Selective utilization of nonhomologous end-joining and homologous recombination DNA repair pathways during nervous system development

Abstract

The repair of DNA double-strand breaks (DSBs) occurs via nonhomologous end-joining (NHEJ) or homologous recombination (HR). These mechanistically distinct pathways are critical for maintenance of genomic integrity and organismal survival. Although inactivation of either pathway leads to embryonic lethality, here we show selective requirements for each DNA DSB repair pathway at different stages of mammalian nervous system development. DNA damage-induced apoptosis resulting from inactivation of HR (Xrcc2 deficiency) only occurred in proliferating neural precursor cells, whereas disruption of NHEJ (DNA ligase IV deficiency) mainly affected differentiating cells at later developmental stages. Therefore, these data suggest that NHEJ is dispensable for a substantial portion of early development because DSB repair during this period utilizes HR. Moreover, DNA damage-induced apoptosis required the ataxia telangiectasia mutated (Atm) kinase after disruption of NHEJ, but not HR, during neurogenesis. However, embryonic lethality arising from disruption of either repair pathway was rescued by loss of p53 and resulted in specific tumor types reflective of the particular DSB repair pathway inactivated. Thus, these data reveal distinct tissue- and cell-type requirements for each DNA DSB repair pathway during neural development and provide insights for understanding the contributions of DNA DSB responses to disease.

Fig. 1.

Inactivation of NHEJ or HR affects different embryonic stages during mouse development. Widespread apoptosis can be found in the Xrcc2^−/− embryo by E10.5 and is illustrated showing neuroepithelium and adjacent cephalic mesenchyme. (A) Ki67 immunoreactivity (red) marks proliferating cells, and TUNEL staining (green) identifies cells undergoing apoptosis. (B) Although apoptosis is widespread in the proliferative cells of the Xrcc2^−/− embryo at E11.5, it is not detectable in WT or Lig4^−/− embryos until E12.5. In the differentiating hindbrain region at E14.5, Lig4, but not Xrcc2, loss is associated with apoptosis. (Magnification: ×200.)

Fig. 2.

Selective utilization of NHEJ and HR during development. At E14.5, apoptosis is found in the forebrain proliferative VZ of the Xrcc2^−/− embryo, whereas in the Lig4^−/− embryo, TUNEL-positive cells are predominantly located in the postmitotic SVZ (indicated by arrows). (A and B) Magnification: ×200; V, ventricle. (C and E) Doublecortin identifies scattered differentiating cells in the VZ. (D and E) DAPI staining shows the whole embryonic forebrain. (Magnification: ×200.) (F–K) TUNEL-positive cells did not colocalize with doublecortin-positive cells in Xrcc2^−/− embryos (F–H) but did so in Lig4^−/− embryos (I–K). (Magnification: ×1,000.)

Fig. 3.

The DNA damage response in Xrcc2^−/− embryos occurs in G₂/M cells of the VZ. Xrcc2 deficiency leads to p53 ser18 phosphorylation (A) and p53 stabilization (A Inset) in a layer of cells at the margin (arrowheads) of the ventricle (V) of an E14.5 embryo. (B) No p53 phosphorylation or stabilization (Inset) is found in WT embryo. (C) The cartoon represents the characteristic position of the progenitor cell nucleus within the VZ as the cell cycle progresses. (D) Cells that are immunopositive for phosphorylated histone H3 staining (green signal) indicate cells in mitosis, and BrdU-positive cells (red signal) mark those in S phase (arrowheads). The VZ and the postmitotic SVZ are marked.

Fig. 4.

Atm is not required for DNA damage signal transduction after Xrcc2 loss. (A) Analysis of apoptosis was done by using TUNEL staining in E14.5 using WT, Lig4^−/−, Lig4^−/−Atm^−/−, Xrcc2^−/−, Xrcc2^−/−Atm^−/−, and Xrcc2^−/−p53^−/− embryos. Tuj1 immunostaining identifies the differentiating neural cell populations. The merged composites are an overlay of Tuj1, TUNEL, and DAPI staining. (B) The amount of apoptosis was quantified in the VZ and the SVZ of the E14.5 nervous system from the indicated mutants. (C) Xrcc2^−/− mice can survive on a p53^−/− or p53^+/− background, whereas Atm loss confers no survival advantage. (D) Phosphorylation (ser1981) of Atm was found in developing brain tissue from Lig4^−/− but not Xrcc2^−/−; f, forebrain; h, hindbrain isolated from E13.5. Specificity of the Atm antibodies is shown using WT and Atm^−/− E15.5 embryonic brain (b) after ionizing radiation exposure.

Fig. 5.

Inactivation of HR results in multiple tumor types. (A) A Kaplan–Meier curve shows survival of mice with various combinations of Xrcc2 and p53 mutant alleles and also shows Xrcc2^+/− is not haploinsufficient for tumors as Xrcc2^+/−p53^−/− mice developed tumors of the same kind and at the same rate as p53^−/− littermates. (B) A medulloblastoma staining immunopositive for the neuronal marker Tuj1 and a sarcoma staining immunopositive for desmin were isolated from a 10-week-old Xrcc2^−/−p53^−/− mouse. Arrows indicate the medulloblastoma within the cerebellar molecular layer. (C) Lymphomas from Xrcc2^−/−p53^−/− mice were CD8-immunopositive, indicative of a thymoma. Spectral karyotyping reveals a number of chromosomal rearrangements from a primary Xrcc2^−/−p53^−/− thymoma. (D) Image shows the spectral color image on the left and the pseudocolor chromosome on the right. (D Inset) Spectral karyotyping (SKY) analysis from a pro-B cell lymphoma from Lig4^−/−p53^−/− mice showing a typical translocation involving t (12, 15) that affects IgH and c-Myc.