Fig 1.
Expression of GPR116 in adult mice.
(A-C) Representative fluorescent images (of 5 images per organ from 4 examined animals) of mCherry reporter mice, showing GPR116 expression at 8 weeks of age in the heart, pancreas and white adipose tissue (WAT) (A), in the lung (B) and renal glomeruli (C). Endothelial cells are stained with anti-CD31, alveolar epithelial type II cells are stained with anti-Pro-SPC, glomerular podocytes are stained with anti-podocin and glomerular mesangial cells are stained with anti-desmin antibodies. Nuclei are counterstained with DAPI. Scale bars: 50 μm (A and B); 20 μm (C).
Fig 2.
Expression of ELTD1 in adult mice.
(A) Representative H&E staining of different organs from 8-week-old mice expressing ß-galactosidase under the control of the Eltd1 promoter (Eltd1lacZ/+). Activity of ß-galactosidase is detected by X-gal blue staining. (B) Representative fluorescent images (of 9 images from 3 examined animals) of renal glomeruli in Eltd1lacZ/+ mice compared with WT mice. Activity of ß-galactosidase is detected by SPiDER-ßGal, endothelial cells are stained with anti-CD31, glomerular podocytes are stained with anti-podocin and glomerular mesangial cells are stained with anti-desmin antibodies. Nuclei are counterstained with DAPI. Scale bars: 200 μm (A); 50 μm (B).
Fig 3.
Expression of GPR116 and ELTD1 in mouse embryos.
(A) Representative fluorescent images (of 18 images from 3 examined animals) of the heart and OFT area in Gpr116-mCherry reporter mice at embryonic day 11.5–12. Endothelial cells are stained with anti-CD31 antibody. (B) Representative fluorescent images (of 18 images from 3 examined animals) of the heart and OFT area in Eltd1lacZ/+ mice at embryonic day 10–10.5. Activity of ß-galactosidase is detected by SPiDER-ßGal, endothelial cells are stained with anti-CD31 antibody. (A, B) Nuclei are counterstained with DAPI. As, aortic sack; Da, dorsal aorta; Ta, truncus arteriosus; Cv, cardinal vein; Aa, arch arteries; A, atrium; V, ventricle; T, trachea; O, oesophagus. Scale bars: 200 μm.
Fig 4.
Life expectancy and body weight of GPR116-ELTD1 double deficient mice.
(A) Kaplan-Meyer survival curve of Gpr116-/-;Eltd1-/-, Gpr116+/+;Eltd1-/- and Gpr116-/-;Eltd1+/+ mice (n ≥ 11 per group). (B) Bodyweight of Gpr116-/-;Eltd1-/- mice compared to Gpr116-/-;Eltd1+/+, Gpr116-/-;Eltd1+/-, Gpr116+/+;Eltd1-/-,Gpr116+/-;Eltd1-/- and wild-type mice (Gpr116+/+;Eltd1+/+) at 2 and 4 weeks of age. (*, P≤0.05; **, P≤0.01; ***, P≤0.001). (C) A representative image of a Gpr116-/-;Eltd1-/- mouse compared with a control littermate (Gpr116+/-;Eltd1-/-) at 4 weeks of age. (D) Genotype of offspring from intercrosses of Gpr116-/-;Eltd1+/- and Gpr116+/-;Eltd1-/- mice at E18.5 and P0, respectively.
Fig 5.
Defects in large arteries and the cardiac outflow tract in GPR116-ELTD1 double deficient embryos (E18.5).
(A) H&E staining of thoracic sections from two individual Gpr116-/-;Eltd1-/- embryonic mice with enlarged views of the areas marked by dashed lines shown below. Asterisks indicate the position of the ventricle septum defect. LV, left ventricle; RV, right ventricle. (B) Ventral view of large vessels in a Gpr116-/-;Eltd1-/- embryonic mouse and a control littermate (Gpr116-/-;Eltd1+/+) with large arteries marked by dashed lines. A schematic of arteries is shown below. Arrow points to the position where the right subclavian artery usually branches off. 1, Aortic arch; 2, Innominate artery; 3, Right subclavian artery; 4, Left common carotid artery; 5, Ascending thoracic aorta; 6, pulmonary artery. (C) Schematic of the embryonic remodeling process of the branchial arch arteries into the aortic arch and the great vessels in normal configuration (E10.5 and E18.5) as well as in disease examples showing aberrant right subclavian artery, interrupted aortic arch and double aortic arch. RSA and LSA, right and left subclavian artery; RCA and LCA, right and left common carotid artery; A, aorta; da, ductus arteriosus; PT, pulmonary trunk. (D) Representative ventral view of large vessels from a Gpr116-/-;Eltd1-/- embryonic mouse and a control littermate (Gpr116+/-;Eltd1-/-) with arteries indicated by dashed lines. A schematic of arteries is shown below. 1. Ascending aorta; 2. Aortic arch; 3. Pulmonary artery; 4. Right subclavian artery; 5. Right common carotid artery; 6. Left common carotid artery; 7. Left subclavian artery. 2, 3 and 7 are highlighted in yellow indicating interrupted aortic arch. (E) H&E staining of thoracic cross sections (from inferior (left) to superior (right)) from a Gpr116-/-;Eltd1-/- embryonic mouse with enlarged views of the dashed line areas shown below. The aorta is connected to the right ventricle, which leads to double outlet right ventricle (DORV). LV, left ventricle; RV, right ventricle. (F) H&E staining of thoracic cross sections (from inferior (left) to superior (right)) from a Gpr116-/-;Eltd1-/- embryonic mouse with enlarged views of the areas indicated by a dash line shown below, exhibiting a double aortic arch with a retro-esophageal segment. Os, oesophagus; T, trachea; A, aorta. Scale bars: 200 μm (A, E, F).
