Loss of Aspm causes increased apoptosis of developing neural cells during mouse cerebral corticogenesis

Madoka Tonosaki; Akira Fujimori; Takeshi Yaoi; Kyoko Itoh

doi:10.1371/journal.pone.0294893

. 2023 Nov 29;18(11):e0294893. doi: 10.1371/journal.pone.0294893

Loss of Aspm causes increased apoptosis of developing neural cells during mouse cerebral corticogenesis

Madoka Tonosaki ¹, Akira Fujimori ², Takeshi Yaoi ¹, Kyoko Itoh ^1,^*

Editor: Carlos Oliva³

PMCID: PMC10686469 PMID: 38019816

Abstract

Abnormal spindle-like microcephaly associated (ASPM) is a causative gene of primary autosomal recessive microcephaly. Microcephaly is considered to be a consequence of a small brain, but the associated molecular mechanisms are not fully understood. In this study, we generated brain-specific Aspm knockout mice to evaluate the fetal brain phenotype and observed cortical reduction in the late stage of murine cortical development. It has been reported that the total number of neurons is regulated by the number of neural stem and progenitor cells. In the Aspm knockout mice, no apparent change was shown in the neural progenitor cell proliferation and there was no obvious effect on the number of newly generated neurons in the developing cortex. On the other hand, the knockout mice showed a constant increase in apoptosis in the cerebral cortex from the early through the late stages of cortical development. Furthermore, apoptosis occurred in the neural progenitor cells associated with DNA damage. Overall, these results suggest that apoptosis of the neural progenitor cells is involved in the thinning of the mouse cerebral cortex, due to the loss of the Aspm gene in neocortical development.

Introduction

Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder, which is characterized by microcephaly, present at birth, and nonprogressive mental retardation. Microcephaly is the consequence of a small, but architecturally normal brain, and it is the cerebral cortex that shows the greatest size reduction. There are at least seven MCPH loci, and abnormal spindle-like microcephaly associated (ASPM) gene has been identified as the most common cause of MCPH: the MCPH5 gene [1–3]. It has been proposed that ASPM is a major determinant of cerebral cortical size among primates, including humans [4–6]. The function of the Aspm protein in neural progenitor cell expansion, as well as its localization to the mitotic spindle and midbody [7,8], suggest that it regulates brain development by a cellular division-related mechanism. In addition, the Aspm protein also causes a massive loss of germ cells, resulting in a severe reduction in testis [9] and ovary size [10], accompanied by reduced fertility.

ASPM participates in the spindle organization, spindle positioning and cytokinesis process in all dividing cells. Aspm accumulates at the spindle poles during the M phase (metaphase) and regulates cell proliferation [7,11]. During neurogenesis, the majority of neurons and glia in the mammalian neocortex arise from the division of neural progenitor cells (NPCs) in the ventricular zone of the brain. Primary NPCs have a specific pattern of mitotic activity, that is, symmetrical and asymmetrical division. Each symmetrical division increases the progenitor cell number by generating two progenitor cells per division. Asymmetric neurogenic divisions produce one neuron and one progenitor cell. In the developing mammalian cortex, the division fate of a cell appears dependent upon the orientation of the mitotic spindle and hence the position of the cleavage furrow with respect to the apical surface of the neuroepithelium. Therefore, mutations in the ASPM gene are a cause of mitotic aberrations during neurogenesis. It has been shown that the loss of Aspm causes abnormal mitosis, leading to a prolonged cell cycle [12] and increased immature neuronal differentiation associated with abnormal symmetric division [13] in the neural stem and progenitor cells in mice during cerebral cortical formation.

At present, it is unknown whether or not the loss of Aspm causes apoptosis of NPCs or neurons in cortical development. In one report, the expression of Aspm was shown to be downregulated in the ventricular zone of irradiated fetal mouse brain, as well as in irradiated neurosphere cultures [14], which suggested that the suppression of Aspm by ionizing radiation could be the initial molecular target leading to a sequential microcephaly formation. When DNA damage induced by DNA double-strand breaks is too severe to repair normally, cells will undergo apoptosis. It has been reported that, in cultured cells, the suppression of Aspm increases DNA double-strand breaks under radiation exposure [15]. However, it is unclear whether or not Aspm deficiency increases apoptosis in brain tissue in normal environments.

In the present study, we showed that the loss of Aspm causes increased apoptosis, occurring most frequently in the early stages (E12.5) in NPCs and neurons, associated with DNA damage during brain formation, using multiple indicators of apoptosis (activation of caspases (cleaved caspase-3 (CC3)-positive cells), DNA fragmentation (TUNEL-positive cells), and nuclear condensation).

Materials and methods

Animals

All of the animal experiments conducted in this study were approved by the Institutional Review Board for Biomedical Research using Animals at Kyoto Prefectural University of Medicine, and the animals were handled according to the Institutional Guidelines and Regulations. In order to obtain NesCre;Aspm^flox/+ mice, Nestin-cre (C57BL/6-Tg(Nes-Cre)1Kag, Center for Animal Resources and Development (CARD), CARD-ID 650) mice [16] were mated with Aspm^flox/flox mice [9]. Brain specific Aspm knockout mice (NesCre;Aspm^flox/flox, Aspm cKO) were generated by mating Aspm^flox/flox mice and NesCre;Aspm^flox/+ mice and detected by genotyping.

Genotyping

Mouse genotyping DNA was isolated from tail tissue followed by digestion at 95°C in 180 μL 50 mM NaOH for 10 min by heat inactivation, mixed with 20 μL 1 M Tris-HCl pH 8.0 and the supernatant was used for PCR. PCR was performed using the primer pairs to detect floxed Aspm and wild-type alleles were detected by PCR amplification using primer set 1 (Forward; 5’-CAGGAGGAGTACATTGATGAAACT-3’ and Reverse; 5’- TGTAAGGGTTTGGACTGGATATTT-3’). These primers amplify a 165 bp wildtype band and a 199 bp targeted (floxed Aspm) band. The Cre alleles were also detected by PCR amplification using the primer set 2 (Forward; 5’-TCGATGCAACGAGTGATGAG-3’ and Reverse; 5’-TCGGCTATACGTAACAGGG-3’) that amplified a 480 bp band.

The PCR conditions employed were as follows. After DNA denaturation at 94°C for 5 s, there were 38 cycles at 98°C for 5 s, followed by 55°C for 5s, and 72°C for 11 s (SapphireAmpFast PCR Master Mix (TakaraBio)).

EdU and BrdU labeling

For the pulse-chase experiments, 5-Bromo-2’-deoxyuridine (BrdU dissolved in PBS, 1 mg/mL, 500 μL/mouse) or 5-ethynyl-2’-deoxyuridine (EdU dissolved in PBS, 500 μg/mL, 500 μL/mouse) was injected intraperitoneally and chased for the number of times specified.

Histological and immunofluorescence analyses

For the preparation of the cryosections, the embryonic brains were extracted from the dams after deep anesthesia induced by intraperitoneal injection of pentobarbital solution (pentobarbitaol sodium (Kyoritsu Seiyaku, Japan)/saline, 100 mg/20 mL/1 kg body weight), fixed with 4% paraformaldehyde at 4°C for 6 hours, washed with PBS 3 times every hour and cryoprotected with 30% sucrose in PBS at 4°C overnight. The brains were then embedded in O.C.T. compound (Sakura Finetek Japan Co., Ltd.), frozen in dry ice powder and stored at -80°C. Cryosections were prepared using a cryomicrotome (LEICA CM1850, Leica, Japan) at 16 μm- (E12.5) or 10 μm-thickness (E14.5 and E16.5). Immunofluorescence staining of the cryosections was performed using the following primary antibodies; anti-Tbr1 (rabbit polyclonal, 1:500, ab31940, Abcam, Cambridge, UK), anti-Ctip2 (rat monoclonal, 1:500, ab18465, Abcam), anti-Satb2 (mouse monoclonal, 1:50, ab51502, Abcam), anti-Tbr2 (rabbit polyclonal, 1:500, ab23345, Abcam), anti-phospho-histone H3 (mouse monoclonal, 1:100, 05–598, millipore), anti-phospho-histone H3 (rabbit polyclonal, 1:100, 06–570, millipore), anti-γH2AX (mouse monoclonal, 1:100, 05–636, Millipore), anti-cleaved caspase-3 (rabbit polyclonal, 1:600, 9661S, Cell Signaling Technology, Danvers, MA, USA), anti-Tuj1 (mouse monoclonal, 1:100, 2G10, 05–559, upstate), anti-NeuN (rabbit polyclonal, 1:500, ab104225, Abcam), anti-DCX (rabbit polyclonal, 1:500, ab18723, Abcam), anti-Nestin (mouse monoclonal, 1:50, ab6142, Abcam), anti-Pax6 (rabbit polyclonal, 1:300, PRB-278P, Biolegend), anti-Sox2 (rabbit polyclonal, 1:100, AB5603, millipore), anti-Aspm (rabbit polyclonal, 1:500, del5 gift from Dr. Fujimoto [10]), and anti-γ-tubulin (mouse monoclonal, 1:1000, T6557, Sigma-Aldrich, Japan).

After three washes with PBS, the samples were incubated with Alexa488-, Alexa546- or Alexa647- conjugated anti-rabbit antibody (1:1000, Life Technologies, Japan), Alexa546- or Alexa647- conjugated anti-mouse antibody (1:1000, Life Technologies, Japan), or Alexa488 conjugated anti-rat antibody (1:1000, Life Technologies, Japan) for 1 h at room temperature. After three washes with PBS, the samples were incubated with DAPI solution (1.0 μg/mL in PBS) or YOYO1 solution (0.5 μg/mL in PBS) for 10 min at room temperature. The samples were then embedded with SlowFade (ThermoFisher, Japan).

The TUNEL reaction was carried out using the terminal deoxynucleotidyl transferase (TdT) (in situ cell death detection kit, TMR red, Roche, USA) according to the manufacturer’s instructions. For the double-staining (TUNEL with neural markers) procedures, TUNEL staining was followed by immunostaining with the antibody against anti-NeuN, Tuj1, DCX, Tbr2 or Pax6. In order to stain BrdU incorporation, sections were rinsed with PBS to rehydration for 10 min at room temperature and incubated in 2 N HCl for 30 min at 37°C to denature the DNA, then washed with PBST (0.1% Tween/PBS) for 10 min 3 times. The sections were blocked for 2 hours at room temperature with a solution of 10% normal goat serum and 0.3% Tween in PBS. The sections were stained with anti-BrdU antibody (mouse monoclonal, 1:200, Bu-33 B2531, Sigma-Aldrich, Japan). In order to detect EdU incorporation, a Click-iT EdU cell proliferation kit (ThermoFisher scientific, USA) was used. All of the images acquired were obtained with a confocal laser microscope (Olympus FV1000).

For Aspm and γTub immunostaining, Z-stacks (1 μm steps × 3, 8-bit, 1024 ×1024 resolution) were observed through a Plan-Apochromat 63×/1.40 Oil DIC M27 using a ZEISS LSM 900 inverted confocal microscope. The brightness and contrast of all of the images were adjusted, and the maximum intensity projections of the z-stack images were taken using Fiji/ImageJ, version 1.53b.

Image analysis

The images acquired with the Olympus FluoView FV1000 confocal microscope were processed with ImageJ/Fiji to adjust color and contrast. In order to conduct a quantitative analysis of the immunoreactive cells, at least three independent brain samples were analyzed at each embryonic stage (at E12.5, E14.5 and E16.5). The thickness of the cerebral cortex at E12.5, E14.5 and E16.5 and/or layer marker positive regions, which were clearly detectable with Satb2, Ctip2 and Tbr1 positive cells at E16.5 (Fig 1), was measured at three positions (left, center and right) within 250 μm-wide dorsal cortex regions, and the average thickness was calculated. In order to analyze the total number of immunoreactive cells, the cells were counted in each layer in each brain within the 250 μm-wide cortex regions, followed by calculation of the mean value obtained in three sections (Figs 2 and 3). Furthermore, measurements per genotype were calculated as the mean of at least three mice. In order to identify specific neural progenitors, apical progenitor cells (APs) were defined as the phospho-histone H3 (pHH3) positive cells located beneath the surface of the lateral ventricle, and basal progenitor cells (BPs) were defined as the pHH3 positive cells localized in the basal region of the ventricular zone (Fig 2) [17]. In order to analyze the distribution of the EdU positive cells in cerebral cortices at E16.5, the cortical layers were divided into 9 (WT) or 8 (cKO) identical bins of 100 pixels width using ImageJ (Fiji), and the number of cells in each bin was counted manually (WT: bin1-2 (ventricular zone (VZ)/subventricular zone (SVZ)), bin3-5 (intermediate zone (IZ)), bin6-9 (cortical plate (CP)); cKO: bin1-2 (VZ/SVZ), bin3-4 (IZ), and bin5-8 (CP)) (Fig 3). In order to measure the γH2AX positive cells, we used ImageJ plugin “Analyze Particles” and selected a signal size within 3.00–200.00 to remove the particles that were too small or big, and thus regarded as false positive signals. In order to analyze the distribution of TUNEL, γH2AX, CC3 positive cells and nuclear condensation, the cortical layers were divided into two (VZ, preplate (PP) at E12.5) or three layers (VZ, IZ and CP at E14.5, E16.5) and the total number was counted in each layer of the whole cortical regions, and the ratio (percentage) of the cells present in each layer was calculated. The density (cell number/area) was calculated to compare the cell density among the developmental brains.

Fig 1 — (A-C) Representative YOYO1 stained images of the embryonic cortices of the WT and Aspm cKO mice (NesCre;*Aspm*^flox/flox) at E12.5, E14.5 and E16.5. The scale bars represent 500 μm. (A’-C’) Magnification of the boxed area in (A-C). The scale bars represent 50 μm. (D) Quantification of the thickness per unit area (A’-C’) (250 μm radial columns) *: P < 0.05 (E12.5: WT 151.1 ± 2.0 μm, cKO 155.1 ± 6.5 μm, P = 0.5922, E14.5: WT 238.0 ± 13.5 μm, cKO 226.9 ± 5.8 μm, P = 0.4913, E16.5: WT 482.4 ± 7.5 μm, cKO 427.1 ± 15.5 μm, P = 0.0325, Student’s t-test, values represent mean ± SEM, n = 3 mice per genotype, ns: not significant) (E) SOX2 and DAPI stained image of E16.5 mouse cerebral cortex. The two head arrows indicate the CP, VZ and cortical wall. (F) Quantification of cortical wall and the CP region were defined based on YOYO1 staining and that of VZ was defined based on SOX2 staining. The IZ was defined as the value obtained by subtracting the CP and VZ from cortical wall. (VZ, WT 85.54 ± 2.2 μm, cKO 70.59 ± 2.1 μm, P = 0.0104; IZ, WT 229.29 ± 4.9 μm, cKO 210.55 ± 9.3 μm, P = 0.1490; CP, WT 159.51 ± 6.4 μm, cKO 140.03 ± 9.0 μm, P = 0.0908, Student’s t-test, values represent mean ± SEM, n = 3 mice per genotype) (G-I) Representative images of the WT and Aspm cKO Satb2, Ctip2 and Tbr1, respectively. Scale bar represents 50 μm. (J-L) Quantification of the cell number of the upper layer marker Satb2, the middle layer maker Ctip2 and the deep layer marker Tbr1, respectively, per unit area G-I (250μm radial columns). (J: WT 314.0 ± 23.4 cells, cKO 266.7 ± 36.2 cells, P = 0.3336, K: WT 392.7 ± 19.3 cells, cKO 327.7 ± 26.8 cells, P = 0.1205, L: WT 416.0 ± 14.7 cells, cKO 377.3 ± 17.9 cells, P = 0.1702, Student’s t-test, values represent mean ± SEM, n = 3 mice per genotype, ns: not significant).

Fig 2 — (A) Representative images of proliferative cells in the WT and Aspm cKO mice, which were positive for pHH3, in the developing cortex at E12.5, E14.5 and at E16.5. Scale bar: 50 μm. (B) The distribution of mitotic APs and BPs in the developing cortex of the WT and Aspm cKO mice. (AP, E12.5: P = 0.5659, E14.5: P = 0.9142, E16.5: P = 0.7655; BP, E12.5: P > 0.9999, E14.5: P = 0.2962, E16.5: P > 0.9999, Student’s t-test, values represent mean ± SEM, n = 3 mice per genotype) (C) Representative images of proliferative cells in developing cortex from the WT and Aspm cKO mice at E12.5, E14.5 or at E16.5 one hour after the EdU (at E12.5) or BrdU (at E14.5 or E16.5) injections. Scale bar: 50 μm. (D) The number of EdU- or BrdU-positive cells in the embryonic cortex of the WT and Aspm cKO mice. (E12.5: P = 0.9772 (WT: n = 3, cKO: n = 3), E14.5: P = 0.6676 (WT: n = 4, cKO: n = 9), E16.5: P = 0.6392 (WT: n = 7, cKO: n = 6), Student’s t-test, values represent mean ± SEM).

