Fig. 2
Complete deletion of exons in Rhbdf1 results in growth retardation, brain hemorrhage, and cardiac hypertrophy (A5). a Exome-Seq showing loss of exons 2 through 18 in Rhbdf1−/− homozygous mutant mice generated using the definitive-null strategy. The red rectangle shows the exons lost in the mutant mice. b PCR of tail genomic DNA with all four primers shows that wildtype mice have one wildtype band (268-bp PCR product), heterozygous-null mice carry a wildtype band (268 bp) and a mutant band (546 bp), and homozygous-null mice carry one mutant band (546 bp). NTC, no template control. New England Biolabs 1 kb DNA Ladder was used as a molecular marker. Samples were run in duplicate. c Images of representative postnatal day 28 Rhbdf1−/− (left) and Rhbdf1+/− (right) male littermates. d Kaplan–Meier survival analyses showing that the lifespan of Rhbdf1−/− homozygous-null female (left panel) and male (right panel) mice is significantly shorter than that of heterozygous-null littermate controls. e Body weights of Rhbdf1−/− female (n = 4) and male (n = 3) mice compared with those of Rhbdf1+/− littermates (n = 3 for each sex) on postnatal day 21. Data represent mean ± S.D.; ***p < 0.001. f Brain hemorrhaging in Rhbdf1−/− mice. Upper left panel: multifocal hemorrhages (arrows) were observed in Rhbdf1−/− mice. All other panels: H&E-stained coronal sections of postnatal day 21 Rhbdf1−/− brain showing prominent hemorrhages in the brain stem, hippocampus, and hypothalamus. g Enlarged hearts in both female (left panel) and male (right panel) Rhbdf1−/− mice. Enlarged hearts were observed in pups older than 3 weeks of age. When we measured heart and body weights of Rhbdf1−/− (n = 3 females; n = 4 males) and Rhbdf1+/− (n = 4 females; n = 5 males) littermates at postnatal day 21, the heart-to-body-weight ratio (HW/BW) was significantly higher Rhbdf1−/− mice relative to Rhbdf1+/− littermates. Data represent mean ± S.D.; *p < 0.05, ***p < 0.001. h Cardiac complications in Rhbdf1−/− mice on postnatal day 21. Masson’s trichrome staining of heart sections from a 3-week-old Rhbdf1−/− mouse showing interventricular septum and left ventricular fibrosis. Notably, both perivascular and interstitial fibrosis were observed in Rhbdf1−/− mice