Histopathology reveals correlative and unique phenotypes in a high-throughput mouse phenotyping screen

Abstract

The Mouse Genetics Project (MGP) at the Wellcome Trust Sanger Institute aims to generate and phenotype over 800 genetically modified mouse lines over the next 5 years to gain a better understanding of mammalian gene function and provide an invaluable resource to the scientific community for follow-up studies. Phenotyping includes the generation of a standardized biobank of paraffin-embedded tissues for each mouse line, but histopathology is not routinely performed. In collaboration with the Pathology Core of the Centre for Modeling Human Disease (CMHD) we report the utility of histopathology in a high-throughput primary phenotyping screen. Histopathology was assessed in an unbiased selection of 50 mouse lines with (n=30) or without (n=20) clinical phenotypes detected by the standard MGP primary phenotyping screen. Our findings revealed that histopathology added correlating morphological data in 19 of 30 lines (63.3%) in which the primary screen detected a phenotype. In addition, seven of the 50 lines (14%) presented significant histopathology findings that were not associated with or predicted by the standard primary screen. Three of these seven lines had no clinical phenotype detected by the standard primary screen. Incidental and strain-associated background lesions were present in all mutant lines with good concordance to wild-type controls. These findings demonstrate the complementary and unique contribution of histopathology to high-throughput primary phenotyping of mutant mice.

Keywords: High-throughput phenotyping; Histopathology; Mouse; Pathology.

Fig. 1

Examples of the knockout first conditional ready allele designs used and illustration of the clinical phenotyping pipeline. Acot6^{tm1a(KOMP)Wtsi} contained a promoter-driven selectable marker (neo) (A), whereas Adam17^{tm1a(EUCOMM)Wtsi} contained a promoterless selectable marker (B) (mouse Ensembl release GRCm38.p1). The alleles were expected to be null alleles, but assessment of the degree of knockdown and the extent of off-target effects on nearby genes was not carried out systematically. (C) The Sanger Institute MGP clinical (blue) and pathology (green) phenotyping pipeline showing tests performed between 4 and 16 weeks of age. Seven male (M) and seven female (F) mutant mice were processed through this pipeline for each allele screened. The pipeline was controlled by processing seven male and seven female wild-type mice per week. CBC, complete blood count.

Fig. 2

Distribution of the number of phenotypic abnormalities. Distribution of the number of phenotypic hits for each of the 50 mutant lines of mice analyzed, split by hits detected during clinical phenotyping (green bars) and histopathology assessment (red bars). No phenotypic abnormalities were detected in 17 of the 50 mutant mouse lines studied. Of the remaining 33 mutant mouse lines, the total number of phenotypic abnormalities ranged from 1 to 45 per line.

Fig. 3

Histopathology complements clinical phenotypes from the primary screen. Lrig^−/− mice were annotated to have abnormal skin (data from male mice is shown) (Fisher’s exact test adjusted P-value=2.09e–10). The number and proportion of affected mice in knockout and wild-type baseline controls is presented (A). Abnormal skin with scaly foci (arrows) was observed in Lrig^−/− mice (B). Histopathology revealed epidermal hyperplasia (arrow) and hyperkeratosis (arrowhead) (C). Mcph1^−/− mice were annotated as infertile. Ovarian hypoplasia with absence of folliculogenesis was observed in Mcph1^−/− females (D, top right panel); ovary from a wild-type mouse with growing follicles (arrows) is shown (D, top left panel). Seminiferous tubule vacuolation with lack of germ cells (arrows) was observed in Mcph1^−/− males (D, bottom right panel); normal testis with abundant developing germ cells (arrows) is shown from a wild-type mouse (D, bottom left panel). Tbc1d10a^−/− mice were annotated to have decreased circulating low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol (LDL data from male mice is shown). LDL cholesterol is significantly decreased in knockout mice compared with wild-type mice (E) [adjusted P=0.012, male knockout effect estimated: −0.35±0.10 mM (±standard error)]. The wild-type data, collected as local controls (phenotyped in the same week), are shown in the figure as the boxplot, whereas the green band shows the natural variation in the parameter as assessed by the 95% confidence intervals in control mice of that genetic background. Blood fat parameters between WT and knockout mice were compared using a mixed model statistical model. Histopathology revealed absent or minimal hepatic lipidosis in Tbc1d10a^−/− mice (F, bottom panel). Marked hepatic lipidosis is evident in wild-type mice (F, top panel).

Fig. 4

Histopathology identified novel phenotypes in lines with clinical phenotype annotations from the primary screen. Benign proliferative lesion of the brown fat (hibernoma) in Efna^−/− mice (A, right panel, arrows); normal brown fat from a wild-type mouse is shown (A, left panel). Sertoli cell vacuolation in Abdh5^−/− mice (B, right panel, arrows); normal seminiferous tubules from a wild-type mouse testis is shown (B, left panel). The clinical phenotype annotations from the primary screen for both Efna^−/− and Abhd5^−/− lines were skeletal abnormalities.

Fig. 5

Histopathology identified novel phenotypes in lines with no recorded clinical phenotype annotations from the primary screen. Seminiferous tubule degeneration and atrophy (arrows, top right panel) with markedly reduced density or absence of sperm in the epididymal duct (arrows, bottom right panel) in Smyd4^−/− mice. Normal seminiferous tubules with abundant developing germ cells (arrow, top left panel) and sperm store in the epididymal duct (arrows, bottom left panel) are shown in a wild-type mouse. Infertility or reduced fecundity was not observed in this line.