Fig 3 — (A, D) Co-staining images of Tbr1 (green), EdU (red) and YOYO1 (blue) in the WT and Aspm cKO mice at E14.5 (A) and E16.5 (D) cortices two days after the EdU injections (E12.5 and E14.5, respectively). Scale bar: 50 μm. (B, E) The number of EdU-positive cells per 250 μm. *P < 0.05 (B: WT 162.0 ± 6.1, cKO 169.7 ± 11.6, P = 0.5889, E: WT 228.7 ± 6.9, cKO 174.7 ± 11.4, P = 0.0154, Student’s t-test, values represent mean ± SEM, n = 3 mice per genotype) (C) Comparison of the distribution of the EdU-positive cells among the cortical layers (cortical layers × genotype interaction F(2, 12) = 0.3488 P < 0.7125; two-way ANOVA with Tukey’s multiple comparisons test; ns: not significant, values represent mean ± SEM, n = 3 mice per genotype). (F) Histogram of the EdU-positive cells, which were shown in C. (bins × genotype interaction F(8, 36) = 2.893 P = 0.0135; two-way ANOVA with Tukey’s multiple comparisons test; **P < 0.01, values represent mean ± SEM, n = 3 mice per genotype). (G) Comparison of the distribution of the EdU-positive cells among the cortical layers (cortical layers × genotype interaction F(2, 12) = 27.82 P < 0.0001; two-way ANOVA with Tukey’s multiple comparisons test; ****P < 0.0001, ns: not significant, values represent mean ± SEM, n = 3 mice per genotype).

For P7 brains (S1 Fig), a Nikon Ti Eclipse confocal microscope were used, and the images were processed with ImageJ/Fiji to adjust color and contrast. Whole brain images were obtained by tiling and stitching (6×6) the images captured with a 20× lens (2048×2048 pix). The high magnification images were cropped from the whole brain images.

Statistical analysis

Each value was obtained from at least three independent samples. All data are shown as mean ± SEM. Prism 6 (Graph-Pad Software) was used for the analysis. For comparisons between the WT and cKO, we employed the unpaired Student’s t-test. Two-way ANOVA with Tukey’s multiple comparisons test was conducted for comparison with the progenitor cell types, bins or cortical layers × genotype. The significance of the comparisons is represented on the graphs by asterisks; *P < 0.05; **P < 0.01; ***P < 0.001 and ****P < 0.0001, respectively.

Results

Cerebral cortical reduction in Aspm cKO was exhibited at the late embryonic stage

First, we generated brain specific Aspm knockout mouse (NesCre;Aspm^flox/flox, Aspm cKO) (S2 Fig) in order to examine whether the morphology of the embryonic cerebral cortex was affected by the loss of Aspm. Cortical thickness did not significantly change between the WT and cKO mice at E12.5 and E14.5 (E12.5, WT 151.1 ± 2.0 μm, cKO 155.1 ± 6.4 μm, P = 0.5922; E14.5, WT 238.0 ± 13.5 μm, cKO 227.0 ± 5.8 μm, P = 0.4913). However, a morphological difference was observed at E16.5, with a decrease in cortical thickness (WT 482.4 ± 7.5 μm, cKO 427.1 ± 15.5 μm, P = 0.0325) (Fig 1A–1D).

We further analyzed how the changes were related to the layer construction of the cerebral cortex at E16.5. First, there was a significant decrease in the thickness of the VZ in Aspm cKO (Figs 1C’, 1E and 1F and S3). Although the thickness of IZ or CP showed a slight decrease, the significance did not reach the level of significance as compared to WT (Figs 1C’, 1E and 1F and S4).

The CP is composed of six layers, and each layer consists of neurons with different birthdates. Therefore, we examined the CP in more detail using specific markers for each cortical layer. In the mouse brains lacking Aspm, the number of cells immunoreactive for the upper layer marker (Satb2-positive, II/III and IV layer), the middle layer marker (Ctip2-positive, layer V) and for the deep layer marker (Tbr1-positive, layer VI) showed a slight decrease, but no significant differences, when compared to those of the WT mice, (Fig 1G–1L), suggesting that a normal cytoarchitecture of the cerebral cortex was formed even with a loss of Aspm.

These findings suggest that Aspm is important for NPC maintenance, not for neurogenesis or differentiation from NPCs to neurons at all embryonic stages.

Loss of Aspm did not affect the proliferation of NPCs

Next, we conducted assays to determine whether or not the thinning of total cortical thickness and VZ thickness caused by Aspm deficiency is related to changes in the neural progenitor cell proliferation. In order to identify specific neural progenitors, apical progenitor cells (APs) were defined as the phospho-histone H3 (pHH3) positive cells located beneath the surface of the lateral ventricle, and basal progenitor cells (BPs) were defined as the pHH3 positive cells localized in the basal region of the ventricular zone (Fig 2) [17]. Immunofluorescence studies for pHH3, which is the mitotic marker, revealed no significant differences in the number of APs and BPs positive for pHH3 between the WT and Aspm cKO mice (Fig 2A and 2B).

Furthermore, similar to the pHH3 results, we examined the abundance of mitotic cells by thymidine analog labeling assay (EdU or BrdU). Mitotic cells in the S phase of the cell cycle were observed 1 hr after an intraperitoneal administration of EdU or BrdU in the basal VZ (Fig 2C and 2D). In both cases, there were no obvious differences in the mitosis of the NPCs between the WT and cKO mice at the early corticogenesis (at E12.5), the middle (at E14.5) or the late corticogenesis stages (at E16.5).

These results suggest that loss of Aspm does not affect the proliferation of the NPCs, but might affect NPCs pool during mouse neurogenesis.

Loss of Aspm causes reduction in mid to late born neurons

In order to determine whether the loss of Aspm affects the generation of early- or late-born neurons, we performed an EdU-birthdating assay. Pregnant mice were given a single dose of EdU at E12.5 or E14.5, and two days later (i.e., E14.5 or E16.5, respectively), the abundance of EdU-positive cells was measured. The E12.5-labeled cells showed no significant differences between the WT and cKO mice (Fig 3A and 3B). The number of EdU-labeled cells with Tbr1-immunoreactivity in the CP, as well as the number of EdU-labeled cells in the IZ and VZ showed no significant difference between the WT and Aspm cKO brains at E12.5 (Fig 3C). In contrast, the number of cells labeled at E14.5 was significantly reduced, with most of them located in the VZ/SVZ or IZ areas in the cKO mice (Fig 3D and 3E), arguing that the main cell population affected by the loss of ASPM were NPCs rather than neurons. There was a significant reduction in the number of EdU-labeled cells distributed in the IZ, but not in the VZ and CP (Fig 3F and 3G). Although the most of the EdU-labeled cells in the CP showed immunoreactivity for Satb2, late born neurons, there were no significant differences in ratio of Tbr1⁺/EdU⁺, Ctip2⁺/EdU⁺, and Satb2⁺/EdU⁺ cells in the cerebral wall between the WT and Aspm cKO brains (S5 Fig).

These findings suggested that the loss of Aspm induced a reduction in the number of mid to late born neurons without any aberrant expression of cerebral layer-specific transcription factors in the developing cortex, which might be due to affected NPCs pool.

Loss of Aspm led to an increase in apoptosis of NPCs

We conducted assays to determine whether the occurrence of apoptosis was related to the cortical thinning observed in association with the loss of Aspm (Fig 4). In the TUNEL assay for measuring apoptosis, TUNEL-positive cells were distributed in the VZ and PP at E12.5 (Fig 4A, 4B and 4J) and in the VZ, IZ and CP at E14.5 and E16.5 (Fig 4C–4F and 4J). Although the TUNEL-positive cells were significantly increased in the developing cortex in the cKO mice throughout corticogenesis (Fig 4G), postnatal brains showed no differences from WT brains (S1 Fig). The numerical density of the TUNEL-positive cells in the developing cortex (TUNEL index) decreased as the embryonic stage proceeded (Fig 4H and 4I). Furthermore, the TUNEL-index was significantly higher in the VZ at E12.5 and 14.5, but not at E16.5 in the cKO mice as compared to WT mice (Fig 4J). Thus, we found an increased number of apoptosis instances in the developing cortex obtained from Aspm cKO mice and the apoptosis was higher in the VZ at the early to the middle phase of the corticogenesis, compared with the later corticogenesis.

Fig 4 — (A-F) Representative double staining images (TUNEL and the neuronal marker Tuj1) of the WT and Aspm cKO embryonic cortices at E12.5 (A, B), E14.5 (C, D) and E16.5 (E, F). Scale bars represent 50 μm. (G-I) TUNEL index (I: *Num/Area*) is the number of TUNEL-positive cells (G: *Num*) in a cerebral cortex divided by the unit area (H: *Area*). (G: E12.5 *P =* 0.0124, E14.5 *P =* 0.0151, E16.5 *P =* 0.0117; H: E12.5 *P =* 0.1941, E14.5 *P =* 0.5675, E16.5 *P =* 0.5402; I: E12.5 P = 0.0063, E14.5 *P =* 0.0007, E16.5 *P =* 0.0010, Student’s t-test, *P < 0.05; **P < 0.01 and ***P < 0.001, values represent mean ± SEM, n = 3 mice per genotype) (J) Distribution of the TUNEL index in the developing cortex divided into the VZ, IZ, PP and/or CP. (E12.5: cortical layers × genotype interaction F(1, 8) = 5.709 P = 0.0439, E14.5: cortical layers × genotype interaction F(2, 12) = 11.96 P = 0.0014, E16.5: cortical layers × genotype interaction F(2, 12) = 2.471 P = 0.1263; two-way ANOVA with Tukey’s multiple comparisons test; *P < 0.05; **P < 0.01; ***P < 0.001 and ****P < 0.0001, ns: not significant, n = 3 mice per genotype).

In order to confirm the features of the apoptosis, we employed nuclear morphology, including nuclear condensation and DNA fragmentation, and the molecular signatures were evaluated by immunohistochemistry for CC3 (S6 Fig). The CC3-positive cells and/or nuclear condensation were significantly increased in the cKO mice at E12.5, distributed in the VZ and PP. The CC3-positive cells and/or nuclear condensation were significantly increased at E14.5 and E16.5 as well. Furthermore, a significant increase in the numerical density of CC3-positive cells (CC3 index) was revealed in the VZ at E14.5 and in the IZ at E16.5 in samples obtained from the developing cortex of the cKO mice. The numerical density of nuclear condensation (nuclear index) showed a significant increase in the VZ at E14.5 in cKO mice.

Next, we conducted tests to determine whether the apoptosis was induced in neural progenitor cells, intermediate progenitors, or neurons by the loss of Aspm (S7 Fig). TUNEL-positive cells expressed the neuronal markers NeuN, Tuj1, and DCX (S7A–S7C and S7A’–S7C’ Fig), but not the APs marker Pax6 or BPs marker Tbr2 (S7D–S7E and S7D’–S7E’ Fig). In CC3-positive cells, the expression was consistent with the neural progenitor cell marker Nestin (S7F and S7F’ Fig).

Judging from these results, we conclude that Aspm expression is required for the maintenance of neural progenitor cells and neurons and plays a critical role in during neurogenesis.

DNA-damaged cells are increased along with the loss of Aspm in developing cortex

Next, we conducted assays to determine whether DNA damage has relevance to increased apoptosis in developing cortex along with the loss of Aspm. Immunofluorescence for the DNA damage marker, γH2AX, revealed that the number of DNA-damaged cells significantly increased at E12.5 and E14.5, that is, in the early- and middle-phase of corticogenesis in cKO mice (Fig 5).

Fig 5 — (A-F) Representative immunofluorescence images (γH2AX (red) and YOYO1 (blue) of embryonic cortex in the WT and Aspm cKO mice at E12.5 (A, B), E14.5 (C, D) and E16.5 (E, F). The number of γH2AX-positive cells were counted in the developing cortex. Arrows indicate γH2AX-positive cells (red). Scale bars represent 500 μm. (G) The number of γH2AX positive cells in cerebral cortex (E12.5: WT1.00 ± 0.33 cells, cKO 3.89 ± 0.80 cells P = 0.0294, E14.5 WT 4.67 ± 0.69 cells, cKO 8.00 ± 0.39 cells P = 0.0137, E16.5: WT 3.11 ± 1.87 cells, cKO 4.67 ± 0.88 cells P = 0.4936, Student’s t-test, *P < 0.05, values represent mean ± SEM, n = 3 mice per genotype) (H) The area of cerebral certex which cell number was counted. (E12.5: WT 0.12 ± 0.0 mm², cKO 0.16 ± 0.01 mm², P = 0.0027, E14.5: WT 0.31 ± 0.04 mm², cKO 0.30 ± 0.01 mm², P = 0.8369, E16.5: WT 0.50 ± 0.04 mm², cKO 0.48 ± 0.01 mm², P = 0.5989, Student’s t-test, **P < 0.01, values represent mean ± SEM, n = 3 mice per genotype) (I) γH2AX index (Num/Area) is the number of γH2AX-positive cells (G) in a cerebral cortex divided by the unit area (H). (E12.5: WT 8.27 ± 3.04 cells/mm², cKO 23.89 ± 4.79, P = 0.0521, E14.5: WT 15.87 ± 4.56 cells/mm², cKO 26.32 ± 0.94, P = 0.0880, E16.5: WT 6.21 ± 3.93 cells/mm², cKO 9.78 ± 1.62, P = 0.4484, ns: not significant, values represent mean ± SEM, n = 3 mice per genotype) (J) Distribution of γH2AX index in the developing cortex divided into the VZ, IZ, PP and/or CP. (E12.5: cortical layers × genotype interaction F(1, 8) = 3.387 P = 0.1030, E14.5: cortical layers × genotype interaction F(2, 12) = 0.2642 P = 0.7722, E16.5: cortical layers × genotype interaction F(2, 12) = 0.4965 P = 0.6206; two-way ANOVA with Tukey’s multiple comparisons test; *P < 0.05, ns: not significant, n = 3 mice per genotype).

The numerical density of γH2AX-immunoreactive cells (γH2AX index) tended to increase in the VZ, PP, IZ and in the CP from the early to the late phases of corticogenesis in the cKO mice brains, although the difference did not reach the level of significance. This tendency is consistent with previous results regarding apoptosis. Furthermore, the double immunofluorescence of γH2AX with the neural progenitor cell marker, Pax6, revealed that the cells with DNA damage were NPCs expressing Pax6 (S8 Fig). However, Pax6 expression was not observed in cells with high nuclear condensation. This result suggests that the Pax6 protein was not detected in NPCs with strong DNA damage due to the loss of Aspm. In addition, the thickness of VZ and the number of NPCs, immunoreactive for PAX6, showed a decrease at E16.5 in cKO compared to WT (S3 Fig).

Altogether, these results suggest that the loss of Aspm causes an increase in the number of cells with DNA damage in developing murine cortex, resulting in a reduction of VZ at the later corticogenesis stage.

Discussion

In this study, we compared brain-specific Aspm-knockdown mice with normal control mice and the results provided important insights into how the loss of Aspm causes abnormal brain formation in developing cortex. These findings suggest that the microcephaly associated with MCPH5 might be aggravated by increased apoptosis in NPCs, but not by aberrant proliferation of NPCs during cortical development, although we can’t exclude that there are other ways of aberrant division such as altered ratio of self-renewal vs. neurogenic divisions.

Loss of Aspm reduced cerebral cortical thickness with a thinning of all cortical layers

The developing cerebral wall exhibited a significant reduction in thickness at E16.5 in the cKO mice, which was associated with a significant thinning of the ventricular zone and a reduction in cell numbers in all of the cortical layers (Fig 1). These findings suggest that the thinning of the cortex might be due to poor neurogenesis at mid to late embryonic stages and the reduction in VZ-thickness at E16.5 might impair further gliogenesis (Figs 1F and S3). Reduced brain size is thought to be due to a reduced number of total neurons caused by decreased proliferation of neural progenitors during fetal development [3,4]. Fish et al. [13] reported that Aspm-downregulation, using RNA interference, induced the alteration in the cleavage plane orientation in neuroepithelial cells of embryonic telencephalon, followed by a reduction in the number of neuroepithelial progenitor cells and microcephaly. In the present study, we did not find any aberrant cleavage plane orientation in the neuroepithelial cells and the mitosis of the NPCs was not affected in the cortex of the cKO mice when we focused on the S and M phase of the cell cycle (Fig 2). Therefore, it is highly likely that cell loss might occur not only in NPCs, but also in postmitotic neurons, i.e., neuroblasts in the VZ/SVZ, migrating neurons in the IZ or immature neurons after reaching the CP. These observations suggest that this cell loss could play some role in building the normal cortical architecture, except for uniform thinning, as observed in the cKO mice. It is worth noting that the EdU-birthdating assay revealed that the number of cells which were born at E14.5 was significantly reduced, with a significant reduction in IZ from the cKO mice (Fig 3), which indicated that the loss of Aspm might reduce the number of mid to later born neurons in IZ. It has been reported that the deletion of Aspm induced prematurely exit of the cell cycle and early differentiation of the cerebellar granule neuron progenitors, resulting in a decrease of terminally differentiated granule neurons in the internal granule layer [18]. Although the present analyses were conducted in regard to the cerebral cortical development related to the loss of Aspm, the reduced mid to later born neurons might have been induced by impaired mitotic progression due to an earlier exit of the cell cycle with the accelerated differentiation of NPCs.

