Requirement of Mouse BCCIP for Neural Development and Progenitor Proliferation

Multiple DNA repair pathways are involved in the orderly development of neural systems at distinct stages. The homologous recombination (HR) pathway is required to resolve stalled replication forks and critical for the proliferation of progenitor cells during neural development. BCCIP is a BRCA2 and CDKN1A interacting protein implicated in HR and inhibition of DNA replication stress. In this study, we determined the role of BCCIP in neural development using a conditional BCCIP knock-down mouse model. BCCIP deficiency impaired embryonic and postnatal neural development, causing severe ataxia, cerebral and cerebellar defects, and microcephaly. These development defects are associated with spontaneous DNA damage and subsequent cell death in the proliferative cell populations of the neural system during embryogenesis. With in vitro neural spheroid cultures, BCCIP deficiency impaired neural progenitor's self-renewal capability, and spontaneously activated p53. These data suggest that BCCIP and its anti-replication stress functions are essential for normal neural development by maintaining an orderly proliferation of neural progenitors

Essential Roles of BCCIP in Mouse Embryonic Development and Structural Stability of Chromosomes

BCCIP is a BRCA2- and CDKN1A(p21)-interacting protein that has been implicated in the maintenance of genomic integrity. To understand the in vivo functions of BCCIP, we generated a conditional BCCIP knockdown transgenic mouse model using Cre-LoxP mediated RNA interference. The BCCIP knockdown embryos displayed impaired cellular proliferation and apoptosis at day E7.5. Consistent with these results, the in vitro proliferation of blastocysts and mouse embryonic fibroblasts (MEFs) of BCCIP knockdown mice were impaired considerably. The BCCIP deficient mouse embryos die before E11.5 day. Deletion of the p53 gene could not rescue the embryonic lethality due to BCCIP deficiency, but partially rescues the growth delay of mouse embryonic fibroblasts in vitro. To further understand the cause of development and proliferation defects in BCCIP-deficient mice, MEFs were subjected to chromosome stability analysis. The BCCIP-deficient MEFs displayed significant spontaneous chromosome structural alterations associated with replication stress, including a 3.5-fold induction of chromatid breaks. Remarkably, the BCCIP-deficient MEFs had a ∼20-fold increase in sister chromatid union (SCU), yet the induction of sister chromatid exchanges (SCE) was modestly at 1.5 fold. SCU is a unique type of chromatid aberration that may give rise to chromatin bridges between daughter nuclei in anaphase. In addition, the BCCIP-deficient MEFs have reduced repair of irradiation-induced DNA damage and reductions of Rad51 protein and nuclear foci. Our data suggest a unique function of BCCIP, not only in repair of DNA damage, but also in resolving stalled replication forks and prevention of replication stress. In addition, BCCIP deficiency causes excessive spontaneous chromatin bridges via the formation of SCU, which can subsequently impair chromosome segregations in mitosis and cell division

Search for eccentric black hole coalescences during the third observing run of LIGO and Virgo

Despite the growing number of confident binary black hole coalescences observed through gravitational waves so far, the astrophysical origin of these binaries remains uncertain. Orbital eccentricity is one of the clearest tracers of binary formation channels. Identifying binary eccentricity, however, remains challenging due to the limited availability of gravitational waveforms that include effects of eccentricity. Here, we present observational results for a waveform-independent search sensitive to eccentric black hole coalescences, covering the third observing run (O3) of the LIGO and Virgo detectors. We identified no new high-significance candidates beyond those that were already identified with searches focusing on quasi-circular binaries. We determine the sensitivity of our search to high-mass (total mass M>70 M⊙) binaries covering eccentricities up to 0.3 at 15 Hz orbital frequency, and use this to compare model predictions to search results. Assuming all detections are indeed quasi-circular, for our fiducial population model, we place an upper limit for the merger rate density of high-mass binaries with eccentricities 0<e≤0.3 at 0.33 Gpc−3 yr−1 at 90\% confidence level

Ultralight vector dark matter search using data from the KAGRA O3GK run

Among the various candidates for dark matter (DM), ultralight vector DM can be probed by laser interferometric gravitational wave detectors through the measurement of oscillating length changes in the arm cavities. In this context, KAGRA has a unique feature due to differing compositions of its mirrors, enhancing the signal of vector DM in the length change in the auxiliary channels. Here we present the result of a search for U(1)B−L gauge boson DM using the KAGRA data from auxiliary length channels during the first joint observation run together with GEO600. By applying our search pipeline, which takes into account the stochastic nature of ultralight DM, upper bounds on the coupling strength between the U(1)B−L gauge boson and ordinary matter are obtained for a range of DM masses. While our constraints are less stringent than those derived from previous experiments, this study demonstrates the applicability of our method to the lower-mass vector DM search, which is made difficult in this measurement by the short observation time compared to the auto-correlation time scale of DM

BCCIP knock-down causes apoptotic cell death and reduction of cell proliferation capacity in the neocortices progenitor cells.

