An XRCC4 mutant mouse, a model for human X4 syndrome, reveals interplays with Xlf, PAXX, and ATM in lymphoid development
- Université de Paris, Imagine Institute, Laboratory “Genome Dynamics in the Immune System”, INSERM UMR 1163, F-75015, France
- Equipe Labellisée Ligue Nationale Contre le Cancer, F75015, France
- Université de Paris and Université Paris-Saclay, Inserm, LRP/iRCM/IBFJ CEA, UMR Stabilité Génétique Cellules Souches et Radiations, F-92265, France
- Université de Paris, Imagine Institute, Transgenesis facility, INSERM UMR 1163, F-75015, France
- Institute for Integrative Biology of the Cell (I2BC), Institute Joliot, CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, F-91198, France
- Sorbonne Université, Muséum National d'Histoire Naturelle, CNRS UMR 7590, Institut de Minéralogie, de Physique des Matériaux et de Cosmochimie, F-75005, France
Figures
Figure 1 with 1 supplement
X4 and Lig4 expression and impaired non-homologous end-joining (NHEJ) in Xrcc4M61R mice.
(A) Schematic representation of CRISPR/Cas9-driven homologous recombination strategy to generated Xrcc4M61R knock-in mouse model. (B) 5′UTR to 3′UTR Xrcc4 RT-PCR in mouse thymocyte extracts from four littermates. The lower transcript represents the splicing out of exon 3 as described (Gao et al., 1998). (C) WB analysis from mouse whole tissue extracts. (D) Western blot (WB) analysis from mouse embryonic fibroblast (MEF) extracts. (E) WB analysis from mouse whole tissue extracts. WB were performed at least three times with two animals per genotypes. (F) MEFs’ sensitivity to DNA double-strand break (DSB)-inducing agent phleomycin. Statistical tests for Xrcc4M61R vs. WT (gray *) and Xrcc4M61R vs. Xrcc4-/- (black *). (G) Relative survival of CD3/CD28-activated mature T cells following irradiation and 4 hr recovery. Statistical analysis for Xrcc4M61R vs. WT (gray *) and Xrcc4M61R vs. Nhej1-/- (green *).
Figure 1—figure supplement 1
Nucleotide sequence of two Xrcc4 transcripts in Xrcc4M61R mice.
(A) Sequence alignment of WT and upper/lower Xrcc4 transcripts (Figure 1B) showing M61R and silent mutations on the full-length (upper) form and the splicing out of exon 3 on the lower form. (B) Nucleotide sequence traces of upper and lower Xrcc4 transcripts.
Figure 2
Xrcc4M61R mice exhibit modest T cell development defect in the thymus phenocopying Nhej1-/- mice.
(A) Thymus cellularity of WT, Xrcc4M61R, and Nhej1-/- (Xlf) 6–9-week-old mice. (B) Immunostaining of thymus cellular populations. Various relevant populations are highlighted in black gates. (C) Quantification of DN3A thymocyte subpopulation (DN3 CD28-CD25+) gated from total DN3 thymocytes (CD4-CD8-CD44-CD25+). (D) Thymocyte apoptosis analysis after 20 hr of culture. AnnexinV+/7AAD+ apoptotic cells are highlighted in black gates. (E) Quantification of thymocyte apoptosis (AnnexinV+/7AAD+ cells) after 20 hr of culture. (F) Quantitative RT-PCR analysis of TP53 target genes Cdkn1a (encoding P21), Bbc3 (encoding PUMA), and Bax in thymocyte extracts. There is no statistical difference between Xrcc4M61R and Nhej1-/- in the expression of these TP53 target genes. (G) Illustrative representation of principal component analysis/unsupervised hierarchical clustering (PCA/HC) analysis of mTRAJ-mTRAV combinations in thymus according to PROMIDISα. (H) Immunostaining of bone marrow cellular populations. V(D)J recombination-dependent developmental stages are highlighted within red rectangles. Various relevant populations are highlighted in black gates. Quantification of B-cell population (B220+) (I) and immature B-cell population (CD19+ B220+ IgM+) (J) from total bone marrow.