Loss of Aspm is involved in apoptosis induction during embryonic neocortex formation, not in postnatal brain

The knockout of Aspm in NPCs increased apoptosis in all of the stages of cortical development compared to normal brains (Fig 4), but postnatal brains showed no differences from normal brains (S1 Fig). Considering reports [12,19,20] that Aspm contributes to the stabilization of symmetric divisions in mitotic NPCs, it is likely that the expression of Aspm in NPCs is indispensable for maintaining the degree of stemness which enables neuron production. We found that the frequency of apoptosis was exclusively higher in the VZ in the early to the mid corticogenesis stages, resulting in a reduction of the neural progenitor cell pool required for neurogenesis (Figs 1F and S3).

Aspm-deficient cerebellar granule neuron progenitors show impaired mitotic progression, altered patterns of division orientation and differentiation, and increased DNA damage, which causes progenitor attrition through apoptosis [18]. Similar mechanisms might be involved in the abnormal corticogenesis in our Aspm cKO.

Jayaraman et al. [21] reported that loss of Wdr62, Aspm or both impairs centriole duplication, correlating with the severity of microcephaly, and leads to a loss of centrosomes and cilia in the developing brain, and accordingly, microcephaly has been described as a ‘centriolopathy’. They showed an expansion of basal progenitors beyond the germinal zones at mid-neurogenesis, suggesting regulation of cell fate leading to precocious formation of basal progenitors rather than merely mitotic progression in Emx1-Cre; Aspm-flox. In addition, they showed little-to-no apoptosis in the Aspm-/- mice brains. We did not find any remarkable delamination of basal progenitor cells beyond the germinal zones at E14.5, using birthdate analyses (Fig 3). Furthermore, the number of apoptotic cells was significantly higher in all stages of corticogenesis in our Aspm cKO mice brains (Figs 4, 5 and S6), suggesting that the microcephaly might be followed by cell loss via apoptosis rather than proliferation and/or cell fate dysregulation. The reasons for these different results are unknown, but they might have been due to differences in the Aspm KO mice and experiment methods employed, including the ages or markers analyzed.

Johnson MB et al. [22] published an excellent study showing that Aspm KO ferrets, a gyrencephalic animal, exhibited severe microcephaly, reflecting reduced cortical surface area without any significant change in cortical thickness, as in human patients. The mutant ferret fetal cortex displayed a massive premature displacement of ventricular radial glial cells (VRG), the most undifferentiated cell type, to the outer subventricular zone (OSVZ), and a more differentiated progenitor, resulting in a loss of "cortical units". This finding is similar to that in another previous report [21]. Unfortunately, we did not find remarkable displaced progenitors, or severe microcephaly in the present Aspm cKO brains, which is most likely because of that mice, agyrencephalic animal, have few OSVG, Tbr2-immunoreactive BPs.

Aspm is required for stabilization of symmetric NPC division

It has been reported that Aspm expression and BRCA1 expression are related. For example, in experiments using cultured cancer cells, when Aspm expression was suppressed, BRCA1 expression was also decreased and the reverse has also been confirmed [23–25]. Furthermore, both BRCA1 and BRCA2, DNA damage repair factors, are important for the homologous recombination (HR)-mediated DNA damage repair pathway [26,27]. Recently, HR has been reported to be a DNA damage repair pathway that repairs DNA damage caused by symmetric division of NPC [27]. Symmetric division of NPC is a mode of division seen early in the cortical formation process [20]. Based on these observations, we propose the hypothesis that the DNA damage that occurs in the early phase of brain formation is repaired via HR by DNA damage repair molecules such as BRCA1 and BRCA2.

Since our data showed that the loss of Aspm expression results in a greater number of apoptosis instances in NPC during early cortical formation (Figs 4 and S6), it is worth speculating how Aspm can stabilize symmetric divisions in NPC, through centrosome biogenesis and/or spindle organization and positioning during mitosis. Novorol et al. reported that there was increased apoptosis in MCPH models, including Aspm knockdown zebrafish [28], which appeared to be secondary to the mitotic defects, such as abnormal prometaphase progression. In the current study, we only examined the proliferation of NPCs only during the S and M phases, but we found no abnormalities despite the loss of Aspm, and thus, it is likely that DNA-damage may induce early cell death after the metaphase or postmitotic neurons.

Conclusion

The results of this study demonstrated that the loss of Aspm is involved in increasing the number of apoptosis instances during cerebral cortical development. The increase in apoptosis occurred in both NPCs and neurons, particularly from the early stages of cortical formation, and gradually decreased in the late stages. These findings suggest that Aspm deficiency can induce apoptotic loss of NPCs　and immature neurons, leading to decreased NSCs pool and neuronal loss and ultimately to phenotypic change (i.e., cortical thinning) as found in MCPH.

Supporting information

S1 Fig. TUNEL staining of postnatal mouse brain at P7.

(A-B) representative TUNEL staining images of WT and Aspm cKO postnatal brains at P7. Scale bars represent 1 mm. (A’-B’) Higher magnification of the boxed area in A and B, respectively. Arrowheads indicated TUNEL-positive cells.

(TIF)

Click here for additional data file.^{(2.1MB, tif)}

S2 Fig. Aspm cKO and wild type mice.

This figure shows the Aspm cKO and wild type mice used in this study (see Materials and Methods). (A) The mouse Aspm gene targeting construct (B) Genotyping of the conditional Aspm knockout mouse (C) Immunostaining for Aspm (red), γTub (centrosome; green) and DNA (blue) in the NPCs of the wild type and conditional knockout (NesCre;Aspm^f/f) E14.5 mice brains.

(TIF)

Click here for additional data file.^{(856.6KB, tif)}

S3 Fig. Thickness of the ventricular zone in developing mouse cortex.

(A-C) Representative SOX2 staining images (green) of embryonic cortex in the WT and Aspm cKO mice at E12.5 (A), E14.5 (B) and E16.5 (C). (D, E) Quantification of the thickness of the ventricular zone and the cell number immunoreactive for SOX2 at E12.5, E14.5 and E16.5. Arrows indicate the thickness measurements. *: P < 0.05, ns: not significant (D: E12.5: P = 0.6180, E14.5: P = 0.8887, E16.5: P = 0.0104, D: E12.5: P = 0.2012, E14.5: P = 0.4038, E16.5: P = 0.0842, Student’s t-test, values represent mean ± SEM, n = 3 mice per genotype).

(TIF)

Click here for additional data file.^{(467.2KB, tif)}

S4 Fig. Cortical layer thickness in developing mouse cortex.

(A-C) Representative immunofluorescence images (Tbr1 (green) and YOYO1 (blue)) of embryonic cortex in the WT and Aspm cKO mice at E12.5 (A), E14.5 (B) and E16.5 (C). (D, E) Quantification of the thickness of the cortical wall and cortical layer (PP at E12.5 or CP at E14.5 and E16.5). Arrows indicate the thickness measurements. *: P < 0.05, ns: not significant (D: E12.5: P = 0.5922, E14.5: P = 0.4913, E16.5: P = 0.0325, E: E12.5: P = 0.2291, E14.5: P = 0.1897, E16.5: P = 0.0906, Student’s t-test, values represent mean ± SEM, n = 3 mice per genotype).

(TIF)

Click here for additional data file.^{(912.5KB, tif)}

S5 Fig. Identification of EdU labeled cells by neural marker.

(A-C) Representative images of EdU birthdating assay in WT E16.5 cortices two days after EdU injections (E14.5). Scale bar: 50 μm. (D-F) Higher magnification of the boxed area in A-C. Scale bar: 50 μm. Yellow-filled arrowheads indicated Tbr1⁺/EdU⁺, Ctip2⁺/EdU⁺ or Satb2⁺/EdU⁺ double positive cells, respectively and white-open arrowheads indicated EdU single positive cells. (G-I) Quantitative assessment of cell type of EdU positive cells at E16.5 two days after EdU labeling at E14.5. (G: WT n = 492 cells, 8.7 ± 2.0%, cKO n = 408 cells, 9.9 ± 3.5%, P = 0.7606, H: WT n = 592 cells, 3.4 ± 1.8%, cKO n = 451 cells, 3.4 ±1.8%, P = 0.3744, I: WT n = 458 cells, 76.6 ± 12.8%, cKO n = 460 cells, 86.9 ± 6.0%, P = 0.5079. Student’s t-test, values represent mean ± SEM, n = 3 per genotype).

(TIF)

Click here for additional data file.^{(2MB, tif)}

S6 Fig. Immunohistochemistry for cleaved caspase 3 and nuclear condensation.

(A-F) Representative immunohistochemistry images (cleaved caspase 3 (CC3) and nuclear condensation of the embryonic cortex at E12.5 (A, B), E14.5 (C, D) and E16.5 (E, F) in the WT and Aspm cKO mice. The number of CC3-positive cells and/or nuclear condensation were counted in developing cortex, separated into the ventricular zone (VZ), the preplate (PP), the intermediate zone (IZ), and the cortical pate (CP). The scale bars represent 100 μm. (A’-F’) Higher magnification of the boxed area in (A-F). Arrows indicated condensed nuclei, which were shown as YOYO1 higher intense singals. The scale bars represent 50 μm. (G, H) CC3 index (H: Num/Area) is the number of CC3-positive cells (G: Num) in a cerebral cortex section divided by the unit area (Area). (G: E12.5 P = 0.0475, E14.5 P = 0.0075, E16.5 P = 0.0015; H: E12.5 P = 0.0055, E14.5 P = 0.0076, E16.5 P = 0.0045, Student’s t-test, *P < 0.05; **P < 0.01, values represent mean ± SEM, n = 3 mice per genotype). (I) Distribution of the CC3 index in the developing cortex divided into the VZ, IZ, PP and/or CP (E12.5: cortical layers × genotype interaction F(1, 8) = 0.8502 P = 0.3835, E14.5: cortical layers × genotype interaction F(2, 12) = 1.437 P = 0.2757, E16.5: cortical layers × genotype interaction F(2, 12) = 2.748 P = 0.1041; two-way ANOVA with Tukey’s multiple comparisons test; *P < 0.05; **P < 0.01, ns: not significant, n = 3 mice per genotype) (J, K) Nuclear index (K: Num/Area) is the number of nuclear condensation instances (J: Num) in a cerebral cortex section divided by the unit area (Area). (J: E12.5 P = 0.0380 E14.5 P = 0.0405, E16.5 P = 0.0036; K: E12.5 P = 0.0051, E14.5 P = 0.0693, E16.5 P = 0.0016, Student’s t-test, *P < 0.05; **P < 0.01, values represent mean ± SEM, n = 3 mice per genotype). (L) Distribution of Nuclear index in the developing cortex divided into the VZ, IZ, PP and/or CP (E12.5: cortical layers × genotype interaction F(1, 8) = 2.250 P = 0.1720, E14.5: cortical layers × genotype interaction F(2, 12) = 16.77 P = 0.0003, E16.5: cortical layers × genotype interaction F(2, 12) = 2.384 P = 0.1343; two-way ANOVA with Tukey’s multiple comparisons test; *P < 0.05; **P < 0.01; ***P < 0.001 and ****P < 0.0001, ns: not significant, n = 3 mice per genotype).

(TIF)

Click here for additional data file.^{(2.4MB, tif)}

S7 Fig. TUNEL staining and neuronal makers of the Aspm cKO mice.

Representative double immunofluorescence images of TUNEL and cell type-specific markers (NeuN (A, A’), TuJ1 (B, B’), DCX (C, C’), Tbr2 (D, D’) or Pax6 (E, E’)) in the embryonic cortex of Aspm cKO mice at E14.5. Scale bar: 100 μm (see Materials and Methods). Most of the TUNEL-positive cells were immunoreactive for neuronal markers NeuN (A’) and Tuj1 (B’) (filled arrowhead) and immature neuronal marker DCX (C’). In contrast, no double labeling was detected for the TUNEL or the intermediate progenitor cell marker Tbr2 (D’) or neural progenitor cell marker Pax6 (E’) (open arrowheads). Representative double immunofluorescence images of cleaved caspase 3 (CC3) and neural progenitor cell marker (Nestin (F, F’)) in the embryonic cortex of Aspm cKO mice at E14.5, in which the filled arrowhead indicated Nestin/CC3 double positive cells. Scale bar: 10 μm.

(TIF)

Click here for additional data file.^{(3MB, tif)}

S8 Fig. DNA damages occurred in NPCs of Aspm cKO.

(A) Representative double immunofluorescence images of the DNA damage marker (γH2AX) and the neural progenitor cell marker (Pax6) in the murine embryonic cortex of Aspm cKO mice at E14.5. (B-E) Higher magnification of the boxed area in (A). In γH2AX-positive cells, Pax6 was positive in NPCs with weak nuclear condensation (filled arrowheads), although Pax6 was negative in cells with a high nuclear condensation (open arrowheads). Scale bar: 100 μm (A), 10 μm (B-E).

(TIF)

Click here for additional data file.^{(2MB, tif)}

S1 Raw images. Raw images of genotyping result (S2 Fig).

This is an original full-size image of an agarose gel in which the genotyping PCR products from the transgenic individuals have been size fractionated (3% agarose/TAE, 100V, 20min). Each lane corresponds to one individual. PCR products to detect Aspm^flox and Cre were loaded in the top and bottom lanes, respectively. f and + indicate floxed and wild-type alleles, respectively. In the bottom lanes, + and—indicate the presence or absence of the Cre cassette. S2B Fig shows the cropped image of a set of lanes labeled WT and cKO.

(PDF)