Table 1.
Frequencies of malformation observed in Gpr116-/-;Eltd1-/-, Gpr116+/+;Eltd1-/- and Gpr116-/-;Eltd1+/+ E18.5 embryos.
Fig 6.
Cardiac hypertrophy, vascular malformations and splenomegaly in 4–12 weeks old GPR116-ELTD1 double deficient mice.
(A) Organ to body weight ratios in Gpr116-/-;Eltd1-/- mice compared to Gpr116-/-;Eltd1+/+, Gpr116+/+; Eltd1-/- and wild-type mice at 4 weeks of age (n = 8 per group; *, P≤0.05; **, P≤0.01; ***, P≤0.001). (B) Representative H&E staining of hearts from a Gpr116-/-;Eltd1-/- mouse compared to control mice (Gpr116+/-;Eltd1-/- and Gpr11+/+;Eltd1-/-) at 4 weeks of age. (C) Cardiac MRI analysis of Gpr116-/-;Eltd1-/- mice compared to littermate control mice (Gpr116+/+;Eltd1-/- or Gpr116+/-;Eltd1-/-) at 4 and 8 weeks of age, respectively (*, P≤0.05; n.s., not significant.) (see also S1 and S2 Movies). (D) Micro-CT scanning of a vascular corrosion cast from WT and Gpr116-/-;Eltd1-/- mice at 4 weeks (left panels) and 12 weeks (right panels) showing the large vessels. Asterisk indicates a common outlet of left and right carotid artery. Arrow indicates a fistula between the left subclavian artery and the left superior vena cava branch. 1. Aortic arch; 2. Innominate artery; 3. Right subclavian artery; 4. Right common carotid artery; 5. Left common carotid artery; 6. Left subclavian artery; 7. Left superior vena cava; 8. Right superior vena cava (see also S3–S6 Movies).
Fig 7.
GPR116-ELTD1 double deficient mice developed glomerular thrombotic microangiopathy.
(A) Giemsa staining of blood smear from dKO mice and controls (insets show enlarged images). (B) Analyses of erythrocyte numbers, packed cell volumes and mean corpuscular volumes (MCV) as well as hemoglobin and plasma lactate dehydrogenase (LDH) levels in Gpr116-/-;Eltd1-/- mice compared to WT, Gpr116-/-;Eltd1+/+ and Gpr116+/+;Eltd1-/- mice at 4 weeks of age. (n = 8 per group; *, P≤0.05; **, P≤0.01; ***, P≤0.001). (C) Upper panel: Protein content of urine from Gpr116-/-;Eltd1-/- mice and control littermates (Gpr116+/+;Eltd1-/-, Gpr116-/-;Eltd1+/+, Gpr116-/-;Eltd1+/-) at 7 and 12 weeks of age analyzed by Coomassie Brilliant Blue stain of urinary protein separated by SDS-PAGE. Bovine serum albumin (BSA) is used as standard. Lower panel: Plasma urea concentration in dKO mice and controls at 4 weeks of age. (D) Goldner staining of renal glomeruli sections from Gpr116-/-;Eltd1-/- and control (Gpr116+/-;Eltd1-/-) mice at 4 weeks of age. (E) PAS-staining of renal glomeruli sections from Gpr116-/-;Eltd1-/- and control (Gpr116-/-;Eltd1+/+ and Gpr116+/+;Eltd1-/-) mice at 4 weeks of age. (F) Representative transmission electron micrographs of renal glomeruli from Gpr116-/-;Eltd1-/- and control (Gpr116+/-;Eltd1-/-) mice at 4 weeks of age. Arrow indicates the glomerular filtration barrier in the healthy control littermate. Double arrow indicates the expanded subendothelial zone. Scale bars: 50 μm (A, D, E); 2500 nm (F).