Xrcc2 is required for genetic stability, embryonic neurogenesis and viability in mice - PubMed (original) (raw)

Xrcc2 is required for genetic stability, embryonic neurogenesis and viability in mice

B Deans et al. EMBO J. 2000.

Abstract

Repair of DNA damage by homologous recombination has only recently been established as an important mechanism in maintaining genetic stability in mammalian cells. The recently cloned Xrcc2 gene is a member of the mammalian Rad51 gene family, thought to be central to homologous recombination repair. To understand its function in mammals, we have disrupted Xrcc2 in mice. No Xrcc2(-/-) animals were found alive, with embryonic lethality occurring from mid-gestation. Xrcc2(-/-) embryos surviving until later stages of embryogenesis commonly showed developmental abnormalities and died at birth. Neonatal lethality, apparently due to respiratory failure, was associated with a high frequency of apoptotic death of post- mitotic neurons in the developing brain, leading to abnormal cortical structure. Embryonic cells showed genetic instability, revealed by a high level of chromosomal aberrations, and were sensitive to gamma-rays. Our findings demonstrate that homologous recombination has an important role in endogenous damage repair in the developing embryo. Xrcc2 disruption identifies a range of defects that arise from malfunction of this repair pathway, and establishes a previously unidentified role for homologous recombination repair in correct neuronal development.

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Figures

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Fig. 1. Disruption of Xrcc2. (A) Diagram of the wild-type Xrcc2 genomic locus, targeting construct and the targeted locus. Homologous recombination of the targeting vector with the Xrcc2 locus results in the replacement of exon III with a neomycin cassette, and the introduction of a _Bam_HI site. Relevant _Bam_HI (B), _Eco_RI (E), _Hin_dIII (H), _Xba_I (X) and _Xho_I (Xh) restriction sites, and the position of the probe used for Southern blot analysis are shown. Exons II and III are indicated by numbered solid boxes. (B) Genomic DNA from ES cell colonies digested with _Bam_HI, and analysed by Southern blotting and hybridization with the probe indicated in (A). Truncation of wild-type 11.1 kb fragment to 6.2 kb confirms correct targeting of an allele. +/+ and +/– represent the genotypes of wild-type (Xrcc2+/+) and targeted (Xrcc2+/–) ES lines, respectively. (C) RT–PCR on total RNA extracts from embryos using Xrcc2 exon II to exon III specific primers, with primers to Gapdh as a control. Xrcc2+/+ and Xrcc2+/–, but not _Xrcc2_–/–, generated a band corresponding to Xrcc2 mRNA. (D) A representative example of PCR genotyping for a litter of E8.5 embryos from an Xrcc2+/– intercross. PCR primers detect neo or exon III in the same reaction, giving products of 280 and 545 bp, respectively. Control lanes shown are a targeted ES cell line (ES +/–), wild-type genomic DNA (129Sv) and a plasmid constructed to mimic the targeted locus (pX2); –/– denotes homozygous disruption of Xrcc2. (E) Double targeting of Xrcc2 in ES cells shown by Southern blot analysis of _Xho_I-digested genomic DNA. HM-1 ES cells (lane 1) underwent sequential gene targeting steps, first with an _Hprt_-based vector (lane 2) and then, following confirmation of correct targeting of an Xrcc2 allele, with the _neo_-based construct as in (A). Both strategies introduce discernible _Xho_I restriction sites, reducing a wild-type fragment from 26.5 kb to 4.2 or 3.4 kb for Hprt or neo, respectively. All targeted clones isolated following transfection with the neo vector (shown for four clones; lanes 3, 4, 5 and 6) had re-targeted the already disrupted allele (n = 8).

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Fig. 2. _Xrcc2_–/– embryos display growth retardation and morphological defects. Xrcc2 null (–/–) and wild-type littermates (+/+) are shown at (A) E9.5, (B) E11.5, (C) E14.5 and (D) E18.5 stages of development. An additional smaller _Xrcc2_–/– fetus, from a separate litter, illustrates the extremities of size observed at E18.5. (E) _Xrcc2_–/– E11.5 embryo with failed neural tube closure, denoted by asterisks. (F) _Xrcc2_-null E14.5 embryo with exencephaly. (G and H) The lungs of _Xrcc2_-null neonates fail to inflate. In comparison with wild-type littermates (G), alveolar size is reduced in haematoxylin–eosin-stained lungs of _Xrcc2_–/– (H) neonates.

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Fig. 3. _Xrcc2_–/– embryonic cells are hypersensitive to radiation and have a high level of chromosomal aberrations. Representative photographs of blastocysts cultured for 6 days following either no irradiation [(A) Xrcc2+/+ and (B) _Xrcc2_–/–] or γ-irradiation (4 Gy) [(C) Xrcc2+/+ and (D) _Xrcc2_–/–]. ICM, inner cell mass; TGC, trophoblast giant cells. (E) Complex chromatid exchange involving at least six chromosomes, including chromosomes 2 (yellow) and 4 (green); centromeres are stained pink. (F) Reciprocal chromosome exchange involving chromosomes 4 (green) and 11 (red); centromeres are stained pink.

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Fig. 4. Extensive cell death in _Xrcc2_–/– E9.5 embryos (sagittal sections) as shown by TUNEL staining (green). (A and B) midbrain and (C and D) dorsal region at the level of the forelimb of stage-matched Xrcc2+/+ (A and C) and _Xrcc2_–/– embryos (B and D).

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Fig. 5. Extensive cell death in the developing CNS of _Xrcc2_–/– E11.5–12.5 embryos. TUNEL staining (DAB) in (A and B) the spinal cord at E12.5, (C and D) the midbrain at E12.5, and (E and F) the hindbrain at E11.5 in sagittal sections of Xrcc2+/+ (A, C and E) and _Xrcc2_–/– (B, D and F) littermates. Nuclei are counterstained with haematoxylin. V, fourth ventricle; VZ, ventricular zone; ML, mantle layer.

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Fig. 6. Extensive apoptosis in _Xrcc2_–/– embryos reflects the spatial and temporal pattern of neurogenesis. TUNEL staining for sagittal sections of the developing cortex of the forebrain at (A and B) E12.5, (C and D) E14.5 and (E and F) E16.5 for wild-type (A, C and E) and _Xrcc2_–/– (B, D and F) embryos. At E16.5, arrows indicate TUNEL-positive nuclei. V, lateral ventricle; VZ, ventricular zone; PPL, primordial plexiform layer; IZ, intermediate zone; CP, cortical plate.

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Fig. 7. Apoptosis in early post-mitotic neurons of _Xrcc2_–/– embryos. Sagittal section of (A and B) the frontal cortex and (C and D) the septal area of the developing forebrain in an E12.0 _Xrcc2_-null embryo, stained with propidium iodide (A and C), and double stained with TUNEL reagents (green) and the anti-β3-tubulin antibody, TUJ1 (red) (B and D). TUNEL-positive nuclei are localized predominantly within the newly generated, TUJ1-staining neuronal cell population of the PPL layer. V, lateral ventricle; VZ, ventricular zone; PPL, primordial plexiform layer.

References

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