Click here for additional data file.^{(10.5MB, pdf)}

Data Availability

Funding Statement

This work was supported by JSPS KAKENHI Grant Numbers JP25293240 (Kyoko Itoh), JP16K19689 and JP18K15683 (Madoka Tonosaki). There was no additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1.Roberts E, Hampshire DJ, Pattison L, Springell K, Jafri H, Corry P, et al. Autosomal recessive primary microcephaly: an analysis of locus heterogeneity and phenotypic variation. J Med Genet. 2002;39: 718–721. doi: 10.1136/jmg.39.10.718 [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Nicholas AK, Swanson EA, Cox JJ, Karbani G, Malik S, Springell K, et al. The molecular landscape of ASPM mutations in primary microcephaly. J Med Genet. 2009;46: 249–253. doi: 10.1136/jmg.2008.062380 [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Thornton GK, Woods CG. Primary microcephaly: do all roads lead to Rome? Trends Genet. 2009;25: 501–510. doi: 10.1016/j.tig.2009.09.011 [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Bond J, Roberts E, Mochida GH, Hampshire DJ, Scott S, Askham JM, et al. ASPM is a major determinant of cerebral cortical size. Nat Genet. 2002;32: 316–320. doi: 10.1038/ng995 [DOI] [PubMed] [Google Scholar]
5.Passemard S, Verloes A, Billette de Villemeur T, Boespflug-Tanguy O, Hernandez K, Laurent M, et al. Abnormal spindle-like microcephaly-associated (ASPM) mutations strongly disrupt neocortical structure but spare the hippocampus and long-term memory. Cortex. 2016;74: 158–176. doi: 10.1016/j.cortex.2015.10.010 [DOI] [PubMed] [Google Scholar]
6.Desir J, Cassart M, David P, Van Bogaert P, Abramowicz M. Primary microcephaly with ASPM mutation shows simplified cortical gyration with antero-posterior gradient pre- and post-natally. Am J Med Genet A. 2008;146A: 1439–1443. doi: 10.1002/ajmg.a.32312 [DOI] [PubMed] [Google Scholar]
7.Higgins J, Midgley C, Bergh A, Bell SM, Askham JM, Roberts E, et al. Human ASPM participates in spindle organisation, spindle orientation and cytokinesis. BMC Cell Biol. 2010;11. doi: 10.1186/1471-2121-11-85 [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Paramasivam M, Chang Y, LoTurco JJ. ASPM and citron kinase co-localize to the midbody ring during cytokinesis. Cell Cycle. 2007;6: 1605–1612. doi: 10.4161/cc.6.13.4356 [DOI] [PubMed] [Google Scholar]
9.Fujimori A, Itoh K, Goto S, Hirakawa H, Wang B, Kokubo T, et al. Disruption of Aspm causes microcephaly with abnormal neuronal differentiation. Brain Dev. 2014;36: 661–669. doi: 10.1016/j.braindev.2013.10.006 [DOI] [PubMed] [Google Scholar]
10.Mori M, Tando S, Ogi H, Tonosaki M, Yaoi T, Fujimori A, et al. Loss of abnormal spindle-like, microcephaly-associated (Aspm) disrupts female folliculogenesis in mice during maturation and aging. Reprod Biol. 2022;22: 100673. doi: 10.1016/j.repbio.2022.100673 [DOI] [PubMed] [Google Scholar]
11.Kouprina N, Pavlicek A, Collins NK, Nakano M, Noskov VN, Ohzeki JI, et al. The microcephaly ASPM gene is expressed in proliferating tissues and encodes for a mitotic spindle protein. Hum Mol Genet. 2005;14: 2155–2165. doi: 10.1093/hmg/ddi220 [DOI] [PubMed] [Google Scholar]
12.Capecchi MR, Pozner A. ASPM regulates symmetric stem cell division by tuning Cyclin e ubiquitination. Nat Commun. 2015;6: 8763. doi: 10.1038/ncomms9763 [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Fish JL, Kosodo Y, Enard W, Paabo S, Huttner WB. Aspm specifically maintains symmetric proliferative divisions of neuroepithelial cells. Proc Natl Acad Sci. 2006;103: 10438–10443. doi: 10.1073/pnas.0604066103 [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Fujimori A, Yaoi T, Ogi H, Wang B, Suetomi K, Sekine E, et al. Ionizing radiation downregulates ASPM, a gene responsible for microcephaly in humans. Biochem Biophys Res Commun. 2008;369: 953–957. doi: 10.1016/j.bbrc.2008.02.149 [DOI] [PubMed] [Google Scholar]
15.Kato TA, Okayasu R, Jeggo PA, Fujimori A. ASPM influences DNA double-strand break repair and represents a potential target for radiotherapy. Int J Radiat Biol. 2011;87: 1189–1195. doi: 10.3109/09553002.2011.624152 [DOI] [PubMed] [Google Scholar]
16.Isaka F, Ishibashi M, Taki W, Hashimoto N, Nakanishi S, Kageyama R. Ectopic expression of the bHLH gene Math1 disturbs neural development. Eur J Neurosci. 1999;11: 2582–2588. doi: 10.1046/j.1460-9568.1999.00699.x [DOI] [PubMed] [Google Scholar]
17.Arai Y, Pulvers JN, Haffner C, Schilling B, Nüsslein I, Calegari F, et al. Neural stem and progenitor cells shorten S-phase on commitment to neuron production. Nat Commun. 2011;2: 154. doi: 10.1038/ncomms1155 [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Williams SE, Garcia I, Crowther AJ, Li S, Stewart A, Liu H, et al. Aspm sustains postnatal cerebellar neurogenesis and medulloblastoma growth in mice. Development. 2015;142: 3921–3932. doi: 10.1242/dev.124271 [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Konno D, Shioi G, Shitamukai A, Mori A, Kiyonari H, Miyata T, et al. Neuroepithelial progenitors undergo LGN-dependent planar divisions to maintain self-renewability during mammalian neurogenesis. Nat Cell Biol. 2008;10: 93–101. doi: 10.1038/ncb1673 [DOI] [PubMed] [Google Scholar]
20.Paridaen JT, Huttner WB. Neurogenesis during development of the vertebrate central nervous system. EMBO Reports. 2014. pp. 351–364. doi: 10.1002/embr.201438447 [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Jayaraman D, Kodani A, Gonzalez DM, Mancias JD, Mochida GH, Vagnoni C, et al. Microcephaly Proteins Wdr62 and Aspm Define a Mother Centriole Complex Regulating Centriole Biogenesis, Apical Complex, and Cell Fate. Neuron. 2016;92: 813–828. doi: 10.1016/j.neuron.2016.09.056 [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Johnson MB, Sun X, Kodani A, Borges-monroy R, Girskis KM, Patel K, et al. Aspm knockout ferret reveals an evolutionary mechanism governing cerebral cortical size. Nature. 2018;556: 370–375. doi: 10.1038/s41586-018-0035-0 [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Bae I, Rih JK, Kim HJ, Kang HJ, Hassan B, Kirilyuk A, et al. BRCA1 Regulates Gene Expression for Orderly Mitotic Progression. Cell Cycle. 2005;4: 1641–1666. doi: 10.4161/cc.4.11.2152 [DOI] [PubMed] [Google Scholar]
24.Zhong X, Liu L, Zhao A, Pfeifer GP, Xu X. The abnormal spindle-like, microcephaly-associated (ASPM) gene encodes a centrosomal protein. Cell Cycle. 2005;4: 1227–1229. doi: 10.4161/cc.4.9.2029 [DOI] [PubMed] [Google Scholar]
25.Xu S, Wu X, Wang P, Cao SL, Peng B, Xu X. ASPM promotes homologous recombination-mediated DNA repair by safeguarding BRCA1 stability. iScience. 2021;24: 102534. doi: 10.1016/j.isci.2021.102534 [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Isono M, Niimi A, Oike T, Hagiwara Y, Sato H, Sekine R, et al. BRCA1 Directs the Repair Pathway to Homologous Recombination by Promoting 53BP1 Dephosphorylation. Cell Rep. 2017;18: 520–532. doi: 10.1016/j.celrep.2016.12.042 [DOI] [PubMed] [Google Scholar]
27.Keil JM, Doyle DZ, Qalieh A, Lam MM, Funk OH, Qalieh Y, et al. Symmetric neural progenitor divisions require chromatin-mediated homologous recombination DNA repair by Ino80. Nat Commun. 2020;11: 3839. doi: 10.1038/s41467-020-17551-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Novorol C, Burkhardt J, Wood KJ, Iqbal A, Roque C, Coutts N, et al. Microcephaly models in the developing zebrafish retinal neuroepithelium point to an underlying defect in metaphase progression. Open Biol. 2013;3: 130065. doi: 10.1098/rsob.130065 [DOI] [PMC free article] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0294893.r001

Decision Letter 0

Carlos Oliva

16 Apr 2023

PONE-D-23-02973Loss of Aspm causes increased apoptosis of developing neuronal cells during mouse cerebral corticogenesisPLOS ONE

Dear Dr. Itoh,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Both reviewers agree that there are several technical issues and problems with data interpretation that need to be addressed, before the article is suited for publication. Please provide a detailed response to reviewers comments in your next submission

Please submit your revised manuscript by May 31 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Carlos Oliva, PhD

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering.

3. Thank you for stating in your Funding Statement:

"This work was supported by JSPS KAKENHI Grant Numbers JP25293240 (Kyoko Itoh), JP16K19689 and JP18K15683 (Madoka Tonosaki)."

Please provide an amended statement that declares *all* the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now. Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement.

Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf.

4. Thank you for stating the following financial disclosure:

"This work was supported by JSPS KAKENHI Grant Numbers JP25293240 (Kyoko Itoh), JP16K19689 and JP18K15683 (Madoka Tonosaki)."

Please state what role the funders took in the study. If the funders had no role, please state: ""The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.""

If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

5. Thank you for stating the following in your Competing Interests section:

"The authors hereby declare no conflict of interest associated with this study."

Please complete your Competing Interests on the online submission form to state any Competing Interests. If you have no competing interests, please state ""The authors have declared that no competing interests exist."", as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now

This information should be included in your cover letter; we will change the online submission form on your behalf.

6. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

7. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

8. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript by Tonosaki et al., entitled "Loss of Aspm causes increased apoptosis of developing neuronal cells during mouse cerebral corticogenesis" described the role of ASPM in embryonic murine brain cortical development, suggesting that the loss of ASPM reduced cortical thickness by increased apoptosis of neural precursor cells and postmitotic neurons. Although most of the experiments suggested this, there are some data interpretation inconsistencies. For example, the authors mentioned that the reduced cortical thickness is due to early neurogenesis since the Tbr1-associated layer was the only one reduced. However, they do not show changes in cortical thickness at early neurogenesis, only at late neurogenesis. Furthermore, as the Tbr1 neurons are the first to be generated, how can they see cortical defects only at E16.5 and not at E12.5 or E13.5? Or, if the changes are only seen at E16.5, why are there no changes in Satb2 neurons? Also, the authors did not cite or discuss important published articles such as Johnson et al., 2018. Nature or Williams et al., 2015. Development, where they mentioned that loss of Aspm is also associated with apoptosis and important for brain development.

Comments:

1. Fig. 1E does not explain how VZ/, IZ, or CP was defined.

2. In Fig. 1F-K it is not explained how the authors defined the areas measured. For example, for SATB2, several cells spread across the cortex are SATB2 positive. Then, what parameter is used by the authors to establish the right quantification area? Quantifying the number of SATB2, CTIP2 and TBR1 positive cells would be more appropriate. Also, the authors did not state the age of the samples analyzed. If the samples are embryonic, how can they define specific cortical layers since they have not been adequately established yet?

3. The authors mentioned, "Since the deep layer neurons are composed of early-born neurons, these findings suggest that the thinning of the cortex in late cortical formation might be due to poor neurogenesis at early embryonal stages". If only early neurogenesis is affected, how later is neurogenesis not? Depleting NPCs at early stages should also affect later neurogenesis as the NPC pool is reduced. Also, it is possible that the loss of ASPM only affects direct neurogenesis but not indirect neurogenesis. However, the authors did not test this possibility.

4. The authors mentioned that "Immunofluorescence studies for pHH3, which is the mitotic marker, revealed no significant differences in the abundance of apical progenitor cells (APs) and basal progenitor cells (BPs) between the WT and Aspm cKO mice (Figs 2A and 2B)". How did the authors determine apical or basal progenitors? The authors assume the nature of these progenitors based on their spatial location. However, it is highly recommended that these experiments be performed using proper cell markers such as Pax6 or Tbr2 and colocalization studies.

5. In Fig. 2C-D, it is unclear whether the absence of proliferative disruption is specific to a particular progenitor population since the authors did not use any cell-specific marker. Therefore, it is highly recommended that these experiments be performed using proper cell markers such as Pax6 or Tbr2 since the authors state that only early neurogenesis is affected.

6. The authors mentioned, "Pregnant mice were given a single dose of EdU at E12.5 or E14.5, and two days later (i.e., E14.5 or E16.5, respectively), the abundance of EdU-positive neurons was measured. Most of the cells, EdU-labeled at E12.5, were observed in the superficial area corresponding to the CP, and EdU-positive cells were found in postmitotic neurons showing Tbr1-immunoreactivity". However, TBR1 immunostaining was not performed. So then, how can the authors ensure that EdU staining corresponds to postmitotic neurons? Also, the authors made similar conclusions on Fig. 3C-F but did not perform immunostainings for specific neuronal populations such as Satb2, Ctip2 or Tbr1.

7. The authors do not have data to support the following statement: "These findings suggest that the loss of Aspm induced a reduction in the number of mid-born neurons under radial migration" since they did not perform neuronal migration experiments.

8. Fig. 4A-H are not cited in the text.

9. In Figure 5, it is unclear why the authors show the immunostainings in low magnification. Also, it is suggested to show gammaH2AX and YOYO1 in separate channels.

10. In the discussion section, the authors state, "The knockout of Aspm in neural progenitor cells increased apoptosis in all stages of cortical development compared to normal brains (Fig 4), but postnatal brains showed no differences from normal brains (data not shown)". Therefore, it is highly recommended that the authors include the postnatal data since it is an essential part of their discussion.

11. It is strongly suggested to add the supplemental material to the manuscript.

Reviewer #2: The manuscript by Tonosaki et al. provides evidences that Aspm (abnormal spindle-like microcephaly associated) is necessary to prevent excessive apoptosis in the developing brain cortex. Mutations in ASPM are among the most common causes of primary microcephaly in humans, and previous studies have focused on the analyzing mechanisms of Aspm in regulating cerebral cortical size. This manuscript originally shows that the elimination of Aspm in the developing telencephalon triggers an increase in cell death, suggesting a different (or additional) function from those previously described. The data presented supports the conclusion to a certain extent, and several major points should be addressed.

1. The Nes-Cre transgenic line used for generating cKOs is not described (information is missing in Methods). It is important to validate the genetic approach. The most widely used Nes-Cre Tg mouse (Tronche et al. Nat Genet 1999; JAX #003771) is known to be insufficient for directing recombination in early embryonic neural cortical progenitors (Liang, et al Biol Open 1:1200-3, 2012 and others), being Nes-Cre inactive in VZ or SVZ cells at E12.5 or even at E14.5. Assessing when Aspm deletion takes place is central because it might change conclusions of the study. For example, the statements “microcephaly associated with MCPH5 is not due to aberrant proliferation of neural progenitor cells during cortical development” (line 337, 207, others), “cerebral cortical reduction in Aspm cKO was exhibited at the late embryonic stage” (173, abstract), as well as discrepancies with previous reports, might arise from the inefficacy of deleting Aspm in VZ progenitors with the genetic approach used

Which was the Nes-Cre Tg line used? The efficacy of Cre recombination in the developing cortex should be tested (with conditional reporters) or cite the corresponding validations.

The downregulation of Aspm, at mRNA or protein levels, should be assessed in the Nes-Cre Aspm-flox/flox used in this study.

2. Some of the major findings should be validated with a complementary analysis in Aspm full knock out embryos (previously used by the authors of this manuscript, Fujimori et al 2014) or in Emx1-Cre; Aspm-flox (Jayaraman et al 2016).

3. Findings and differences with published work (Fish 2006, Jayaraman 2016) should be compared and discussed, taking into consideration animal models used, different ages or markers tested. As an example, upper layers thickness by P10 showed in cited works, is not evident here.

4. The authors propose a reduction in Tbr1+ thickness (line 190, Fig 1H-K). This would benefit from determining differences in cell numbers instead of thickness, in order to assert whether neurogenesis is affected, and considering the effect on thickness is mild.

Importantly, this reduction is not consistent with later results (Fig.3) where birth dating experiments at E12.5 do not show deep layer differences between WT and cKO.

5. In EdU/BrdU labeling experiments (Fig 3) it would be useful to also test for differences in number and distribution of EdU cells at a later timepoint (P5-10) when neurons have completed migration. The reduction in EdU cell numbers seen in the IZ but not CP, which suggests they are later born, would also be supported by Tbr1 co-staining. Authors should also consider adding an EdU pulse at E16.5 to solidly conclude which neurons are affected.

Additionally, in Fig 3 E-F, no EdU + cells are shown in quantification in bins 1-2 (or VZ/SVZ), but they are clearly evident in the images. Distinguishing between EdU cells remaining in VZ or migrating into IZ (with short chase times), coupled with the use of specific markers could help determine whether there is changes in neurogenic divisions. For this point, authors should consider similarities and/or differences with published work.

Minor

6. Examples of DNA damage in both Pax6+ and Pax6- cells are shown in Fig Suppl 4. Since numbers were not quantified, lines 314-315 should be revised. Quantifications of both cell type specific death and DNA damage would increase the robustness of the results.

7. It would be ideal to show the actual data points in the graphs together with mean +/- SEM.

8. Please, rephrase “..observed cortical reduction in the late neurogenesis of murine cortical development” Abstract, line 27.

9. Line 59: mutations in ASPM are a cause (not consequence) of mitotic aberrations during neurogenesis.

10. References for mouse lines are missing in Methods section.

11. Please indicate ages in Figures 1 F-H, 1E, 3 A-F where they are missing so that is easily interpreted.

12. Label the stainings close/within the panels in all figures. They are missing in Figs. 2, 3A-C, 5A-F and Fig.S2 A-F.

13. Line 339 should be modified. The authors show that there is no excessive or decreased proliferation but can’t exclude that there are other ways of aberrant division such as altered ratio of self-renewal vs. neurogenic divisions.

14. Please avoid the use of “cortical areas” as it may get confused with functional areas of the cortex (line 270). I suggest to remove the word “layers” (line 32, abstract)

15. I think it is more common to use embryonic instead of embryonal (176, 190 and others)

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2023 Nov 29;18(11):e0294893. doi: 10.1371/journal.pone.0294893.r002

Author response to Decision Letter 0

8 Jul 2023

Response to reviewer #1’ comments

First of all, thank the reviewer #1 for reviewing our manuscript and for the valuable comments. We have tried to reply all the comments from the reviewer #1 and the details of the changes made are described as follows.

Major Comments:

Although most of the experiments suggested this, there are some data interpretation inconsistencies. For example, the authors mentioned that the reduced cortical thickness is due to early neurogenesis since the Tbr1-associated layer was the only one reduced. However, they do not show changes in cortical thickness at early neurogenesis, only at late neurogenesis.