Figure 3 with 1 supplement
PAXX and ATM are compensatory factors of X4M61R in immune development.
(A, B) Thymus and spleen cellularity of WT, Xrcc4M61R, Xrcc4M61R–Paxx, and Xrcc4M61R–Atm 6–9-week-old mice. (C) Immunostaining of BM and thymus cellular populations. Various relevant populations are highlighted in black gates. (D) Quantification of B220+ subpopulation of total BM. (E) Quantification of DN3A thymocyte subpopulation (DN3 CD28-CD25+) of total DN3 thymocytes (CD4-CD8-CD44-CD25+). (F) Schematic representation of PCR strategy to analyze Tcrb rearrangement according to Abramowski et al., 2018. (G) Autoradiogram of ladder of productive Dβ2-Jβ2 and Vβ10-Dβ2Jβ2 semi-quantitative PCR products revealed by the TCR-Jβ probe. Genomic DNA dilutions are represented with triangles. Germline allele configuration (GL) is revealed by the upper band of Dβ2-Jβ2 PCR. Southern were performed twice with two animals per genotype.
Figure 3—figure supplement 1
Growth retardations of Xrcc4M61R-Paxx and Xrcc4M61R-Atm mice.
(A–C) Photographs of 7-week-old littermates.
Figure 4
Embryonic lethality and neuronal apoptosis of Xrcc4M61R-Nhej1 embryos.
(A) Impaired development of Xrcc4M61R-Nhej1 mice. Actual and expected numbers of Xrcc4M61R-Nhej1 alive pups, E18.5 and E15.5 embryos. (B) Pictures of Xrcc4M61R and Xrcc4M61R-Nhej1 E15.5 embryos. (C) Cleaved-caspase 3 (CC3, red) immunostaining of E15.5 brain slices. Scarce apoptotic cells are detected in the whole dorsal telencephalon from Nhej1-/- and Xrcc4M61R embryos. By contrast, massive neuronal apoptosis is observed in the upper layers of the developing cortex of Xrcc4M61R-Nhej1, Xrcc4-/-, and Nhej1-Paxx DKO embryos. The white boxes indicate the location of the standard window (100 µm wide) spanning from the ventricular to pial surface used to quantify pyknotic nuclei in the dorsal telencephalons (see below). Scale bar: 100 µm. (D) Number of CC3-positive cells in the VZ/SVZ (black)—containing the cycling neural progenitors—and intermediate zone (IZ)/cortical plate (CP) (gray)—containing post-mitotic neurons—of the whole dorsal telencephalons from WT, Nhej1-/-, and Xrcc4M61R embryos. (E) Percentage of apoptotic (pyknotic) nuclei in the ventricular zone (VZ)/sub-ventricular zone (SVZ) (left panel) and the IZ/CP (right panel) found in the standard windows shown in (C). Data were obtained from both hemispheres of three embryos per condition.
Figure 5
Complete V(D)J recombination defect in E18.5 Xrcc4M61R-Xlf embryos.
(A) Immunostaining of E18.5 fetal CD19+ B220+ CD43+ hepatocytes and fetal thymocytes. Various relevant populations are highlighted in black gates. (B) Quantification of intracellular IgM expression in subpopulation of fetal Pro-B cells (CD19+ B220+ CD43+ surfaceIgM- hepatocytes). (C) Quantification of DN3A thymocyte subpopulation (DN3 CD28-CD25+) of total DN3 fetal thymocytes (CD4-CD8-CD44-CD25+). (D) Schematic representation of PCR strategy to analyze Tcrb rearrangement according to Abramowski et al., 2018. (E) Autoradiogram of ladder of productive Dβ2-Jβ2 and Vβ10-Dβ2Jβ2 direct PCR products revealed by the TCR-Jβ probe. Germline allele configuration (GL) is revealed by the upper band of Dβ2-Jβ2 PCR. Southern was performed once with two animal per genotype.
Author response image 1
Additional files
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
An XRCC4 mutant mouse, a model for human X4 syndrome, reveals interplays with Xlf, PAXX, and ATM in lymphoid development
eLife 10:e69353.
https://doi.org/10.7554/eLife.69353
