FBXO7 sensitivity of phenotypic traits elucidated by a hypomorphic allele

Abstract

FBXO7 encodes an F box containing protein that interacts with multiple partners to facilitate numerous cellular processes and has a canonical role as part of an SCF E3 ubiquitin ligase complex. Mutation of FBXO7 is responsible for an early onset Parkinsonian pyramidal syndrome and genome-wide association studies have linked variants in FBXO7 to erythroid traits. A putative orthologue in Drosophila, nutcracker, has been shown to regulate the proteasome, and deficiency of nutcracker results in male infertility. Therefore, we reasoned that modulating Fbxo7 levels in a murine model could provide insights into the role of this protein in mammals. We used a targeted gene trap model which retained 4-16% residual gene expression and assessed the sensitivity of phenotypic traits to gene dosage. Fbxo7 hypomorphs showed regenerative anaemia associated with a shorter erythrocyte half-life, and male mice were infertile. Alterations to T cell phenotypes were also observed, which intriguingly were both T cell intrinsic and extrinsic. Hypomorphic mice were also sensitive to infection with Salmonella, succumbing to a normally sublethal challenge. Despite these phenotypes, Fbxo7 hypomorphs were produced at a normal Mendelian ratio with a normal lifespan and no evidence of neurological symptoms. These data suggest that erythrocyte survival, T cell development and spermatogenesis are particularly sensitive to Fbxo7 gene dosage.

Fig 1. Fbxo7 targeted mice present with anaemia and extramedullary haematopoiesis.

(A) Red blood cell count (RBC); (B) haemoglobin; (C) haematocrit; (D) mean corpuscular haemoglobin concentration (MCHC); (E) mean corpuscular volume (MCV); (F) red blood cell distribution width (RDW); (G) spleen weight; (H) characterisation of splenic erythrocyte development; and (I) circulating reticulocyte; from 15–17 week old Fbxo7^+/+ and Fbxo7^tm1a/tm1a female mice. P values calculated from unpaired two-tailed students t tests with Welch’s correction (A-G, I), or unpaired two-tailed students t tests with Holm-Sidak multiple testing correction (H). The data are representative of at least 3 independent experiments and each symbol represents an individual mouse with the line at the mean and error bars represent standard deviation or standard error of the mean for panel H.

Fig 2. Anaemia in Fbxo7 targeted mice is regenerative with a shorter erythrocyte half-life that is haematopoietic intrinsic.

(A) plasma bilirubin and (B) plasma iron from 15-17-week-old Fbxo7^+/+ and Fbxo7^tm1a/tm1a female mice. (C) In vivo half-life of Fbxo7^+/+ and Fbxo7^tm1a/tm1a erythrocytes, the P value indicated is for each time point. D-H) characterisation of erythrocyte indices from Fbxo7^+/+ and Fbxo7^tm1a/tm1a bone marrow chimeras 10 weeks after reconstitution (female recipient mice). All data representative of three independent experiments, each symbol represents an individual mouse with the line at the mean and error bars represent standard deviation, except for (C) where n = 6 for Fbxo7^+/+ and Fbxo7^tm1a/tm1a with mean ± standard error of the mean. P values from an unpaired two-tailed students t test with Welch’s correction (A, B, D-H) or C two-way repeated measures ANOVA with Sidak post-hoc test for individual time points.

Fig 3. Fbxo7^tm1a mice have reduced circulating T cells with an altered phenotype and impaired T cell development.

(A) Circulating T cell percentage in peripheral blood, (B) blood T cell effector percentage. (C) T cell percentage in the spleen and (D) splenic T cell effector percentage. (E) total thymus cell number and (F) T cell developmental stages in the thymus, DN = CD4/CD8 double negative, DP = CD4/CD8 double positive, CD4 SP = CD4 single positive and CD8 SP = CD8 single positive. All mice were female and 8 weeks old. P values from an unpaired two-tailed students t test with Holm-Sidak multiple testing correction except panel E which is from unpaired two-tailed students t tests with Welch’s correction. All data representative of three independent experiments, each symbol represents an individual mouse with the line at the mean and error bars represent standard error of the mean.

Fig 4. Fbxo7 has a T cell intrinsic role in controlling T cell number in the periphery but not phenotype.

(A) T cell developmental stages in the thymus, DN = CD4/CD8 double negative, DP = CD4/CD8 double positive, CD4 SP = CD4 single positive and CD8 SP = CD8 single positive. (B) T cell percentage in the spleen and (C) splenic T cell effector percentage. P values from an unpaired two-tailed students t test with Holm-Sidak multiple testing correction. All data representative of three independent experiments (spleen 16–20 week old female mice, thymus 12–14 week old male mice), each symbol represents an individual mouse with the line at the mean and error bars represent standard error of the mean.

Fig 5. Increased morbidity after infection of Fbxo7^tm1a mice with Salmonella typhimurium.

(A) Survival curve of 4 female and 4 male Fbxo7^+/+ mice and 3 female and 4 male Fbxo7^tm1a/tm1a mice. (B) body weight changes during the course of infection from mice shown in panel A. (C) spleen weights at day 6 post Salmonella infection of male mice, with 3 Fbxo7^+/+ and 4 Fbxo7^tm1a/tm1a mice. (D) spleen and liver Salmonella counts at day 6 post infection of male mice, with 3 Fbxo7^+/+ and 4 Fbxo7^tm1a/tm1a mice. (E) histopathological image of liver showing a typical necro-inflammatory focus with an oval nodule consisting of eosinophilic necrotic hepatocytes surrounded by a rim of inflammatory cells that are predominantly neutrophil leucocytes, from a male Fbxo7^tm1a/tm1a mouse at day 6. (F) body weight changes during course of infection for male mice deficient for Fbxo7 in T cells (Fbxo7^tm1c/tm1c;CD4-Cre ⁺ 8 mice) and littermate controls (Fbxo7^tm1c/tm1c;CD4-Cre—5 mice). (G) spleen and liver Salmonella counts at day 14 post infection from irradiated female mice reconstituted with Fbxo7^+/+ (10 mice) or Fbxo7^tm1a/tm1a bone marrow (9 mice). (H-L) plasma clinical chemistry parameters at day 6 post infection from male mice (5 mice/genotype) for plasma levels of amylase, lipase, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and ferritin (P values calculated from unpaired two-tailed students t tests with Welch’s correction). For panels C, D and G-L symbols represent individual mice with the line at the mean and error bars representing the standard error of the mean (D and G) or standard deviation (C, H-L). For panel F symbols represent the mean with error bars representing the standard error of the mean.

Fig 6. Defects in the testis and epididymis of Fbxo7^tm1a/tm1a mice.

(A) Image of epididymal spermatozoa smear stained with hemacolor from 12-14-week-old mice. (B) H&E stained section of epididymis from 20-week-old mice. (C) testis weight from 18-19-week-old mice. (D) semithin toloudine blue stained section image of testis from 8-week-old mice. Images are representative of at least two mice per genotype, P value from an unpaired two-tailed students t test with Welch’s correction with each symbol representing a testis, line is at the mean and error bars represent standard deviation.