Furthermore, as the Tbr1 neurons are the first to be generated, how can they see cortical defects only at E16.5 and not at E12.5 or E13.5? Or, if the changes are only seen at E16.5, why are there no changes in Satb2 neurons?

I would greatly appreciate your important suggestions.

According to your suggestions, we prepared the supplemental figure (S6 Fig), which included the results about cortical thickness at E12.5, E14.5 and E16.5. Because the cortical layer at E12.5 was before the formation of the cortical plate, we evaluated it as preplate thickness, which showed immunoreactivity for Tbr1.

In terms of the cortical wall, which is total thickness of cerebral cortex, there was a significant difference at late state of murine brain development. However, the cortical thickness, which is the preplate or the cortical plate, did not show the significant difference at all ages. This result revealed that early cortical thickness did not change. Then we have thought that it was due to the loss of neurons or the reduction in generating neurons.

Page 9, line 196

We further analyzed how the changes were related to the layer construction of the cerebral cortex at E16.5. First, there was a significant decrease in the thickness of the VZ in Aspm cKO (Figs 1C’, 1E and 1F, and S8 Fig). Although the thickness of IZ or CP showed a slight decrease, the significance did not reach the level of significance as compared to WT (Figs 1C’, 1E and 1F, and S6 Fig).

The CP is composed of six layers, and each layer consists of neurons with different birthdates. Therefore, we examined the CP in more detail using specific markers for each cortical layer. In the mouse brains lacking Aspm, the number of cells immunoreactive for the upper layer marker (Satb2-positive, II/III and IV layer), the middle layer marker (Ctip2-positive, layer V) and for the deep layer marker (Tbr1-positive, layer VI) showed a slight decrease, but no significant differences, when compared to those of the WT mice, (Figs 1G-1L), suggesting that a normal cytoarchitecture of the cerebral cortex was formed even with a loss of Aspm.

These findings suggest that the thinning of the cortex in late cortical formation might be due to poor neurogenesis or an excess loss of neurons at all embryonal stages.　

I would greatly appreciate your important suggestions.

As mentioned above and in the Fig 1D, the thickness of cortical wall showed no significant difference until E14.5, and reduction in thickness was observed at E16.5. In addition, we showed that there was no difference in the number of Ctip2, Satb2 and Tbr1-positive neurons at E16.5 (Fig 1G-L).

Thus, we thought that the excess loss of neurons might continuously occur at all embryonal stages. We have demonstrated the significant increase in apoptosis in the developing cerebral cortex at all embryonal stages, however; apoptosis in the VZ was higher at E12.5 and E14,5 than at E16.5 as shown in the Fig 4, it was possible that loss of the neuronal progenitor cells might affect progression of corticogenesis.

Page 10, line 231

Loss of Aspm led to an increase in apoptosis of both NPCs and postmitotic neurons

We conducted assays to determine whether the occurrence of apoptosis was related to the cortical thinning observed in association with the loss of Aspm (Fig 4). In the TUNEL assay for measuring apoptosis, TUNEL-positive cells were distributed in the VZ and PP at E12.5 (Figs 4A, 4B and 4J) and in the VZ, IZ and CP at E14.5 and E16.5 (Figs 4C-4F and 4J). The TUNEL-positive cells were significantly increased in the developing cortex in the cKO mice throughout corticogenesis (Fig 4G). The numerical density of the TUNEL-positive cells in the developing cortex areas (TUNEL index) decreased as the embryonal stage proceeded (Figs 4H and 4I). Furthermore, the TUNEL-index was significantly higher in the VZ at E12.5 and 14.5, but not at E16.5 in the cKO mice as compared to WT mice (Fig 4J). Thus, we found an increased number of apoptosis instances in the developing cortex obtained from Aspm cKO mice and the apoptosis was higher in the VZ at the early to the middle phase of the corticogenesis, compared with the later corticogenesis.

In order to confirm the features of the apoptosis, we employed nuclear morphology, including nuclear condensation and DNA fragmentation, and the molecular signatures were evaluated by immunohistochemistry for CC3 (S2 Fig). The CC3-positive cells and/or nuclear condensation were significantly increased in the cKO mice at E12.5, distributed in the VZ and PP. The CC3-positive cells and/or nuclear condensation were significantly increased at E14.5 and E16.5 as well. Furthermore, a significant increase in the numerical density of CC3-positive cells (CC3 index) was revealed in the VZ at E14.5 and in the IZ at E16.5 in samples obtained from the developing cortex of the cKO mice. The numerical density of nuclear condensation (nuclear index) showed a significant increase in the VZ at E14.5 in cKO mice.

Next, we conducted tests to determine whether the apoptosis was induced in neural progenitor cells, intermediate progenitors, or neurons by the loss of Aspm (S3 Fig). TUNEL-positive cells expressed the neuronal markers NeuN, Tuj1, and DCX (S3 A-C, A’-C’ Figs), but not the APs marker Pax6 or BPs marker Tbr2 (S3 D-E, D’-E’ Figs). In CC3-positive cells, the expression was consistent with the neural progenitor cell marker Nestin (S3 F and F’ Figs).

Judging from these results, we conclude that Aspm expression is required for the maintenance of neural progenitor cells and neurons and plays a critical role during neurogenesis.

3.Also, the authors did not cite or discuss important published articles such as Johnson et al., 2018. Nature or Williams et al., 2015. Development, where they mentioned that loss of Aspm is also associated with apoptosis and important for brain development.

I would greatly appreciate your important suggestions.

As indicated, we have cited in the Discussion.

Page 19, line 431

Page 17, line 398 and 414

It has been reported that the deletion of Aspm induced prematurely exit of the cell cycle and early differentiation of the cerebellar granule neuron progenitors, resulting in a decrease of terminally differentiated granule neurons in the internal granule layer [18].

Comments:

1. Fig. 1E does not explain how VZ/, IZ, or CP was defined.

I would greatly appreciate your important suggestions.

We defined the VZ, IZ, and CP as follows: The CP was defined based on DAPI staining and the VZ was defined based on SOX2 staining. The IZ was defined as the value obtained by subtracting the CP and VZ from the cortical wall, which was mesured DAPI staininig. The thickness was as average of three points (left, center and right within 250 µm-wide dorsal cortex regions, n = 3 brains) because each layer thickness was not uniform.

We have remade the Figure 1, which included the image to show a definition of the VZ, IZ and CP using the SOX2/DAPI staining.

Page 10, line 217

(E) SOX2 and DAPI stained image of E16.5 mouse cerebral cortex. The two head arrows indicate the CP, VZ and cortical wall. (F) Quantification of cortical wall and the CP region were defined based on DAPI staining and that of VZ was defined based on SOX2 staining. The IZ was defined as the value obtained by subtracting the CP and VZ from cortical wall. (VZ, WT 85.54 ± 2.2 μm, cKO 70.59 ± 2.1 μm, P = 0.0104; IZ, WT　229.29 ± 4.9 μm, cKO 210.55 ± 9.3 μm, P = 0.1490; CP, WT 159.51 ± 6.4 μm, cKO 140.03 ± 9.0 μm, P = 0.0908, Student’s t-test, values represent mean ± SEM, n = 3 mice per genotype) (G-I) Representative images of the WT and Aspm cKO Satb2, Ctip2 and Tbr1, respectively. Scale bar represents 50 µm. (J-L) Quantification of the cell number of the upper layer marker Satb2, the middle layer maker Ctip2 and the deep layer marker Tbr1, respectively, per unit area G-I (250μm radial columns). (J: WT 314.0 ± 23.4 cells, cKO 266.7 ± 36.2 cells, P = 0.3336, K: WT 392.7 ± 19.3 cells, cKO 327.7 ± 26.8 cells, P = 0.1205, L: WT 416.0 ± 14.7 cells, cKO 377.3 ± 17.9 cells, P = 0.1702, Student’s t-test, values represent mean ± SEM, n = 3 mice per genotype, ns: not significant).

I would greatly appreciate your important suggestions.

1) As suggested, we have changed the graphs Fig 1I-K to the number of Satb2, Ctip2 and Tbr1 positive cells, respectively.

2) We apologize for the unclear presentation. The samples in the Fig1G-I were images of embryonal day 16.5 (E16.5) brains in order to analysis the cortical layer construction refer to the Fig 1C. We have added the labels refer to the embryonal date to all figures in order to easily recognize the embryonic date.

3) As your suggestion, cortical layers have not been adequately established yet at the embryonal stage. However, the Satb2, Ctip2 and Tbr1 were used for analyzing the cortical layer thickness in the E16.5 Aspm-null cortex in the published report by Fujimori A et al (Brain & Development 36 (2014) 661–669). Therefore, we also conducted the immunostaining such as the Satb2, Ctip2 and Tbr1 to confirm the difference in the layer formation between WT and cKO.

Therefore, we have added to the co-staining image of SOX2 and DAPI to Fig 1(Fig 1E). According to the change, we have changed Fig 1 and Figure legend as follows:

Page 9, line 210

Fig 1. Embryonal brain of Aspm cKO

(A-C) Representative DAPI stained images of the embryonal cortices of the WT and Aspm cKO mice (NesCre;Aspmflox/flox) at E12.5, E14.5 and E16.5. The scale bars represent 500 μm. (A’-C’) Magnification of the boxed area in (A-C). The scale bars represent 50 μm. (D) Quantification of the thickness per unit area (A’-C’) (250 μm radial columns) *: P < 0.05 (E12.5: WT 151.1 ± 2.0 μm, cKO 155.1 ± 6.5 μm, P = 0.5922, E14.5: WT 238.0 ± 13.5 μm, cKO 226.9 ± 5.8 μm, P = 0.4913, E16.5: WT 482.4 ± 7.5 μm, cKO 427.1 ± 15.5 μm, P = 0.0325, Student’s t-test, values represent mean ± SEM, n = 3 mice per genotype, ns: not significant) (E) SOX2 and DAPI stained image of E16.5 mouse cerebral cortex. The two head arrows indicate the CP, VZ and cortical wall. (F) Quantification of cortical wall and the CP region were defined based on DAPI staining and that of VZ was defined based on SOX2 staining. The IZ was defined as the value obtained by subtracting the CP and VZ from cortical wall. (VZ, WT 85.54 ± 2.2 μm, cKO 70.59 ± 2.1 μm, P = 0.0104; IZ, WT　229.29 ± 4.9 μm, cKO 210.55 ± 9.3 μm, P = 0.1490; CP, WT 159.51 ± 6.4 μm, cKO 140.03 ± 9.0 μm, P = 0.0908, Student’s t-test, values represent mean ± SEM, n = 3 mice per genotype) (G-I) Representative images of the WT and Aspm cKO Satb2, Ctip2 and Tbr1, respectively. Scale bar represents 50 µm. (J-L) Quantification of the cell number of the upper layer marker Satb2, the middle layer maker Ctip2 and the deep layer marker Tbr1, respectively, per unit area G-I (250μm radial columns). (J: WT 314.0 ± 23.4 cells, cKO 266.7 ± 36.2 cells, P = 0.3336, K: WT 392.7 ± 19.3 cells, cKO 327.7 ± 26.8 cells, P = 0.1205, L: WT 416.0 ± 14.7 cells, cKO 377.3 ± 17.9 cells, P = 0.1702, Student’s t-test, values represent mean ± SEM, n = 3 mice per genotype, ns: not significant).

4) According to the change, we have changed the description in the Results as follows:

Page 9, line 188

Cerebral cortical reduction in Aspm cKO was exhibited at the late embryonal stage

First, we generated brain specific Aspm knockout mouse (NesCre;Aspmflox/flox, Aspm cKO) (S1 Fig) in order to examine whether the morphology of the embryonal cerebral cortex was affected by the loss of Aspm. Cortical thickness did not significantly change between the WT and cKO mice at E12.5 and E14.5 (E12.5, WT 151.1 ± 2.0 µm, cKO 155.1 ± 6.4 µm, P = 0.5922; E14.5, WT 238.0 ± 13.5 µm, cKO 227.0 ± 5.8 µm, P = 0.4913). However, a morphological difference was observed at E16.5, with a decrease in cortical thickness (WT 482.4 ± 7.5 µm, cKO 427.1 ± 15.5 µm, P = 0.0325) (Figs 1A-1D).

We further analyzed how the changes were related to the layer construction of the cerebral cortex at E16.5. First, there was a significant decrease in the thickness of the VZ in Aspm cKO (Figs 1C’, 1E and 1F, and S8 Fig). Although the thickness of IZ or CP showed a slight decrease, the significance did not reach the level of significance as compared to WT (Figs 1C’, 1E and 1F, and S6 Fig).

The CP is composed of six layers, and each layer consists of neurons with different birthdates. Therefore, we examined the CP in more detail using specific markers for each cortical layer. In the mouse brains lacking Aspm, the number of cells immunoreactive for the upper layer marker (Satb2-positive, II/III and IV layer), the middle layer marker (Ctip2-positive, layer V) and for the deep layer marker (Tbr1-positive, layer VI) showed a slight decrease, but no significant differences, when compared to those of the WT mice, (Figs 1G-1L), suggesting that a normal cytoarchitecture of the cerebral cortex was formed even with a loss of Aspm.

These findings suggest that the thinning of the cortex in late cortical formation might be due to poor neurogenesis or an excess loss of neurons at all embryonal stages.　

I would greatly appreciate your important suggestions.

In order to clarify the point, we have prepared additional supplemental figure (S8 Fig) to show the SOX2 staining for assessment of the thickness of the ventricular zone as NPC pool size at E12.5, E14.5 and E16.5. According to this figure, there was a significant difference between WT and cKO only at E16.5, suggesting that the NPC pool was reduced at the late stage.

We described in Results as follows.

Page 15, line 345

In addition, the thickness of VZ and the number of NPCs, immunoreactive for PAX6, showed a decrease at E16.5 in cKO compared to WT (S8 Fig).

As your suggestion, we did not precisely evaluate that the loss of Aspm affected the direct neurogenesis and/or indirect neurogenesis. The present data is supposed to show the effects on the direct neurogenesis, which is most likely because of that mice, agyrencephalic animal, have fewer indirect neurogenesis.

We described in Discussion as follows:

Page 19, line 431

4.The authors mentioned that "Immunofluorescence studies for pHH3, which is the mitotic marker, revealed no significant differences in the abundance of apical progenitor cells (APs) and basal progenitor cells (BPs) between the WT and Aspm cKO mice (Figs 2A and 2B)". How did the authors determine apical or basal progenitors? The authors assume the nature of these progenitors based on their spatial location. However, it is highly recommended that these experiments be performed using proper cell markers such as Pax6 or Tbr2 and colocalization studies.

I would greatly appreciate your important suggestions.

As suggested, we did not precisely detect apical progenitor cells and basal progenitor cells using specific immunostaining such as Pax6 and Tbr2. However, according to the published report by Arai Y et al (Nat Commun. 2011, ref 17), apical progenitor cells and basal progenitor cells were determined based on the spatial location. Furthermore we did not determine APs and BPs using co-staining such as pHH3/Pax6 and pHH3/Tbr2. However, as mentioned above, the determination of APs and BP based on the spatial location in the published report. So, we thought that it was useful to evaluate APs and BPs based on the spatial location of pHH3 positive cells.

Thus, we have detected them based on the spatial localization and described as follows:

Page 10, line 233

In order to identify specific neural progenitors, apical progenitor cells (APs) were defined as the phospho-histone H3 (pHH3) positive cells located beneath the surface of the lateral ventricle, and basal progenitor cells (BPs) were defined as the pHH3 positive cells localized in the basal region of the ventricular zone (Fig 2) [17] Immunofluorescence studies for pHH3, which is the mitotic marker, revealed no significant differences in the abundance of APs and BPs between the WT and Aspm cKO mice (Figs 2A and 2B).

5.In Fig. 2C-D, it is unclear whether the absence of proliferative disruption is specific to a particular progenitor population since the authors did not use any cell-specific marker. Therefore, it is highly recommended that these experiments be performed using proper cell markers such as Pax6 or Tbr2 since the authors state that only early neurogenesis is affected.

I would greatly appreciate your important suggestions.

I agree with you that these experiments should be performed using proper cell markers such as Pax6 or Tbr2 in order to assess whether direct or indirect neurogenesis was more affected in the present mice brain. However; we suggest that a few Tbr2-immunoreactive BPs, OSVG, exist in mice brain, agyrencephalic animal, and proliferation of neuronal proliferation is unlikely involved in reduction of cortical neurons in Aspm cKO.

6.The authors mentioned, "Pregnant mice were given a single dose of EdU at E12.5 or E14.5, and two days later (i.e., E14.5 or E16.5, respectively), the abundance of EdU-positive neurons was measured. Most of the cells, EdU-labeled at E12.5, were observed in the superficial area corresponding to the CP, and EdU-positive cells were found in postmitotic neurons showing Tbr1-immunoreactivity". However, TBR1 immunostaining was not performed. So then, how can the authors ensure that EdU staining corresponds to postmitotic neurons? Also, the authors made similar conclusions on Fig. 3C-F but did not perform immunostainings for specific neuronal populations such as Satb2, Ctip2 or Tbr1.

