Figure 1.
Body weight and life span of the affected mice.
(A) Body weights of affected male mice compared to their unaffected male siblings (n = 10 each). Values are expressed as mean ± SE. (B) Life span of the affected mice compared to their unaffected siblings (n = 10 each).
Figure 2.
General appearance of the affected mice.
Note the size difference of affected versus unaffected sibling (A). Affected mice at different ages, 4 weeks (B), kyphosis with sharper spine angle in affected mouse and patchy alopecia (C), at 6 months of age (D) and just before death at 7.5 months of age (E).
Figure 3.
Skeletal abnormalities in the affected mice.
(A) Radiographs of affected mouse and unaffected sibling at 26 weeks of age. Yellow bars indicate the position of spine. Scale bar = 1 cm. (B) Micro–CT imaging of the femur trabecular bone in the wild-type and skcm04Jus mice taken at 26 weeks of age. The 3D images of trabecular bone were reconstructed as described in Materials and Methods; scale bar = 1 cm.
Table 1.
Structural parameters for trabecular bone.
Figure 4.
Skin histopathology of the affected mice.
Skin of an affected mouse at age 16 weeks showed hyperkeratosis (arrow) and hyperplasia of the epidermis and thin dermis layer with scanty subcutaneous adipose tissue (A) when compared to a wild-type mouse (B). The hair follicles contained no hair shafts and their upper portions were dilated and filled with keratinized materials in a mutant mouse (C) as compared to the normal hair follicles in a wild-type mouse (D). (H&E, Bar = 200 µm in A and B; 100 µm in C and D).
Figure 5.
Histopathology analysis of amyloid in skin of an affected mouse.
Skin sections were stained with H&E (A) and Congo red (B); and the latter stained section was also observed using a polarizing microscope (C). Note that amyloid deposits appeared eosinophilic by H&E stain, pink-red in Congo red stain, and showed yellow-green birefringence under a polarizing microscope.
Table 2.
Distributions and amyloid type in various tissues of the affected mice using Congo-Red Stain and immunohistochemistry.
Figure 6.
Histopathological analysis of amyloids in different organs of the affected mice.
Amyloid deposition in sinusoids and around the portal vein in liver (A); in red pulp and peri-white pulp area in spleen (B). In kidney, amyloid was found in glomerulus (C) and renal tubules (D). Amyloid was also found in adrenal cortex and medulla (E), islet of Langerhans and around the acinar cells of pancreas (F), salivary glands (G) and myocardium (arrows in H). All sections were stained with Congo red; amyloid deposits appeared pink-red color with this staining. Bar = 100 µm.
Figure 7.
Immunohistochemistry of amyloidosis in affected mice.
Immunohistochemistry analysis of amyloids in liver (upper panel) and in kidney (lower panel) of an affected mouse. Antibodies against AA amyloid (AA), κ light chain (ALκ), and λ light chain (AL λ) were used to differentiate types of amyloid; note progressively increase of amyloid deposition with age. Bar = 50 µm.
Figure 8.
Mapping and molecular analyses of the gene responsible for the phenotypes.
(A) Whole chromosomal mapping using 295 SNP markers. High homozygosity region is circled comprised of consecutive SNPs between SNP rs30814649 (46468726 bp) to SNP rs32491610 (64723695 bp) on chromosome 7. (B) Fine mapping of the candidate region using 52 SNPs on chromosome 7. Complete homozygosity is located between rs32116930 (53918742 bp) and rs32209625 (56317368 bp) (circled area). (C) DNA sequence analysis of mouse Zdhhc13 gene. Nucleotide sequences in exon 12 showing that affected mouse was homozygous for T at position c.1273 (arrow); unaffected parent was heterozygous A/T and wild-type was A/A at the same position.
Table 3.
Real-time quantitative RT–PCR of Zdhhc13 mRNA in mouse tissues.
Figure 9.
(A) Expression of Zdhhc13 in normal adult tissues. GAPDH was used as a loading control, (B) Expression of Zdhhc13 in liver, skin, lung and brain at postnatal (P) days 2, 8, and 30. Again, GAPDH was used as a control, (C) A gene trap vector insertion in intron 1 of Zdhhc13. The open arrow containing a β-geo cassette indicates the location of the gene trap vector, (D) Xgal staining of the p10 gene trap mutant and wild type. Note protein expression in the epithelium of the hair follicles. Sections were counterstained with nuclear fast red. The magnification is 200×, (E) Histopathology of gene traps mice skin, showing the abnormal follicles, lack of hair and thickened epidermis which are similar to ENU mutant mice.
Figure 10.
Palmitoyl-acyl transferase (PAT) activity and IgG light chain palmitoylation in the wild-type and mutant mice.
(A) Acyl-biotin exchange assay showing palmitoylation of huntingtin (Htt) was greatly reduced by the mutant Zdhhc13 as compared to the wild type in the hydroxylamine (NH2OH)-treated group. Low panel was a loading control for huntingtin. Wt: wild Zdhhc13, Mut: mutant Zdhhc13, C: Control: cells transfected with huntingtin alone without co-transfection with Zdhhc13. WB: western blot, (B) HEK 293T cells co-transfected with huntingtin and Zdhhc13 showing expression of these proteins was approximately even, (C) Levels of IgG light chain palmitoylation in the wild-type and mutant mice. IgG light chain purified from serum of the wild and mutant mice were labeled with S-palmitoylation using acyl-biotin exchange method. IgG light chain treated with hydroxylamine showed reduced palmitoylated signals in mutant mice as compared to the wild type (p = 0.001, n = 3, t test). Low panel was a loading control.