Neurogenesis requires TopBP1 to prevent catastrophic replicative DNA damage in early progenitors

Abstract

The rapid proliferation of progenitors during neurogenesis requires a stringent genomic maintenance program to ensure transmission of genetic fidelity. However the essential factors that govern neural progenitor genome integrity are unknown. Here we report that conditional inactivation of mouse TopBP1, a protein linked to DNA replication, and a key activator of the DNA damage response kinase ATR (ataxia telangiectasia and rad3-related) is critical for maintenance of early-born neural progenitors. During cortical development TopBP1 prevented replication-associated DNA damage in Emx1-progenitors which otherwise resulted in profound tissue ablation. Notably, disrupted neurogenesis in TopBP1-depleted tissues was substantially rescued by inactivation of p53 but not of ATM. Our data establish that TopBP1 is essential for preventing replication-associated DNA strand breaks, but is not essential per se for DNA replication. Thus, TopBP1 is crucial for maintaining genome integrity in the early progenitors that drive neurogenesis.

Figure 1. TopBP1 deletion in the nervous system

(a) TopBP1 contains eight BRCA1 C-terminal (BRCT) domains. These encompass an amino terminal TRESLIN/Ticrr interacting domain, a C-terminal ATR-activating, a BACH1-binding region and an ATM phosphorylation site in BRCT domain 5. A conditional TopBP1 allele has LoxP sites flanking exons 3 and 6 and generates an out-of-frame mutation after cre-mediated recombination. (b) TopBP1 protein is absent in P0 TopBP1^Nes-cre brain tissues but is present at normal levels in the thymus and spleen, while Nbs1 and actin levels are similar in all mutant tissues. (c) TopBP1^Nes-cre neonates (asterisks) are initially similar to littermate controls, but become runted by P4. (d) The TopBP1^Nes-cre cerebellum lacks granule neurons resulting in a disorganized Purkinje cell placement as shown at P0 using calbindin staining and at P12 using the neuronal differentiation marker βtubulin III (Tuj1). (e) Quantitative real-time PCR shows TopBP1 deletion occurs by E12.5 in the cortex but not the liver. (f) Western blot analysis of E14.5 TopBP1^Nes-cre tissues shows that TopBP1 is deleted in the cortex but not the liver; p53pSer18 expression is also elevated in TopBP1^Nes-cre tissue. Nbs1 and Ponceau staining are loading and blotting controls, respectively. (g) Apoptosis (arrows) occurs in proliferating cells in the developing cortex at E13.5.

Figure 2. Apoptosis and cortical layering disruption in the TopBP1^Nes-cre brain

(a) Apoptosis in the TopBP1^Nes-cre E16.5 cortex is associated with PCNA+ proliferating cells but not BLBP+ radial glia progenitors. (b) Apical progenitors (arrows) were identified using H3pSer10 immunostaining in E13.5 TopBP1^Nes-cre and control neocortex. (c) Analysis of cortical development in P0 and P10 TopBP1^Nes-cre and WT tissue using Tbr1 and Ctip2 to mark layers VI-IV, Cux1 to identify layers IV-II and Brn2/Satb2 to identify layers V-II reveals disrupted layer development in the TopBP1^Nes-cre cortex. VZ is ventricular zone. Capital Roman numerals indicate the cortical layers.

Figure 3. Dorsal telencephalon progenitors are lost in TopBP1^Emx1-cre mice

(a) Cortices and other structures developing from the dorsal telencephalon are missing in the TopBP1^Emx1-cre brain; shown by Nissl staining at P5. CC is the corpus callosum, hippo is the hippocampus and RMS is the rostral migratory stream. (b) Abundant apoptosis is observed in the E13.5 TopBP1^Emx1-cre neopallial cortex shown using TUNEL and activated caspase-3 staining. Differences in the numbers of apoptotic cells between genotypes calculated using a student’s t-test were significantly different; active caspase 3 positive cells in 1 mm² (± SEM) were; control = 0; TopBP1^Emx1-cre = 734.5 (± 30.58), and TUNEL positive cells in 1 mm² (± SEM) were; control = 7 (± 0.81) and TopBP1^Emx1-cre = 987.5 (± 27.381). (c) Fewer proliferating cells indicated by histone H3pSer10 or BrdU incorporation staining are seen in the E13.5 TopBP1^Emx1-cre neopallial cortex. Differences in the numbers of proliferating cells between genotypes calculated using a t-test were significantly different; BrdU positive cells in 0.27 mm² (± SEM) were; control = 438.4 (±26.48) TopBP1^Emx1-cre = 205.6 (± 10.31) and H3pS10 positive cells in 1 mm² (± SEM) were; control = 91 (± 4.589), TopBP1^Emx1-cre = 27.69 (± 1.707). Widespread DNA damage shown by γH2AX and PCNA immunostaining in regions indicated in white boxes in adjacent panels.

Figure 4. Analysis of cortical development in Lig4^Emx1-cre and Xrcc1^Emx1-cre brain

(a) Nissl staining identifies the Lig4^Emx1-cre and Xrcc1^Emx1-cre cortex (Ctx) and hippocampus (DG and CA1) and shows the relative levels of defective development after Lig4 or Xrcc1 loss. Relative gene deletion (± SEM) was determined in genomic DNA isolated from the cortex using realtime PCR. For Xrcc1; WT = 1.22 (± 0.033), Xrcc1^Emx1-cre = 0.46 (± 0.006). For Lig4; WT = 0.86 (± 0.038), Lig4^Emx1-cre = 0.19 (± 0.004). Tbr2 marks the dentate gyrus (DG). (b) Cortical markers were used to assess development in the Lig4^Emx1-cre and Xrcc1^Emx1-cre cortex. Roman numerals indicate the cortical layers. (c) DNA damage (γH2AX) accumulates in the Lig4^Emx1-cre cortex; DG is the dentate gryus.