I would greatly appreciate your important suggestions.

According to your suggestions, in terms of the birthdating assay at early to middle stage, we have confirmed co-immunostaining of EdU and specific neural maker such as Tbr1 and remade the Fig 3, which includes co-immunostaining for Tbr1, EdU and DAPI images in order to show that the EdU positive cells were postmitotic neurons in cortical plate at E14.5 (Figs 3A and B).

We described as follows:

Page 12, line 263

Most of the cells, EdU-labeled at E12.5, were observed in the superficial area corresponding to the CP, and EdU-positive cells were found in postmitotic neurons showing Tbr1-immunoreactivity. The E12.5-labeled neurons showed no significant differences between the WT and cKO mice (Figs 3A and 3B).

Regarding the double staining ratio, we made supplemental figure (S7 Fig), which revealed that Tbr1/EdU was about 8.7% in WT and 9.9% in cKO, that of Ctip2/EdU was about 3.4% in WT and about 3.4% in cKO, and that of Satb2/EdU was about 76.6% in WT and 86.9% in cKO, respectively (S7 Fig).

We added the description as follows:

Page 12, line 266.

In contrast, the number of cells labeled at E14.5 was significantly reduced, with most of them scattered from the IZ to the CP, in the cKO mice. There was a significant reduction in the number of EdU-labeled cells distributed in the IZ, but not in the VZ and CP (Figs 3C-3F). Although most of the EdU-labeled cells showed immunoreactivity for Satb2, there were no significant differences in ratio of Tbr1+/EdU+, Ctip2+/EdU+, and Satb2+/EdU+ cells in the cerebral wall between the WT and Aspm cKO brains (S7 Fig).

These findings suggested that the loss of Aspm induced a reduction in the number of mid to later born neurons without any aberrant expression of cerebral layer-specific transcription factors in the developing cortex.

As suggested, we have remeasured for the number of EdU-positive cells and remade the Fig 3, which included quantitative results about VZ/SVZ (bin1/bin2). Furthermore, it was performed Edu+/Tbr1+ cells because neurons at the lower CP and subplate (SP) were Tbr1-positive and the border between CP and SP was distinguishable to indicate the cortical layer (CP, IZ and VZ/SVZ) and bin number (Bin1-10).

We also have remade the figure legend as follows:

Page 12, line 276

Fig 3. Neurogenesis during murine corticogenesis.

(A, C) Co-staining images of Tbr1 (green), EdU (red) and DAPI (blue) in the WT and Aspm cKO mice at E14.5 (A) and E16.5 (C) cortices two days after the EdU injections (E12.5 and E14.5, respectively). Scale bar: 50 µm. (B, D) The number of EdU-positive postmitotic cells per 250 µm. *P < 0.05 (B: WT 50.67 ± 3.5, cKO 54.3 ± 7.9, P = 0.6923, D: WT 228.7 ± 6.9, cKO 174.7 ± 11.4, P = 0.0154, Student’s t-test, values represent mean ± SEM, n = 3 mice per genotype) (E) Histogram of the EdU-positive cells, which were shown in C. (bins × genotype interaction F(8, 36) = 2.893 P = 0.0135; two-way ANOVA with Tukey’s multiple comparisons test; **P < 0.01, values represent mean ± SEM, n = 3 mice per genotype). (F) Comparison of the distribution of the EdU-positive cells among the cortical layers (cortical layers × genotype interaction F(2, 12) = 27.82 P < 0.0001; two-way ANOVA with Tukey’s multiple comparisons test; ****P < 0.0001, ns: not significant, values represent mean ± SEM, n = 3 mice per genotype)

I would greatly appreciate your important suggestions.

As your suggestions, we did not perform precise migration assay in the present study. However, most of postmitotic neurons in the IZ are young neurons under radial migration in the developing cerebral cortex.

We described in Discussion as follows:

Page 17, line 391.

Therefore, it is highly likely that cell loss might occur not only in NPCs, but also in postmitotic neurons, i.e., neuroblasts in the VZ/SVZ, migrating neurons in the IZ or immature neurons after reaching the CP.

8.Fig. 4A-H are not cited in the text.

I would greatly appreciate your important suggestions.

We have added the citation in the text appropriately.

Page 13, line 290.

We conducted assays to determine whether the occurrence of apoptosis was related to the cortical thinning observed in association with the loss of Aspm (Fig 4). In the TUNEL assay for measuring apoptosis, TUNEL-positive cells were distributed in the VZ and PP at E12.5 (Figs 4A, 4B and 4J) and in the VZ, IZ and CP at E14.5 and E16.5 (Figs 4C-4F and 4J). The TUNEL-positive cells were significantly increased in the developing cortex in the cKO mice throughout corticogenesis (Fig 4G). The numerical density of the TUNEL-positive cells in the developing cortex areas (TUNEL index) decreased as the embryonal stage proceeded (Figs 4H and 4I). Furthermore, the TUNEL-index was significantly higher in the VZ at E12.5 and 14.5, but not at E16.5 in the cKO mice as compared to WT mice (Fig 4J). Thus, we found an increased number of apoptosis instances in the developing cortex obtained from Aspm cKO mice and the apoptosis was higher in the VZ at the early to the middle phase of the corticogenesis, compared with the later corticogenesis.

9.In Figure 5, it is unclear why the authors show the immunostainings in low magnification. Also, it is suggested to show gammaH2AX and YOYO1 in separate channels.

I would greatly appreciate your important suggestions.

As suggested, we have remade Fig.5 which showed at higher magnification. We showed co-immunostaining images of γH2AX and YOYO1 as well as separated images. According to the change, we have changed Fig 5 and the Figure legend as follows:

9)-1 We have changed the Fig 5.

Page 15, line 351.

Fig 5. Immunohistochemistry for γH2AX

(A-F) Representative immunofluorescence images (γH2AX (red) and YOYO1 (blue) of embryonal cortex in the WT and Aspm cKO mice at E12.5 (A, B), E14.5 (C, D) and E16.5 (E, F). The number of γH2AX-positive cells were counted in the developing cortex. Arrows indicate γH2AX-positive cells (red). Scale bars represent 500 μm. (G) The number of γH2AX positive cells in cerebral cortex (E12.5: WT1.00 ± 0.33 cells, cKO 3.89 ± 0.80 cells P = 0.0294, E14.5 WT 4.67 ± 0.69 cells, cKO 8.00 ± 0.39 cells P = 0.0137, E16.5: WT 3.11 ± 1.87 cells, cKO 4.67 ± 0.88 cells P = 0.4936, Student’s t-test, *P < 0.05, values represent mean ± SEM, n = 3 mice per genotype) (H) The area of cerebral certex which cell number was counted. (E12.5: WT 0.12 ± 0.0 mm2, cKO 0.16 ± 0.01 mm2, P = 0.0027, E14.5: WT 0.31 ± 0.04 mm2, cKO 0.30 ± 0.01 mm2, P = 0.8369, E16.5: WT 0.50 ± 0.04 mm2, cKO 0.48 ± 0.01 mm2, P = 0.5989, Student’s t-test, **P < 0.01, values represent mean ± SEM, n = 3 mice per genotype) (I) γH2AX index (Num/Area) is the number of γH2AX-positive cells (G) in a cerebral cortex divided by the unit area (H). (E12.5: WT 8.27 ± 3.04 cells/mm2, cKO 23.89 ± 4.79, P = 0.0521, E14.5: WT 15.87 ± 4.56 cells/mm2, cKO 26.32 ± 0.94, P = 0.0880, E16.5: WT 6.21 ± 3.93 cells/mm2, cKO 9.78 ± 1.62, P = 0.4484, ns: not significant, values represent mean ± SEM, n = 3 mice per genotype) (J) Distribution of γH2AX index in the developing cortex divided into the VZ, IZ, PP and/or CP. (E12.5: cortical layers × genotype interaction F(1, 8) = 3.387 P = 0.1030, E14.5: cortical layers × genotype interaction F(2, 12) = 0.2642 P = 0.7722, E16.5: cortical layers × genotype interaction F(2, 12) = 0.4965 P = 0.6206; two-way ANOVA with Tukey’s multiple comparisons test; *P < 0.05, ns: not significant, n = 3 mice per genotype)

We have added the description in the Method as follows:

Page 7, line 167.

In order to measure the γH2AX positive cells, we used ImageJ plugin “Analyze Particles” and selected a signal size within 3.00－200.00 to remove the particles that were too small or big, and thus regarded as false positive signals.

10.In the discussion section, the authors state, "The knockout of Aspm in neural progenitor cells increased apoptosis in all stages of cortical development compared to normal brains (Fig 4), but postnatal brains showed no differences from normal brains (data not shown)". Therefore, it is highly recommended that the authors include the postnatal data since it is an essential part of their discussion.

I would greatly appreciate your important suggestions.

As suggested, we made the Supplemental Figure S5 Fig to show the TUNEL staining images of P7 brains as postnatal brains. Although we have evaluated the staining qualitatively, TUNEL-positive cells at the P7 brain were apparently lower than in embyonal brains.

We added the description as follows:

Page 13, line 293.

Although the TUNEL-positive cells were significantly increased in the developing cortex in the cKO mice throughout corticogenesis (Fig 4G), postnatal brains showed no differences from WT brains (S5 Fig).

10)-2 We have prepared the Supplemental Figure S5 Fig.

We also added the supplemental figure legend (S5 Fig legend) as follows:

Page 27, line 600.

S5 Fig. TUNEL staining of postnatal mouse brain at P7

We added the sentense in the Method as follows

Page 8, line 175.

For P7 brains (S5 Fig), a Nikon Ti Eclipse confocal microscope were used, and the images were processed with ImageJ/Fiji to adjust color and contrast. Whole brain images were obtained by tiling and stitching (6×6) the images captured with a 20× lens (2048×2048 pix). The high magnification images were cropped from the whole brain images.

11.It is strongly suggested to add the supplemental material to the manuscript.

I would greatly appreciate your important suggestions.

We described all precise procedures in Materials and Methods of the manuscript even for supplemental figures as well. As mentioned above, the sentence “data not shown” was changed to “S5 Fig” in order to show the actual result.

Response to reviewer #2’ comments

First of all, I would greatly appreciate reviewer’s suggestions and comments on our manuscript. We have tried to revise the former manuscript according to valuable comments.

Refer to Materials

1-1) Which was the Nes-Cre Tg line used? The efficacy of Cre recombination in the developing cortex should be tested (with conditional reporters) or cite the corresponding validations.

I would greatly appreciate your important suggestions.

We used Nestin-cre (C57BL/6-Tg(Nes-Cre)1Kag, Center for Animal Resources and Development (CARD), CARD-ID 650) mice [Isaka, F. et al. Eur.J. Neuroscience 11. 2582-2588, 1999] It was reported to be sufficient for directing recombination in early embryonic neural cortical progenitors.

Since we did not find any changes in proliferation of the neuronal progenitor cells from E12.5 to E16.5, namely during active proliferation stage in corticogenesis, but found increased apoptosis in above stage, we described “the microcephaly associated with MCPH5 might be aggravated by increased apoptosis in NPCs and postmitotic neurons, but not by aberrant proliferation of NPCs during cortical development. (Page 17, line 374, Page 11, 244, others). The loss of postmitotic cells, including neurons and progenitor cells resulted in “cerebral cortical reduction in Aspm cKO was at the late embryonic stage.

We added descriptions in Material and Methods as follows

Page 4, line 81.

In order to obtain NesCre;Aspmflox/+ mice, Nestin-cre (C57BL/6-Tg(Nes-Cre)1Kag, Center for Animal Resources and Development (CARD), CARD-ID 650) mice [16] were mated with Aspmflox/flox mice [9]. Brain specific Aspm knockout mice (NesCre;Aspmflox/flox, Aspm cKO) were generated by mating Aspmflox/flox mice and NesCre;Aspmflox/+ mice and detected by genotyping.

In addition, we showed a loss of Aspm protein at E14.5 (S1 Fig).

Page 26, line 553.

S1 Fig. Aspm cKO and wild type mice

1-1. The downregulation of Aspm, at mRNA or protein levels, should be assessed in the Nes-Cre Aspm-flox/flox used in this study.

I would greatly appreciate your critical suggestions.

We showed a loss of Aspm protein in the ventricular stem cells of Aspm cKO mice brains at E14.5 by

immunohistochemistry (S1-C Fig).

S1 Fig. Aspm cKO and wildtype mouse

Page 26, line 553.

S1 Fig. Aspm cKO and wild type mice

Refer to Materials

I would greatly appreciate your critical suggestions.

Aspm full knock out embryos (previously used by the authors of this manuscript, Fujimori et al 2014) were CAG-CreTg and different form the present mice. Furthermore, they did not analyze precisely embryonic brains.

Jayaraman et al. reported that loss of Wdr62, Aspm or both results in expansion of basal progenitors beyond the germinal zones at mid-neurogenesis, suggesting regulation of cell fate leading to precocious formation of Tbr2+ cells rather than merely mitotic progression in Emx1-Cre; Aspm-flox (Jayaraman et al 2016). In addition, they showed little-to-no apoptosis in the Aspm-/- mice brains.

We did not find remarkable dislamination of basal progenitor cells beyond the germinal zones at E14.5, using birthdate analyses (Fig.3). Furthermore, the number of apoptotic cells showed significantly higher in all stages of corticogenesis in our Aspm cKO mice brains, suggesting the microcephaly might be followed by cell loss via apoptosis rather than mitotic progression or cell fate dysregulation. The reasons for the different results are unknown but might be derived from the differences in Aspm KO mice, and analyzed methods.

We added in Discussion as follows:

Page 18, line 418.

Jayaraman et al. [21] reported that loss of Wdr62, Aspm or both impairs centriole duplication, correlating with the severity of microcephaly, and leads to a loss of centrosomes and cilia in the developing brain, and accordingly, microcephaly has been described as a ‘centriolopathy’. They showed an expansion of basal progenitors beyond the germinal zones at mid-neurogenesis, suggesting regulation of cell fate leading to precocious formation of basal progenitors rather than merely mitotic progression in Emx1-Cre; Aspm-flox. In addition, they showed little-to-no apoptosis in the Aspm-/- mice brains. We did not find any remarkable delamination of basal progenitor cells beyond the germinal zones at E14.5, using birthdate analyses (Fig 3). Furthermore, the number of apoptotic cells was significantly higher in all stages of corticogenesis in our Aspm cKO mice brains (Figs 4, 5 and S2 Fig), suggesting that the microcephaly might be followed by cell loss via apoptosis rather than proliferation and/or cell fate dysregulation. The reasons for these different results are unknown, but they might have been due to differences in the Aspm KO mice and experiment methods employed, including the ages or markers analyzed.

Refer to discussion

I would greatly appreciate your critical suggestions.

We added in Discussion as follows:

Page 17, line 386.

Fish et al. [13] reported that Aspm-downregulation, using RNA interference, induced the alteration in the cleavage plane orientation in neuroepithelial cells of embryonal telencephalon, followed by a reduction in the number of neuroepithelial progenitor cells and microcephaly. In the present study, we did not find any aberrant cleavage plane orientation in the neuroepithelial cells and the mitosis of the NPCs was not affected in the cortex of the cKO mice when we focused on the S and M phase of the cell cycle (Fig 2). Therefore, it is highly likely that cell loss might occur not only in NPCs, but also in postmitotic neurons, i.e., neuroblasts in the VZ/SVZ, migrating neurons in the IZ or immature neurons after reaching the CP. These observations suggest that this cell loss could play some role in building the normal cortical architecture, except for uniform thinning, as observed in the cKO mice.

Page 18, line 418.

Jayaraman et al. [21] reported that loss of Wdr62, Aspm or both impairs centriole duplication, correlating with the severity of microcephaly, and leads to a loss of centrosomes and cilia in the developing brain, and accordingly, microcephaly has been described as a ‘centriolopathy’. They showed an expansion of basal progenitors beyond the germinal zones at mid-neurogenesis, suggesting regulation of cell fate leading to precocious formation of basal progenitors rather than merely mitotic progression in Emx1-Cre; Aspm-flox. In addition, they showed little-to-no apoptosis in the Aspm-/- mice brains. We did not find any remarkable delamination of basal progenitor cells beyond the germinal zones at E14.5, using birthdate analyses (Fig 3). Furthermore, the number of apoptotic cells was significantly higher in all stages of corticogenesis in our Aspm cKO mice brains (Figs 4, 5 and S2 Fig), suggesting that the microcephaly might be followed by cell loss via apoptosis rather than proliferation and/or cell fate dysregulation. The reasons for these different results are unknown, but they might have been due to differences in the Aspm KO mice and experiment methods employed, including the ages or markers analyzed.

