Robust elimination of genome-damaged cells safeguards against brain somatic aneuploidy following Knl1 deletion

The brain is a genomic mosaic shaped by cellular responses to genome damage. Here, we manipulate somatic genome stability by conditional Knl1 deletion from embryonic mouse brain. KNL1 mutations cause microcephaly and KNL1 mediates the spindle assembly checkpoint, a safeguard against chromosome missegregation and aneuploidy. We find that following Knl1 deletion, segregation errors in mitotic neural progenitor cells give rise to DNA damage on the missegregated chromosomes. This triggers rapid p53 activation and robust apoptotic and microglial phagocytic responses that extensively eliminate cells with somatic genome damage, thus causing microcephaly. By leaving only karyotypically normal progenitors to continue dividing, these mechanisms provide a second safeguard against brain somatic aneuploidy. Without Knl1 or p53-dependent safeguards, genome-damaged cells are not cleared, alleviating microcephaly, but paradoxically leading to total pre-weaning lethality. Thus, mitotic genome damage activates robust responses to eliminate somatic mutant cells, which if left unpurged, can impact brain and organismal fitness.

a CC3 immunostaining showed extensive apoptosis in E15.5 cKO cortex. b Pyknotic cells (arrowheads) revealed by DAPI staining (magenta) were immunopositive for CC3 (green), indicating that they were apoptotic (scale bar: 20µm). c Analysis of S-phase cells labeled by a 4-hour pulse of CldU and M-phase cells labeled by pHH3 revealed a significant increase in M-phase cells in the E15.5 cKO cortex, suggesting incompletion or delayed completion of mitosis (mean, two-tailed unpaired t test, n=3 animals). Some pHH3+ cells were abventricular (open arrowheads). d The increase in pHH3 staining was present at E13.5, preceding widespread apoptosis (mean, two-tailed unpaired t test, n=3 animals). e Analysis of E16.5 cortex following a 24-hour pulse of EdU. EdU+ migrating neurons (red arrowheads) were present in the IZ of ctrl but largely absent from cKO (scale bar: 20µm).
a Aggregate assessment of ERCC spike-in standards in UMI RNA-seq revealed excellent correlation over a broad dynamic range. The vast majority of genes from all 16 experimental samples were within the dynamic range of UMI RNA-seq. TPM, transcripts per million. b ddRT-PCR of p53 target gene Bbc3 (Puma) in cKO compared to ctrl (mean, two-tailed unpaired t test, ctrl: n=6, cKO: n=7 animals). C Immunostaining revealed VZ cells labeled by heterochromatic DSB marker pKAP1 in E15.5 cKO (scale bar: 20µm). d Co-immunostaining in E15.5 cKO revealed p53 activation (green) in SOX2+ and EOMES+ NPCs (solid arrowheads), but largely not in RBFOX3+ neurons (open arrowheads, scale bar: 20µm). e DNA damage markers, γH2AX and p53, were widespread in cKO VZ at E13.5, when the majority of cells in the cortical wall were NPCs, and preceding massive apoptosis in cKO (scale bar: 50µm).
a Additional examples of frequent lagging and bridged chromosomes (open arrowheads), and micronuclei (solid arrowheads) in anaphase NPCs in E15.5 cKO cortex (scale bar: 2µm). b Lagging chromosomes (open arrowheads) were present in the midzone labeled by phospho-Vimentin (pVIM, yellow). c Missegregated chromosomes and micronuclei (open arrowheads) in E15.5 cKO cortex were immunopositive for DNA damage marker γH2AX (green, and arrows). d The proportion of metaphases with γH2AX foci was significantly increased in cKO compared to ctrl (mean, two-tailed unpaired t test, n=3 animals). e Dissociated E15.5 cortical NPC culture after 2 days in vitro (DIV) and a 12-hour pulse of EdU (red). NPCs cultured from cKO were characterized by DNA bridges (open arrowheads) immunopositive for γH2AX (green). f Single cell electrophoresis comet assay showed a significant increase in comet tailing in cKO compared to ctrl (mean, two-tailed unpaired t test, n=4 animals).
a Dorsal view of ctrl, cKO, and dKO P4 brain (scale bar: 1mm). The significant cortical area loss in cKO compared to littermate control (ctrl) was partially ameliorated in dKO (mean, ANOVA with post hoc t test, ctrl: n=6, cKO: n=3, dKO: n=4 animals). b Cortical layer marker analysis at P4 revealed no change in BCL11B+ (L5, red) neurons (scale bar: 100µm). The reduction in CUX1+ (L2-4, green) neurons in cKO compared to ctrl was partially ameliorated in dKO. c NPC marker analysis of E15.5 cortex revealed no significant decrease in SOX2+ apical progenitors or EOMES+ intermediate progenitors in dKO compared to ctrl (mean, two-tailed unpaired t test, n=3 animals). d Violin plot of all differentially expressed genes from cKO compared to ctrl, and corresponding FDR from dKO compared to ctrl. The majority of differentially expressed genes from cKO compared to ctrl were not differentially expressed in dKO compared to ctrl. e Analysis by ddRT-PCR validated that cKO expression changes in p53dependent genes Ano3, Eda2r, and Pvt1 were corrected to levels similar to ctrl in dKO (mean, ANOVA with post hoc t test, ctrl: n=7, cKO: n=4, dKO: n=4 animals). Bbc3 (Puma) expression was not significantly changed in dKO compared to ctrl (ctrl: n=6, cKO: n=7, dKO: n=5 animals). f Violin plots of all significantly upregulated (red) and downregulated (blue) genes from cKO compared to ctrl, and corresponding log fold change (log FC) from dKO compared to ctrl. The majority of up-and downregulated genes from cKO compared to ctrl were not differentially expressed in dKO compared to ctrl (log FC shifted towards 0).
a Additional examples of lagging and bridged chromosomes (open arrowheads), and micronuclei (solid arrowheads) in anaphase NPCs in E15.5 dKO cortex (scale bar: 2µm). The fraction of anaphase mitoses with missegregation was significantly increased in dKO similar to cKO (mean, ANOVA with post hoc t test, n=4 animals). b Single cell electrophoresis comet assay showed a significant increase in comet tailing in dKO compared to ctrl (mean, ANOVA with post hoc t test, n=4 animals). c Immunostaining revealed no activation of p53 in E15.5 dKO cortex. d Immunostaining of E15.5 cortex revealed inefficient clearance of cells labeled by heterochromatic DSB marker pKAP1 in dKO (scale bar: 100µm). a AIF1 (IBA1, green) immunostaining revealed morphologic activation of microglia in E15.5 cKO and dKO cortex (scale bar: 50µm). b ADGRE1+ (green) microglia were immunopositive for the proliferation marker, KI67 (red), in both E15.5 ctrl and dKO cortex (scale bar: 50µm). c By birth (P0), ADGRE1+ microglia have returned to resting morphology and were mononuclear (open arrowheads) in the cKO (scale bar: 100µm).   All significantly up-and down-regulated genes from cKO vs. ctrl (FDR < 0.05)