, but not significantly different at E17.5 (P) (n = 3–4 pairs, 3–4 sections analyzed per brain). Number of DAPI+ cells counted per section in wild type vs. Wdr621-21/1-21: 1,075 vs. 934 (E13.5); 3,059 vs. 3,350 (E17.5). Error bars represent s.e.m; ★p < 0.05, ★★p < 0.01, ★★★p < 0.005; NS: not significant (two-tailed Student’s t-test) Scale bar: (A,C) 20 μm; (G,I,K,L) 30 μm.
Figure 3
Figure 3. Defective centrosome inheritance in Wdr621-21/1-21 neocortex.
(A,B) Immunofluorescence staining of murine neocortical progenitors (A) (prometaphase) and human induced pluripotent stem cells (iPSCs) (B) (metaphase) with antibodies specific to WDR62 and γ-tubulin. (C) Experimental procedure to visualize centrosomes with mother centrioles of different ages. Centrosomes replicate once per cell cycle, giving rise to two centrosomes, each consisting of one mother and one daughter centriole transiently labeled via in utero electroporation with Kaede-CETN1 that is photoconvertable from green to red fluorescence. After the first replication, new centrosomes harbor one mother (red) and one daughter (green) centriole. After the second -and subsequent- cycles, centrosomes retaining the mother (red) and a newly synthesized (green) centriole appear yellow whereas those inheriting the daughter (green) and a newly synthesized centriole are green-fluorescent. (D,E) Representative images of the cortical wall of E18.5 WT (D) and Wdr621-21/1-21 (E) littermates electroporated with Keade-CETN1 at E15.5 and photoconverted at E16.5. Insets are digital magnification images of the areas delineated by dotted squares depicting centrosomes that are green-only, red-only, or yellow-fluorescent. Some cells show protein accumulation in the cytoplasm as well (See also Supplementary Figure 4). The cortical wall was divided into three equally sized bins (1: VZ/SVZ; 2: IZ; 3: cortical plate). (F,G) Quantification of the percentage of green-, yellow-, or red- (F) and of green- or yellow-fluorescent centrosomes, as a percentage of all centrosomes, within each bin (G). Wdr621-21/1-21 brains harbor significantly more green centrosomes in bin 1 and fewer in bin 3, and fewer yellow centrosomes in bin 1 and more in bin 3. (H,I) Distribution of green- and yellow centrosomes. Wdr621-21/1-21 brains harbor a higher percentage of yellow centrosomes in bin 3 (H). In bin 1, and in bins 2 and 3, they also harbor, respectively, a higher and a lower percentage of green centrosomes (I). (number of centromeres counted: 7,156 [WT] vs. 6,960 [Wdr621-21/1-21] from 6 sections per brain from three pairs of brains). Error bars represent s.e.m; ★p < 0.05, ★★p < 0.01, ★★★p < 0.005; NS: not significant (two-tailed Student’s t-test). Scale bar: (A) 12.5 μm; (B) 20 μm; (E) also applies to (D) 50 μm.
Figure 4
Figure 4. Abnormalities in neuronal differentiation in Wdr621-21/1-21 brains.
(A–I) Neuronal differentiation defects in Wdr621-21/1-21 brains revealed by in situ hybridization and immunofluorescence staining with markers of post-mitotic cortical neurons. (A) Representative images of coronal brain sections from wild type and Wdr621-21/1-21 brains processed with in situ hybridization for Satb2 at the stages indicated. Wdr621-21/1-21 brains show an expansion of Satb2 mRNA expression domain compared with wild type. (B–E) Representative immunofluorescence images of coronal sections from wild type and Wdr621-21/1-21 brains at P3 stained with an antibody specific to SATB2 (B). Quantification of SATB2+ cells within each of 10 equally sized bins in relation to the total number of SATB2+ cells demonstrates normal distribution (C). Quantification of cortical neurons that are SATB2+, in relation to the total number of cortical cells (DAPI+) within each bin, demonstrates an increase in number of SATB2+ cells in upper (bins 2–4) and deep layers (bins 8 and 9, mostly layer 6) (D). The overall fraction of SATB2+ neurons is increased in Wdr621-21/1-21 brains compared with wild type (n = 4 pairs, average number of DAPI+ cells counted per section: 5,469 [wild type] vs. 5,320 [Wdr621-21/1-21]) (E). (F–I) Representative immunofluorescence images of coronal sections from wild type and Wdr621-21/1-21 brains at P3 stained with antibodies specific to TBR1 (F) or CTIP2 (H). Quantification of the fraction of TBR1+ or CTIP2+ neurons, in relation to the total number of cortical cells (DAPI+), shows that the percentage of TBR1+ cells is increased in Wdr621-21/1-21 brains compared with wild type (n = 3 pairs, average number of DAPI+ cells counted per section: 3,341 [wild type] and 3,207 [Wdr621-21/1-21]) (G), whereas the percentage of CTIP2+ cells is decreased (I) (n = 4 pairs, average number of DAPI+ cells counted per section: 3,518 [wild type] vs. 3,504 [Wdr621-21/1-21]). Error bars represent s.e.m; ★p < 0.05, ★p < 0.01, ★★★p < 0.005; NS: not significant (two-tailed Student’s t-test). Scale bar: (A) shown on each panel): 0.5 mm; (B,E,G) 50 μm.