Refer to Fig 1

Importantly, this reduction is not consistent with later results (Fig.3) where birth dating experiments at E12.5 do not show deep layer differences between WT and cKO.

I would greatly appreciate your critical suggestions.

According to the suggestion, we revised Fig 1.

Page 9, line 210

Fig 1. Embryonal brain of Aspm cKO

Page 9, line 188

We further analyzed how the changes were related to the layer construction of the cerebral cortex at E16.5. First, there was a significant decrease in the thickness of the VZ in Aspm cKO (Figs 1C’, 1E and 1F, and S8 Fig). Although the thickness of IZ or CP showed a slight decrease, the significance did not reach the level of significance as compared to WT (Figs 1C’, 1E and 1F, and S6 Fig).

The CP is composed of six layers, and each layer consists of neurons with different birthdates. Therefore, we examined the CP in more detail using specific markers for each cortical layer. In the mouse brains lacking Aspm, the number of cells immunoreactive for the upper layer marker (Satb2-positive, II/III and IV layer), the middle layer marker (Ctip2-positive, layer V) and for the deep layer marker (Tbr1-positive, layer VI) showed a slight decrease, but no significant differences, when compared to those of the WT mice, (Figs 1G-1L), suggesting that a normal cytoarchitecture of the cerebral cortex was formed even with a loss of Aspm.

These findings suggest that the thinning of the cortex in late cortical formation might be due to poor neurogenesis or an excess loss of neurons at all embryonal stages.

Refer to Fig3

5-1. In EdU/BrdU labeling experiments (Fig 3) it would be useful to also test for differences in number and distribution of EdU cells at a later timepoint (P5-10) when neurons have completed migration. The reduction in EdU cell numbers seen in the IZ but not CP, which suggests they are later born, would also be supported by Tbr1 co-staining. Authors should also consider adding an EdU pulse at E16.5 to solidly conclude which neurons are affected.

I would greatly appreciate your critical suggestions.

We agree with you that the reduction in EdU-labeled cell numbers seen in the IZ but not CP, which suggests they are later born neurons (later than E14.5).

From the S7 Fig, around 90% of EdU-positive cells showed immunoreactivity for Satb2, showing that they might be later-born neurons

Since we could not generate new animals, we could not analyze EdU pulse study at E16.5.

5-2. Additionally, in Fig 3 E-F, no EdU + cells are shown in quantification in bins 1-2 (or VZ/SVZ), but they are clearly evident in the images. Distinguishing between EdU cells remaining in VZ or migrating into IZ (with short chase times), coupled with the use of specific markers could help determine whether there is changes in neurogenic divisions. For this point, authors should consider similarities and/or differences with published work.

I would greatly appreciate your important suggestions.

We described as follows:

Page 12, line 263

We added the description as follows:

Page 12, line 266.

In contrast, the number of cells labeled at E14.5 was significantly reduced, with most of them scattered from the IZ to the CP, in the cKO mice. There was a significant reduction in the number of EdU-labeled cells distributed in the IZ, but not in the VZ and CP (Figs 3C-3F). Although most of the EdU-labeled cells showed immunoreactivity for Satb2, late born neurons, there were no significant differences in ratio of Tbr1+/EdU+, Ctip2+/EdU+, and Satb2+/EdU+ cells in the cerebral wall between the WT and Aspm cKO brains (S7 Fig).

We also have remade the figure legend as follows:

Page 12, line 276

Fig 3. Neurogenesis during murine corticogenesis.

Minor

Refer to S4 Fig

6． Examples of DNA damage in both Pax6+ and Pax6- cells are shown in Fig Suppl 4. Since numbers were not quantified, lines 314-315 should be revised. Quantifications of both cell type specific death and DNA damage would increase the robustness of the results.

I would greatly appreciate your critical suggestions.

According to your suggestions, we omitted ‘increase’ from the description.

Page 15, line 341.

Furthermore, the double immunofluorescence of γH2AX with the neural progenitor cell marker, Pax6, revealed that the cells with DNA damage were neural progenitor cells expressing Pax6 (S4 Fig).

7． It would be ideal to show the actual data points in the graphs together with mean +/- SEM.

I would greatly appreciate your critical suggestions.

We added actual data in figure legends.

Refer to Abstract

8． Please, rephrase “..observed cortical reduction in the late neurogenesis of murine cortical development” Abstract, line 27.

I would greatly appreciate your critical suggestions.

We rephrased the sentence as follows:

Page 1, line 27

In this study, we generated brain-specific Aspm knockout mice to evaluate the fetal brain phenotype and observed cortical reduction in the late stage of murine cortical development.

9． Line 59: mutations in ASPM are a cause (not consequence) of mitotic aberrations during neurogenesis.

I would greatly appreciate your critical suggestions.

We changed the sentence as follows:

Page 3, line 58.

Therefore, mutations in the ASPM gene are a cause of mitotic aberrations during neurogenesis.

10． References for mouse lines are missing in Methods section.

I would greatly appreciate your critical suggestions.

We added in Materials and Methods as follows:

Page 4, Line 82.

11． Please indicate ages in Figures 1 F-H, 1E, 3 A-F where they are missing so that is easily interpreted.

I would greatly appreciate your critical suggestions.

We added age in Fig 1F-H, and Fig 3A-F.

12． Label the stainings close/within the panels in all figures. They are missing in Figs. 2, 3A-C, 5A-F and Fig.S2 A-F.

I would greatly appreciate your critical suggestions.

We labeled the staining in Figs. 2, 3A-C, 5A-F and Fig.S2 A-F.

Fig 2

Fig 3

Fig 5

S2 Fig

13． Line 339 should be modified. The authors show that there is no excessive or decreased proliferation but can’t exclude that there are other ways of aberrant division such as altered ratio of self-renewal vs. neurogenic divisions.

I would greatly appreciate your critical suggestions.

We modified the description as follows:

Page 17, line 374

These findings suggest that the microcephaly associated with MCPH5 might be aggravated by increased apoptosis in NPCs and postmitotic neurons, but not by aberrant proliferation of NPCs during cortical development, although we can’t exclude that there are other ways of aberrant division such as altered ratio of self-renewal vs. neurogenic divisions.

14． Please avoid the use of “cortical areas” as it may get confused with functional areas of the cortex (line 270). I suggest to remove the word “layers” (line 32, abstract)

I would greatly appreciate your critical suggestions.

We removed the ‘areas” as follows:

Page 1, line 30.

On the other hand, the knockout mice showed a constant increase in apoptosis in the cerebral cortex from the early, through the late stages of cortical development.

Page 13, line 295

The numerical density of the TUNEL-positive cells in the developing cortex (TUNEL index) decreased as the embryonal stage proceeded (Figs 4H and 4I).

15． I think it is more common to use embryonic instead of embryonal (176, 190 and others)

I would greatly appreciate your critical suggestions.

We changed “embryonic” to “embryonal” throughout the manuscript.

Attachment

Submitted filename: Response to the reviewers_2_ki_0612.docx

Click here for additional data file.^{(4.5MB, docx)}

PLoS One. doi: 10.1371/journal.pone.0294893.r003

Decision Letter 1

Carlos Oliva

11 Aug 2023

PONE-D-23-02973R1Loss of Aspm causes increased apoptosis of developing neuronal cells during mouse cerebral corticogenesisPLOS ONE

Dear Dr. Itoh,

Thank you for submitting the revised version of your manuscript to PLOS ONE. After careful consideration, we feel that you still need to make some corrections before your paper is ready for publication. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during this second round of review process. Important to notice that you do not need to perform new experiments, but have to make corrections regarding data presentation (use of repetitive images among other points raised) and interpretation of some results.

Please submit your revised manuscript by Sep 25 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Carlos Oliva, PhD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: The reviewer appreciates that the authors have improved the manuscript, including most of our comments and suggestions. However, there are still important issues to be addressed.

The main issue is that the authors misinterpret or overstate their conclusions. Positively, they found reductions in total cortical (Fig. 1D) and VZ thickness (Fig. 1F), probably due to NPC apoptosis (Fig. 4), as they did not find changes in proliferation (Fig. 2) or neurogenesis (Fig. 1G-L). Although the authors showed a decrease in EdU-positive cells (Fig. 3D), these are mostly located in the VZ/SVZ or IZ areas, arguing that the main cell population affected by the loss of ASPM are NPCs rather than neurons. Indeed, in Figure 4, they found an increase in TUNEL+ cells exclusively at the VZ/SVZ.

Thus, I strongly suggest that the authors adjust their conclusions to the data presented in their manuscript and to the limitations of the experimental approach, avoiding overstating their conclusions.

Other comments:

1. "Line 207-208: These findings suggest that the thinning of the cortex in late cortical formation might be due to poor neurogenesis or an excess loss of neurons at all embryonal stages."

The data presented here does not allow the authors to conclude this as the only cortical layer affected is the ventricle zone, suggesting that ASPM is important for NPC maintenance, not neurogenesis or differentiation from NPCs to neurons. Indeed, the IZ or CP areas are unaffected by the ASPM loss.

2. "Lines 232-238: Next, we conducted assays to determine whether or not the thinning of the deep cortical layer caused by Aspm deficiency is related to changes in the neural progenitor cell proliferation. In order to identify specific neural progenitors, apical progenitor cells (APs) were defined as the phospho-histone H3 (pHH3) positive cells located beneath the surface of the lateral ventricle, and basal progenitor cells (BPs) were defined as the pHH3 positive cells localized in the basal region of the ventricular zone (Fig 2) [17]. Immunofluorescence studies for pHH3, which is the mitotic 237 marker, revealed no significant differences in the abundance of APs and BPs between the WT and Aspm cKO mice (Figs 2A and 2B)."

First, the authors do not show the thinning of the deep cortical layer. They showed the thinning of total cortical thickness and also of the VZ only. These data also argue that ASPM is important for NPC maintenance rather than neurogenesis.

Then, using the experimental approach presented, the authors can not assess the abundance of APs or BPs, as they did not use specific cell markers and pHH3 only labels cells during mitosis, which are quite few compared with the total AP or BP population. They can infer whether there are changes in NPCs positive for pHH3 only using their spatial location but not changes in NPC pools.

3. "Lines 263-265: Most of the cells, EdU-labeled at E12.5, were observed in the superficial area corresponding to the CP, and EdU-positive cells were found in postmitotic neurons showing Tbr1-immunoreactivity. The E12.5-labeled neurons showed no significant differences between the WT and cKO mice (Figs 3A and 3B). In contrast, the number of cells labeled at E14.5 was significantly reduced, with most of them scattered from the IZ to the CP, in the cKO mice."

It was impossible to check this statement as there is no graph showing changes in VZ/IZ/CP distribution in EdU-labeled cells at E12.5.

Also, EdU-labeled cells at E14.5 data showed that EdU+ cells are preferentially located in the IZ, where it is possible to find Tbr2+ intermediate progenitors. Although several cells are located at the CP, there are also located at the VZ. Then, it is not correct to assume that they are postmitotic neurons.

Reviewer #2: In this revised version of manuscript, the authors have addressed the major points of my previous review. The authors have included additional data which have improved the presentation.

I have a few observations that should be addressed in the final version

1. Some images are repetitively used in different figures of the manuscript.

The photos illustrating the E16.5 cortex of wild type and Aspm-cKOs shown in Fig.1C, in Fig.3C, in Fig.S6C and in Fig.S7 A,D are exactly the same pictures. In some instances, they are labeled as DAPI, in others as YOYO1.

Please replace the images to avoid repetitive use of the same representative photos.

2. Line 232-233. Revise the phrase “….the thinning of the deep cortical layer caused by Aspm deficiency ….”. This is contradictory with the results in Fig.1 I,L that shows that deep layer Tbr1+ is not significantly different in mutants.

3. The Suppl. Figures should be renumbered according to the order they are first mentioned in the text.

4. I suggest to use the term “embryonic”, instead of the term “embryonal”. It looks that my comment in the previous review was not understood correctly.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

PLoS One. 2023 Nov 29;18(11):e0294893. doi: 10.1371/journal.pone.0294893.r004

Author response to Decision Letter 1

29 Aug 2023

Reviewer #1: The reviewer appreciates that the authors have improved the manuscript, including most of our comments and suggestions. However, there are still important issues to be addressed.

I would greatly appreciate your critical comments. Because of the limitations of the experimental approach, it is difficult to conclude that loss of Aspm mainly affected NPCs or postmitotic immature neurons. According to your suggestions, we revised the descriptions as follows.

Page 9, line 207

These findings suggest that Aspm is important for NPC maintenance, not for neurogenesis or differentiation from NPCs to neurons at all embryonic stages.

Page 11, line 245

These results suggest that loss of Aspm does not affect the proliferation of the NPCs, but might affect NPCs pool during mouse neurogenesis.

Page 12, line 275

Page 21, line 471

These findings suggest that Aspm deficiency can induce apoptotic loss of NPCs and immature neurons, leading to decreased NSCs pool and neuronal loss and ultimately to phenotypic change (i.e., cortical thinning) as found in MCPH.

Other comments:

1. "Line 207-208: These findings suggest that the thinning of the cortex in late cortical formation might be due to poor neurogenesis or an excess loss of neurons at all embryonal stages."

I would greatly appreciate your critical comments. According to your suggestions, we revised the descriptions as follows.

Page 9, line 207

These findings suggest that Aspm is important for NPC maintenance, not for neurogenesis or differentiation from NPCs to neurons at all embryonic stages.

2. "Lines 232-238: Next, we conducted assays to determine whether or not the thinning of the deep cortical layer caused by Aspm deficiency is related to changes in the neural progenitor cell proliferation. In order to identify specific neural progenitors, apical progenitor cells (APs) were defined as the phospho-histone H3 (pHH3) positive cells located beneath the surface of the lateral ventricle, and basal progenitor cells (BPs) were defined as the pHH3 positive cells localized in the basal region of the ventricular zone (Fig 2) [17]. Immunofluorescence studies for pHH3, which is the mitotic marker, revealed no significant differences in the abundance of APs and BPs between the WT and Aspm cKO mice (Figs 2A and 2B)."

I would greatly appreciate your critical comments. According to your suggestions, we revised the descriptions as follows.

Page 11, line 245

These results suggest that loss of Aspm does not affect the proliferation of the NPCs, but might affect NPCs pool during mouse neurogenesis.

It was impossible to check this statement as there is no graph showing changes in VZ/IZ/CP distribution in EdU-labeled cells at E12.5.

I would greatly appreciate your critical comments. According to your suggestions, we assessed VZ/IZ/CP distribution in EdU-labeled cells at E12.5.

Page 12, line 265

The number of EdU-labeled cells with Tbr1-immunoreactivity in the CP, as well as the number of EdU-labeled cells in the IZ and VZ showed no significant difference between the WT and Aspm cKO brains at E12.5 (Fig 3C).

Figure 3 was revised.

Fig 3. Neurogenesis during murine corticogenesis

(A, D) Co-staining images of Tbr1 (green), EdU (red) and YOYO1 (blue) in the WT and Aspm cKO mice at E14.5 (A) and E16.5 (D) cortices two days after the EdU injections (E12.5 and E14.5, respectively). Scale bar: 50 µm. (B, E) The number of EdU-positive cells per 250 µm. *P < 0.05 (B: WT 162.0 ± 6.1, cKO 169.7 ± 11.6, P = 0.5867, E: WT 228.7 ± 6.9, cKO 174.7 ± 11.4, P = 0.0154, Student’s t-test, values represent mean ± SEM, n = 3 mice per genotype) (C) Comparison of the distribution of the EdU-positive cells among the cortical layers (cortical layers × genotype interaction F(2, 12) = 0.3488 P < 0.7125; two-way ANOVA with Tukey’s multiple comparisons test; ns: not significant, values represent mean ± SEM, n = 3 mice per genotype). (F) Histogram of the EdU-positive cells, which were shown in C. (bins × genotype interaction F(8, 36) = 2.893 P = 0.0135; two-way ANOVA with Tukey’s multiple comparisons test; **P < 0.01, values represent mean ± SEM, n = 3 mice per genotype). (G) Comparison of the distribution of the EdU-positive cells among the cortical layers (cortical layers × genotype interaction F(2, 12) = 27.82 P < 0.0001; two-way ANOVA with Tukey’s multiple comparisons test; ****P < 0.0001, ns: not significant, values represent mean ± SEM, n = 3 mice per genotype).

I would greatly appreciate your critical comments. According to your suggestions, we revised the description as follows:

Page 12, line 267, 275

In contrast, the number of cells labeled at E14.5 was significantly reduced, with most of them located in the VZ/SVZ or IZ areas in the cKO mice (Figs 3D and 3E), arguing that the main cell population affected by the loss of ASPM were NPCs rather than neurons.

