Fig. 1: Discovery of an Ovol2 mutation responsible for a severe impairment of lymphopoiesis.

A Manhattan plot. -Log₁₀ P-values plotted vs. the chromosomal positions of mutations identified in the G1 founder of the affected pedigree. (Insets) Representative flow cytometry plots of B220+ and CD3+ peripheral blood lymphocytes in wild-type (WT) mice and mice with the boh phenotype. Numbers adjacent to outlined regions indicate percent cells in each (mean ± SD) (n = 7 boh/boh, 36 WT littermates). B The frequency of peripheral blood B cells from third generation (G3) descendants of a single ENU-mutagenized male mouse, with REF ( + / + ), HET (+/boh), or VAR (boh/boh) genotypes for Ovol2 (n = 7 boh/boh, 49 boh/+, 36 WT littermates). C OVOL2 topology. Schematic of OVOL2^boh point mutation which results in substitution of cysteine to tyrosine at position 120 of total 274 amino acids in OVOL2 protein. Numbers are amino acid positions. D The frequency of peripheral blood B cells from 12-wk-old Ovol2^C120Y/− mice generated by the CRISPR/Cas9 system (n = 11 C120Y/−, 21 C120Y/+ or +/−, 20 WT littermates). E Numbers of splenocytes and the indicated cell populations in the spleens of 12-wk-old Ovol2^C120Y/− and WT littermates (n = 5 mice/genotype). F–M Thymocyte developmental defects and T cell functional defects in Ovol2^C120Y/− mice. F, G Numbers (F) and representative flow cytometry plots (G) of thymocyte populations in 12-wk-old Ovol2^C120Y/− mice and WT littermates. Numbers adjacent to outlined regions indicate percent cells in each (mean ± SD) (n = 5 mice/genotype). (H) CD44 expression on thymic and peripheral blood T cells in 12-wk-old Ovol2^C120Y/− mice and WT littermates (n = 4 (thymus) or 3 (peripheral blood) C120Y/− mice, 7 WT littermates). I–K Frequency of CD3+ (I), CD4+ (J), and CD8+ (K) T cells in the peripheral blood (n = 11 C120Y/−, 21 C120Y/+ or +/−, 19 WT littermates). L Serum ova-specific IgG in mice immunized with T cell-dependent antigen alum-ova (n = 15 C120Y/−, 19 C120Y/+ or +/−, 10 WT littermates). Data presented as absorbance at 450 nm. M Quantitative analysis of the βgal-specific cytotoxic T cell killing response in mice immunized with rSFV-βgal (n = 8 C120Y/−, 22 C120Y/+ or +/−, 20 WT littermates). N Serum NP-specific IgM in mice immunized with T cell-independent antigen NP-Ficoll (n = 8 C120Y/−, 22 C120Y/+ or +/−, 20 WT littermates). Data presented as absorbance at 450 nm. O NK cell cytotoxicity against MHC class I-deficient (β2m^−/−) target cells (n = 9 C120Y/−, 21 C120Y/+ or +/−, 20 WT littermates). Data are representative of one experiment (A, B, M, O) or two independent experiments (D, E–L, N). Data points represent individual mice and means ± SD are indicated. P-values were determined by one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons (B, D, I–O) or two-tailed Student’s t-test (E, F, H). +/+, C120Y/+, and C120Y/− indicate WT, Ovol2^C120Y/+, and Ovol2^C120Y/− genotypes, respectively. MFI Mean fluorescence intensity, DN Double-negative, DP Double-positive, SP Single-positive, CTL Cytotoxic T lymphocyte.