<p>The brain tissues form E15.5 embryos were subjected to IHC and H&E staining analyses. In all panels: CP - cortical plate; IZ - intermediate zone; VZ - ventricular zone. (<b>A</b>) illustrates the magnified views of H&E staining of littermate <i>BCCIP-CON</i> and <i>BCCIP-CKD</i> brain sections. Representative of clusters of pyknotic cell nuclei in the <i>BCCIP-CKD</i> ventricular zone are indicated by arrows. The right panels of 6A show the enlarged images of selected areas of the middle panels. (<b>B</b>) Quantification of clusters of pyknotic nuclei. (<b>C</b>) The apoptotic cells from the same were detected by TUNEL staining. (<b>D</b>) Quantification of TUNEL staining. (<b>E</b>) Proliferative cells were detected by anti-Ki67 staining (column 1), and BrdU incorporation (column 2) at E15.5 (about 2 days after the GFAP-Cre is expressed). (<b>F</b>) Quantification of Ki67 staining. (<b>G</b>) Quantification of BrdU staining. Error bars are standard deviation. White bars: BCCIP-CON; Gray bars: <i>BCCIP-CKD</i>. The asterisks indicate the statistic significance between the <i>BCCIP-CON</i> and <i>BCCIP-CKD</i>, *: P<0.05; **: P<0.01; ***: P<0.001.</p

BCCIP knock-down increased apoptosis in the mitotic proliferating region of <i>BCCIP-CKD</i> neocortices.

<p>(A) βIII-tubulin staining identifies the differentiating neural cell populations. Analysis of apoptosis was done by cleaved-caspase 3 (c-C3) staining in E14.5 using <i>BCCIP-CON</i> and <i>BCCIP-CKD</i> embryos. The merged composites are overlay of βIII-tubulin and DPAI staining. (<b>B</b>) Ki67 staining identifies the proliferating mitotic region. Analysis of apoptosis was done by cleaved-caspase 3 (c-C3) staining in E14.5 using <i>BCCIP-CON</i> and <i>BCCIP-CKD</i> embryos. (<b>C</b>) Apoptosis was analyzed by TUNEL assay in E14.5 using <i>BCCIP-CON</i> and <i>BCCIP-CKD</i> embryos. (<b>D</b>) Quantification of TUNEL staining. The amount of apoptosis was quantified in the VZ/SVZ of E14.5 embryos. (<b>E</b>) Quantification of cleaved-caspase 3 staining. The amount of apoptosis was quantified in the VZ/SVZ of E14.5 embryos. CP: cortical plate. VZ/SVZ: ventricular zone/subventricular zone. White bars: <i>BCCIP-CON</i>; Gray bars: <i>BCCIP-CKD</i>. The merged composites are overlay of Ki67 and DAPI staining. CP: cortical plate. VZ/SVZ: ventricular zone/subventricular zone. *: P<0.05; **: P<0.01; ***: P<0.001.</p

BCCIP knockdown leads to reduced BrdU incorporation and increased apoptosis in neural progenitor cells.

<p>BrdU incorporation and TUNEL assays on single cell suspension of the primary spheroid cultures were performed to assess the proliferation and apoptosis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030638#s4" target="_blank">Materials and Methods</a> for details). (<b>A</b>) shows representative images of BrdU and TUNEL staining. (<b>C</b>) and (<b>B</b>) show the quantification of BrdU staining and TUNEL staining. We scored 500 cells for each cell line after 7 days culture. In all panels, white bars: <i>BCCIP-CON</i>; gray bars: <i>BCCIP-CKD</i>. *: P<0.05; **: P<0.01; ***: P<0.001. Asterisks indicate significant differences and n indicates the number of individual neuroprogenitor cell lines analyzed.</p

Mechanisms by which BCCIP deficiency lead to proliferation defect of the progenitor cells during brain development.

<p>Mechanisms by which BCCIP deficiency lead to proliferation defect of the progenitor cells during brain development.</p

Histological analysis of cerebellum and sagittal brain sections of <i>BCCIP-CON</i> and <i>BCCIP-CKD</i> mice.

<p>(<b>A</b>) Histology of cerebellum of <i>BCCIP-CON</i> (top row) and <i>BCCIP-CKD</i> (bottom row) mice at age P56. Column 1 shows Hematoxylin and Eosin (H&E) staining of cerebellum at magnification of 40×. Columns 2–4 show the immuno-histochemical staining for Calbindin (D-28K) that is specific for Purkinje cells (brown color in column 2), NeuN that is specific for neurons (brown color in column 3), and for GFAP is specific for glial cells (brown color in column 4). ML: molecular layers; GCL: granule cell layer; PC: Purkinje cells. (<b>B</b>) Histology of cerebrum of <i>BCCIP-CON</i> and <i>BCCIP-CKD</i> mice at age P56. Column 1 shows the H&E staining at 40× magnification. Columns 2 and 3 show the reduced neuron density in BCCIP-CKD mice as visualized by anti-NeuN staining. Columns 4 shows reduced density of glial cells (stained with anti-GFAP) in the <i>BCCIP-CKD</i> mice. Hippo: hippocampus; DG: dentate gyrus; CA1, CA2 and CA3: pyramidal cell layer of the hippocampus; Cx: cortex; SGZ: subgranular zone.</p