Figure 5. Defective neurogenesis after TopBP1 inactivation requires p53, but not Atm signaling

(a) The hippocampal defects in TopBP1^Nes-cre brain involve p53 signaling as TopBP1^Nes-cre;p53^−/− mice show partial rescue of hippocampal dentate gyrus development as shown using calbindin immunostaining to assess histology at postnatal day 5 (P5). In contrast to p53, Atm loss failed to rescue defective TopBP1^Nes-cre hippocampal development. Despite recovered development, TopBP1 loss nonetheless resulted in persistent DNA damage as demonstrated by γH2AX foci. (b) Analysis of apoptosis using TUNEL or proliferation using H3pSer10 indicates that p53, but not Atm signaling is involved in the TopBP1^Nes-cre phenotype. (c) Cortices that fail to develop in TopBP1^Emx1-cre mice are rescued by coincident loss of p53 (red-hatched outlines). However, the hippocampal formation is not rescued in the TopBP1^Emx1-cre;p53^−/− brain. (d) Although substantial cortical rescue occurs in TopBP1^Emx1-cre;p53^−/− mice, cortical layering shows disorganization as indicated using Cux1 or Tbr1 and Ctip2 co-staining. (e) DNA damage is present throughout the P5 TopBP1^Emx1-cre;p53^−/− cortices (arrows) and γH2AX foci are frequently observed in PCNA-positive proliferating cells (arrowhead); P5 is postnatal day 5. DG is dentate gyrus.

Figure 6. TopBP1-deficiency during neurogenesis results in DNA strand break accumulation in cortical progenitors

(a) Embryonal neural cells isolated from TopBP1^Nes-cre and TopBP1^Emx1-cre accumulate significant DNA damage compared to wild type controls. Comet analysis of acutely isolated cells from E13.5 TopBP1^Nes-Cre (dark blue; six individual TopBP1^Nes-cre embryo isolates and brown is data from a TopBP1^Nes-cre;p53^−/− embryo) or TopBP1^Emx1-cre;p53^−/− (light blue; six individual embryos) dorsal telencephalon show accumulated DNA damage in situ compared to controls (mauve; 14 individual embryo isolates); a minimum of 300 comet-tail moments were measured for each sample and mean comet-tail moments for each replicate are shown. Mean comet tail moments of irradiated P6 cerebellar granule neurons at increasing doses (orange) are shown in comparison as a standard to quantify DNA strand breaks. Inset scatterplots indicate actual comet tail moment values of 100 representative cells analyzed from the respective genotypes. The inset boxplot indicates the mean comet tail moment of all replicates within each genotype. (b) Cortical progenitors at E11.5 and E12.5 are significantly more susceptible to DNA damage-induced apoptosis than at those at E14.5. Quantitation of TUNEL positive cells was determined in proportion to propidium iodide-stained cells in the neocortex after 0.2Gy of radiation. VZ is ventricular zone.

Figure 7. Analysis of DNA damage responses in TopBP1-depleted cells

(a) The radiation-induced DNA damage response was investigated using tamoxifen (4-OHT)-induced deletion of TopBP1 in mouse embryonic fibroblasts (MEFs): WT are TopBP1^+/+;Cre^TM and KO are TopBP1^LoxP/LoxP;Cre^TM. TopBP1, Chk2, p53(p-ser18), Chk1(p-ser317) and actin indicate the result of probing with the respective antibodies. Activation of Chk2 is indicated by mobility shift (arrow). Ponceau staining and actin immunostaining indicates protein loading. Asterisk indicates decreased Chk1(p-ser317) immunostaining after TopBP1 inactivation. (b) Cerebellar extracts or astrocytes were from wild type or TopBP1^Nes-cre mice and were immunoblotted as described above for MEFs, but also included antibodies recognizing total Chk1 and glial fibrillary acidic protein (GFAP); longer exposure reveals clear GFAP staining in the cerebellum (not shown). Trace TopBP1 signal in replication-defective null-astrocytes is from meningeal fibroblast contamination. Radiation was 4Gy with 2h recovery. (c) TopBP1^Nes-cre;p53^−/− astrocytes show elevated levels of endogenous DNA damage indicated by γH2AX immunostaining (arrows). Astrocyte purity is shown by GFAP immunostaining. (d) DNA replication in control and TopBP1^Nes-cre:p53^−/− astrocytes was determined using EdU pulsing after BrdU pre-labeling. TopBP1 deficiency markedly compromised DNA replication as shown by a significant reduction of EdU uptake; *= p<0.001. (e) DNA repair in TopBP1^Nes-cre;p53^−/− astrocytes determined by quantifying γH2AX foci removal after IR (n=50 cells); R0 and R240 represent recovery after 0 and 240 minutes. (f) DNA repair was assessed after IR (10 Gy) and camptothecin (14μM) using the alkaline comet assay; Tdp1^−/− primary astrocytes were established from tyrosyl-DNA phosphodiesterase- 1 null mice. R60 represents recovery after 60 minutes.