Figure 5
Figure 5. WDR62 is essential for mitotic cycle progression in human fibroblasts and mouse embryonic neocortical progenitors.
(A–D) Characterization of kindred NG1406 with WDR62-associated microcephaly. (A) Pedigree structure depicting a second-cousin consanguineous union. Arrow indicates the index case. (B,C) Coronal T1-weighted images demonstrate pachygyria (B) and 3D reconstruction of computed tomography scanned images show metopic synostosis (C) in the index case. (D) Chromatograms obtained via Sanger sequencing analysis of the two affected patients, their healthy sibling, and their parents, along with wild type control DNA (as indicated). Sanger sequencing confirmed exome sequencing findings of a 4-bp deletion in exon 23 of WDR62 resulting in a frameshift (D955AfsX112; designated WDR62exon23). The mutation was confirmed as being homozygous in the two affected siblings and heterozygous in their parents and was absent from the healthy sibling. (E–I) Analyses of fibroblasts from family NG1406. (E,F) Fibroblast cultures established from the heterozygous parents and the two affected siblings immunostained with antibodies specific to WDR62 and γ-tubulin demonstrate dynamic expression of WDR62 during the mitotic cycle. In heterozygous cells (E), WDR62 is detected at the spindle poles in prometaphase through telophase; in WDR62exon23homozygous cells (F), WDR62 remains diffusely distributed. (G–I) Distribution of mitotic cells in different phases of the mitotic cycle in synchronized cultures. Increased fraction of mitotic cells in prometaphase and anaphase, and decreased fraction of mitotic cells in cytokinesis are observed in WDR62exon23homozygous compared with heterozygous cultures (G). The fraction of cells undergoing mitosis is increased in homozygous cultures (H). Fewer cells proceed to metaphase following nocodazole-mediated arrest at prometaphase and subsequent release in normal culture conditions in WDR62exon23homozygous as compared with heterozygous cultures (I). (J,K) Quantification of the distribution of cells in each phase of the mitotic cycle along the ventricular surface (VS) of the developing neocortex (stained with DAPI; J) reveals more mitotic cells in prometaphase in Wdr621-21/1-21embryos compared with wild type (K). Arrows in (J) indicate mitotic cells along the VS, which are digitally magnified in the corresponding insets. Error bars represent s.e.m; ★p < 0.05, ★★p < 0.01, ★★★p < 0.005 (two-tailed Student’s t-test). Scale bar: (E,F) 5 μm; (J) 30 μm.
Figure 6
Figure 6. WDR62 interacts with AURKB and the Chromosome Passenger Complex.
(A,B) Immunofluorescence staining of fibroblasts at prometaphase with antibodies specific to WDR62, AURKB, and survivin showing partial and transient co-localization of WDR62 with each protein. (C–H) The WDR62exon23 homozygous mutation affects levels of centromeric AURKB and survivin. (C,D) Representative images of heterozygous (top) and homozygous (bottom) fibroblasts stained with AURKB (C, prophase) or survivin (metaphase, D) (red; shown in grayscale) and FITC-conjugated CREST (green; shown in grayscale). (E,F) Schematic representation of method used to quantitate AURKB (E) and survivin (F) intensity. AURKB or survivin and CREST intensity was calculated by subtracting mean fluorescence intensity from a background (B) region (E,F) small circles) in the AURKB or survivin (red) and CREST (green) channel from the mean fluorescent intensity of total AURKB, survivin or CREST in the chromatin region (dotted outline on the merged images). The AURKB/CREST or survivin/CREST ratio was calculated according to the formula shown in the figure. (G,H) Box plot graphs of AURKB/CREST (G) and survivin/CREST (H) ratios in early prophase, prophase, prometaphase and metaphase. WDR62exon23 homozygous fibroblasts show significantly decreased levels of AURKB at prophase (G) and significantly increased levels of survivin at metaphase (H). Quantification of AURKB or survivin intensity using ImageJ was normalized to the CREST control (n = 20 to 35 cells per phase per genotype, *P < 0.05 by Mann-Whitney U test). (I) Immunofluorescence staining of fibroblasts shows partial and transient localization of WDR62 (red) in prometaphase kinetochores (CREST). (J,K) MCPH-associated mutant forms of WDR62 display reduced interaction with AURKB. Co-immunoprecipitation of WT (J) and mutant WDR62 (K) with AURKB. (L) Knockdown of WDR62 and AURKB orthologs in Drosophila. Expression of Wdr62-IR with prospero-Gal4 results in dramatically reduced brain size in 3rd instar larvae; aurb-IR has no effects on brain size. Combined Wdr62-IR and aurb-IR results in formation of clusters of abnormally enlarged neuroblasts, stained with Miranda (red); nuclei are stained with DAPI (blue). Images are 3D-projection of identical Z-sections. Scale bar (A,B) 5 μm; (C,D) 50 μm; (I) 10 μm; (L) 200 μm.
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