Page 12, line 275

Reviewer #2: In this revised version of manuscript, the authors have addressed the major points of my previous review. The authors have included additional data which have improved the presentation.

I have a few observations that should be addressed in the final version

1. Some images are repetitively used in different figures of the manuscript.

Please replace the images to avoid repetitive use of the same representative photos.

I would greatly appreciate your critical comments. According to your suggestions, we revised the figures.

Fig 1C: images of both WT and cKO were replaced.

Fig 1A’,B’, and C’: DAPI was changed to YOYO.

Fig 3 was revised:

DAPI was changed to YOYO.

Fig 3D (previous Fig 3C): images of both WT and cKO were replaced.

Fig 3B was changed and Fig 3C was added, and Fig numbering was changed due to insertion of a new data (Fig 3C)

Fig S6C was not changed.

S7 Fig A and D were replaced. The labels for EdU and Tbr1 were reversed and have been corrected.

I would greatly appreciate your critical comments. According to your suggestions, we revised as follows:

Page 10, line 232

3. The Suppl. Figures should be renumbered according to the order they are first mentioned in the text.

I would greatly appreciate your important comments. According to your comments, we renumbered the Suppl. Figures according to the order.

S5 Fig was changed to S1 Fig.

S1 Fig was changed to S2 Fig.

S8 Fig was changed to S3 Fig.

S6 Fig was changed to S4 Fig.

S7 Fig was changed to S5 Fig.

S2 Fig was changed to S6 Fig.

S3 Fig was changed to S7 Fig.

S4 Fig was changed to S8 Fig.

4. I suggest to use the term “embryonic”, instead of the term “embryonal”. It looks that my comment in the previous review was not understood correctly.

I would greatly appreciate your important comments. According to your suggestions, we revised to “embryonic”, instead of the term “embryonal”.

Attachment

Submitted filename: Response_Reviewer.docx

Click here for additional data file.^{(19KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0294893.r005

Decision Letter 2

Carlos Oliva

7 Nov 2023

PONE-D-23-02973R2Loss of Aspm causes increased apoptosis of developing neuronal cells during mouse cerebral corticogenesisPLOS ONE

Dear Dr. Itoh,

Thank you for submitting your manuscript to PLOS ONE. After this round of revision, both reviewers agreed that most of the previous comments were addressed, however there are still minor critics to consider before publication. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

In particular, one of the reviewers asked to revise some of the conclusions of the manuscript, based on the data presented. Since, it is possible that this will be the last round of revision, I recommend to do a final check of typos in your manuscript. Please submit your revised manuscript by Dec 22 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Carlos Oliva, PhD

Academic Editor

PLOS ONE

Journal Requirements:

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: The authors have significantly improved this manuscript, and their conclusions about NPC apoptosis are well supported by their data. However, the data about neuronal apoptosis is still weak. Thus, I have some minor suggestions to make their conclusions more accurate:

Line 1: replace neuronal by neural

Line 32: eliminate "and postmitotic neurons"

Line 34: eliminate "neuronal"

Line 73: eliminate "and neurons"

Line 295: eliminate "and postmitotic neurons"

Reviewer #2: The comments made to the previous versions of the manuscript were addressed in the revised R2 version

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

PLoS One. 2023 Nov 29;18(11):e0294893. doi: 10.1371/journal.pone.0294893.r006

Author response to Decision Letter 2

7 Nov 2023

I would greatly appreciate the reviewer #1’s critical comments, and I revised the manuscript: PONE-D-23-02973R2 according to the reviewer’s comments.

Line 1: replace neuronal by neural

I replaced neuronal by neural.

Line 32: eliminate "and postmitotic neurons"

I eliminated "and postmitotic neurons"

Line 34: eliminate "neuronal"

I am sorry but I could not find "neuronal" in Line 34.

Line 73: eliminate "and neurons"

I eliminated "and neurons"

Line 295: eliminate "and postmitotic neurons"

I eliminated "and postmitotic neurons"

Attachment

Submitted filename: Response_Reviewer_1108.docx

Click here for additional data file.^{(13KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0294893.r007

Decision Letter 3

Carlos Oliva

13 Nov 2023

Loss of Aspm causes increased apoptosis of developing neural cells during mouse cerebral corticogenesis

PONE-D-23-02973R3

Dear Dr. Itoh,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Carlos Oliva, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0294893.r008

Acceptance letter

Carlos Oliva

17 Nov 2023

PONE-D-23-02973R3

Loss of Aspm causes increased apoptosis of developing neural cells during mouse cerebral corticogenesis

Dear Dr. Itoh:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at customercare@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Carlos Oliva

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. TUNEL staining of postnatal mouse brain at P7.

(TIF)

Click here for additional data file.^{(2.1MB, tif)}

S2 Fig. Aspm cKO and wild type mice.

(TIF)

Click here for additional data file.^{(856.6KB, tif)}

S3 Fig. Thickness of the ventricular zone in developing mouse cortex.

(TIF)

Click here for additional data file.^{(467.2KB, tif)}

S4 Fig. Cortical layer thickness in developing mouse cortex.

(TIF)

Click here for additional data file.^{(912.5KB, tif)}

S5 Fig. Identification of EdU labeled cells by neural marker.

(TIF)

Click here for additional data file.^{(2MB, tif)}

S6 Fig. Immunohistochemistry for cleaved caspase 3 and nuclear condensation.

(TIF)

Click here for additional data file.^{(2.4MB, tif)}

S7 Fig. TUNEL staining and neuronal makers of the Aspm cKO mice.

(TIF)

Click here for additional data file.^{(3MB, tif)}

S8 Fig. DNA damages occurred in NPCs of Aspm cKO.

(TIF)

Click here for additional data file.^{(2MB, tif)}

S1 Raw images. Raw images of genotyping result (S2 Fig).

(PDF)

Click here for additional data file.^{(10.5MB, pdf)}

Attachment

Submitted filename: Response to the reviewers_2_ki_0612.docx

Click here for additional data file.^{(4.5MB, docx)}

Attachment

Submitted filename: Response_Reviewer.docx

Click here for additional data file.^{(19KB, docx)}

Attachment

Submitted filename: Response_Reviewer_1108.docx

Click here for additional data file.^{(13KB, docx)}

Data Availability Statement

[pone.0294893.ref001] 1.Roberts E, Hampshire DJ, Pattison L, Springell K, Jafri H, Corry P, et al. Autosomal recessive primary microcephaly: an analysis of locus heterogeneity and phenotypic variation. J Med Genet. 2002;39: 718–721. doi: 10.1136/jmg.39.10.718 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0294893.ref002] 2.Nicholas AK, Swanson EA, Cox JJ, Karbani G, Malik S, Springell K, et al. The molecular landscape of ASPM mutations in primary microcephaly. J Med Genet. 2009;46: 249–253. doi: 10.1136/jmg.2008.062380 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0294893.ref003] 3.Thornton GK, Woods CG. Primary microcephaly: do all roads lead to Rome? Trends Genet. 2009;25: 501–510. doi: 10.1016/j.tig.2009.09.011 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0294893.ref004] 4.Bond J, Roberts E, Mochida GH, Hampshire DJ, Scott S, Askham JM, et al. ASPM is a major determinant of cerebral cortical size. Nat Genet. 2002;32: 316–320. doi: 10.1038/ng995 [DOI] [PubMed] [Google Scholar]

[pone.0294893.ref005] 5.Passemard S, Verloes A, Billette de Villemeur T, Boespflug-Tanguy O, Hernandez K, Laurent M, et al. Abnormal spindle-like microcephaly-associated (ASPM) mutations strongly disrupt neocortical structure but spare the hippocampus and long-term memory. Cortex. 2016;74: 158–176. doi: 10.1016/j.cortex.2015.10.010 [DOI] [PubMed] [Google Scholar]

[pone.0294893.ref006] 6.Desir J, Cassart M, David P, Van Bogaert P, Abramowicz M. Primary microcephaly with ASPM mutation shows simplified cortical gyration with antero-posterior gradient pre- and post-natally. Am J Med Genet A. 2008;146A: 1439–1443. doi: 10.1002/ajmg.a.32312 [DOI] [PubMed] [Google Scholar]

[pone.0294893.ref007] 7.Higgins J, Midgley C, Bergh A, Bell SM, Askham JM, Roberts E, et al. Human ASPM participates in spindle organisation, spindle orientation and cytokinesis. BMC Cell Biol. 2010;11. doi: 10.1186/1471-2121-11-85 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0294893.ref008] 8.Paramasivam M, Chang Y, LoTurco JJ. ASPM and citron kinase co-localize to the midbody ring during cytokinesis. Cell Cycle. 2007;6: 1605–1612. doi: 10.4161/cc.6.13.4356 [DOI] [PubMed] [Google Scholar]

[pone.0294893.ref009] 9.Fujimori A, Itoh K, Goto S, Hirakawa H, Wang B, Kokubo T, et al. Disruption of Aspm causes microcephaly with abnormal neuronal differentiation. Brain Dev. 2014;36: 661–669. doi: 10.1016/j.braindev.2013.10.006 [DOI] [PubMed] [Google Scholar]

[pone.0294893.ref010] 10.Mori M, Tando S, Ogi H, Tonosaki M, Yaoi T, Fujimori A, et al. Loss of abnormal spindle-like, microcephaly-associated (Aspm) disrupts female folliculogenesis in mice during maturation and aging. Reprod Biol. 2022;22: 100673. doi: 10.1016/j.repbio.2022.100673 [DOI] [PubMed] [Google Scholar]

[pone.0294893.ref011] 11.Kouprina N, Pavlicek A, Collins NK, Nakano M, Noskov VN, Ohzeki JI, et al. The microcephaly ASPM gene is expressed in proliferating tissues and encodes for a mitotic spindle protein. Hum Mol Genet. 2005;14: 2155–2165. doi: 10.1093/hmg/ddi220 [DOI] [PubMed] [Google Scholar]

[pone.0294893.ref012] 12.Capecchi MR, Pozner A. ASPM regulates symmetric stem cell division by tuning Cyclin e ubiquitination. Nat Commun. 2015;6: 8763. doi: 10.1038/ncomms9763 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0294893.ref013] 13.Fish JL, Kosodo Y, Enard W, Paabo S, Huttner WB. Aspm specifically maintains symmetric proliferative divisions of neuroepithelial cells. Proc Natl Acad Sci. 2006;103: 10438–10443. doi: 10.1073/pnas.0604066103 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0294893.ref014] 14.Fujimori A, Yaoi T, Ogi H, Wang B, Suetomi K, Sekine E, et al. Ionizing radiation downregulates ASPM, a gene responsible for microcephaly in humans. Biochem Biophys Res Commun. 2008;369: 953–957. doi: 10.1016/j.bbrc.2008.02.149 [DOI] [PubMed] [Google Scholar]

[pone.0294893.ref015] 15.Kato TA, Okayasu R, Jeggo PA, Fujimori A. ASPM influences DNA double-strand break repair and represents a potential target for radiotherapy. Int J Radiat Biol. 2011;87: 1189–1195. doi: 10.3109/09553002.2011.624152 [DOI] [PubMed] [Google Scholar]

[pone.0294893.ref016] 16.Isaka F, Ishibashi M, Taki W, Hashimoto N, Nakanishi S, Kageyama R. Ectopic expression of the bHLH gene Math1 disturbs neural development. Eur J Neurosci. 1999;11: 2582–2588. doi: 10.1046/j.1460-9568.1999.00699.x [DOI] [PubMed] [Google Scholar]

[pone.0294893.ref017] 17.Arai Y, Pulvers JN, Haffner C, Schilling B, Nüsslein I, Calegari F, et al. Neural stem and progenitor cells shorten S-phase on commitment to neuron production. Nat Commun. 2011;2: 154. doi: 10.1038/ncomms1155 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0294893.ref018] 18.Williams SE, Garcia I, Crowther AJ, Li S, Stewart A, Liu H, et al. Aspm sustains postnatal cerebellar neurogenesis and medulloblastoma growth in mice. Development. 2015;142: 3921–3932. doi: 10.1242/dev.124271 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0294893.ref019] 19.Konno D, Shioi G, Shitamukai A, Mori A, Kiyonari H, Miyata T, et al. Neuroepithelial progenitors undergo LGN-dependent planar divisions to maintain self-renewability during mammalian neurogenesis. Nat Cell Biol. 2008;10: 93–101. doi: 10.1038/ncb1673 [DOI] [PubMed] [Google Scholar]

[pone.0294893.ref020] 20.Paridaen JT, Huttner WB. Neurogenesis during development of the vertebrate central nervous system. EMBO Reports. 2014. pp. 351–364. doi: 10.1002/embr.201438447 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0294893.ref021] 21.Jayaraman D, Kodani A, Gonzalez DM, Mancias JD, Mochida GH, Vagnoni C, et al. Microcephaly Proteins Wdr62 and Aspm Define a Mother Centriole Complex Regulating Centriole Biogenesis, Apical Complex, and Cell Fate. Neuron. 2016;92: 813–828. doi: 10.1016/j.neuron.2016.09.056 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0294893.ref022] 22.Johnson MB, Sun X, Kodani A, Borges-monroy R, Girskis KM, Patel K, et al. Aspm knockout ferret reveals an evolutionary mechanism governing cerebral cortical size. Nature. 2018;556: 370–375. doi: 10.1038/s41586-018-0035-0 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0294893.ref023] 23.Bae I, Rih JK, Kim HJ, Kang HJ, Hassan B, Kirilyuk A, et al. BRCA1 Regulates Gene Expression for Orderly Mitotic Progression. Cell Cycle. 2005;4: 1641–1666. doi: 10.4161/cc.4.11.2152 [DOI] [PubMed] [Google Scholar]

[pone.0294893.ref024] 24.Zhong X, Liu L, Zhao A, Pfeifer GP, Xu X. The abnormal spindle-like, microcephaly-associated (ASPM) gene encodes a centrosomal protein. Cell Cycle. 2005;4: 1227–1229. doi: 10.4161/cc.4.9.2029 [DOI] [PubMed] [Google Scholar]

[pone.0294893.ref025] 25.Xu S, Wu X, Wang P, Cao SL, Peng B, Xu X. ASPM promotes homologous recombination-mediated DNA repair by safeguarding BRCA1 stability. iScience. 2021;24: 102534. doi: 10.1016/j.isci.2021.102534 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0294893.ref026] 26.Isono M, Niimi A, Oike T, Hagiwara Y, Sato H, Sekine R, et al. BRCA1 Directs the Repair Pathway to Homologous Recombination by Promoting 53BP1 Dephosphorylation. Cell Rep. 2017;18: 520–532. doi: 10.1016/j.celrep.2016.12.042 [DOI] [PubMed] [Google Scholar]

[pone.0294893.ref027] 27.Keil JM, Doyle DZ, Qalieh A, Lam MM, Funk OH, Qalieh Y, et al. Symmetric neural progenitor divisions require chromatin-mediated homologous recombination DNA repair by Ino80. Nat Commun. 2020;11: 3839. doi: 10.1038/s41467-020-17551-4 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0294893.ref028] 28.Novorol C, Burkhardt J, Wood KJ, Iqbal A, Roque C, Coutts N, et al. Microcephaly models in the developing zebrafish retinal neuroepithelium point to an underlying defect in metaphase progression. Open Biol. 2013;3: 130065. doi: 10.1098/rsob.130065 [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Loss of Aspm causes increased apoptosis of developing neural cells during mouse cerebral corticogenesis

Madoka Tonosaki

Akira Fujimori

Takeshi Yaoi

Kyoko Itoh

Roles

Abstract

Introduction

Materials and methods

Animals

Genotyping

EdU and BrdU labeling

Histological and immunofluorescence analyses

Image analysis

Fig 1. Embryonic brain of Aspm cKO.

Fig 2. Cell proliferation of NPCs during corticogenesis.

Fig 3. Neurogenesis during murine corticogenesis.

Statistical analysis

Results

Cerebral cortical reduction in Aspm cKO was exhibited at the late embryonic stage

Loss of Aspm did not affect the proliferation of NPCs

Loss of Aspm causes reduction in mid to late born neurons

Loss of Aspm led to an increase in apoptosis of NPCs

Fig 4. TUNEL assay.

DNA-damaged cells are increased along with the loss of Aspm in developing cortex

Fig 5. Immunohistochemistry for γH2AX.

Discussion

Loss of Aspm reduced cerebral cortical thickness with a thinning of all cortical layers

Loss of Aspm is involved in apoptosis induction during embryonic neocortex formation, not in postnatal brain

Aspm is required for stabilization of symmetric NPC division

Conclusion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Carlos Oliva

Roles

Author response to Decision Letter 0

Decision Letter 1

Carlos Oliva

Roles

Author response to Decision Letter 1

Decision Letter 2

Carlos Oliva

Roles

Author response to Decision Letter 2

Decision Letter 3

Carlos Oliva

Roles

Acceptance letter

Carlos Oliva

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases