Fig 1.
Generation, genotyping and general phenotyping of Pdgfd-deficient mice.
(A) Schematic outline of the Pdgfd gene with exon numbers indicated. The target construct was inserted into exon1. The locations for genotyping primer binding sites are indicated by left and right arrows below exon1 and construct. (B) Genotyping PCR showing the presence of the three possible genotypes, Pdgfd+/+, Pdgfd+/- and Pdgfd-/-. The wildtype band is 289 bp, and the knockout band is 387 bp. (C) RT-PCR on heart tissue from Pdgfd+/+ and Pdgfd-/- mice showing the lack of exon1 sequences in Pdgfd mRNA in Pdgfd-/- animals. L19 was used as the reference gene (Pdgfd+/+ n = 3 and Pdgfd-/- n = 3). (D-F) The growth and development of wildtype and Pdgfd-/- animals from homozygote breedings were followed from birth until 16 weeks of age. (D) Observed pups born per litter, from ≥20 litters per genotype. (E) Survival % of pups born during the first week (Pdgfd+/+ n = 137 and Pdgfd-/- n = 108). (F) Weight curves of surviving offspring. Weight of surviving pups was measured every week (0–3 weeks) (Pdgfd+/+ n≥90 and Pdgfd-/- n≥80). Error bars indicating standard deviation in (C-D, F).
Fig 2.
Pdgfd promoter-driven LacZ expression displays a consistent vascular localization pattern.
(A) qPCR analysis on RNA from different tissues from 13 weeks old adult wild-type C57BL/6 mice. Total expression of Pdgfd mRNA measured as % of L19 and normalized to adrenal gland (peak organ). (B-U) X-gal staining (blue) of frozen sections from different tissues from 21 weeks old Pdgfd+/- mice, counterstained with hematoxylin. (B) Adrenal gland. (C) Adrenal cortex. (D) Adrenal medulla. (E) Aorta. (F) Aorta, higher magnification. (G) Heart. (H) Heart, myocardium, higher magnification. (I) Uterus. (J) Uterus, endometrium. (K) Testis. (L) Testis, seminiferous tubules. (M) Kidney. (N) Kidney, glomerulus and afferent arteriole. (O) Renal papilla. (P) Liver. (Q) Liver, bile duct. (R) Liver, blood vessel. (S) Pancreas, endocrine islet indicated by the dashed line. (T) Pancreatic duct (U) Pancreas, blood vessel. Scale bars 20, 50 or 100 μm. Error bars in (A) indicate standard deviation.
Table 1.
PdgfdLacZ expression in selected organs.
Fig 3.
Pdgfd is highly expressed in the cardiac vasculature, particularly in arterial bifurcations.
Analysis of heart, whole mounts and sections from wildtype and Pdgfd+/- mice. (A) Postnatal (day P4) whole mount heart showing X-gal staining (blue) (B) Adult whole mount hearts from 16 weeks old mice Pdgfd+/+ and Pdgfd+/- mice showing X-gal staining. (C) Magnification from (B). (D-G) Representative images of sections from heart showing X-gal staining. (D) Pdgfd+/+, control. (E) Overview of Pdgfd+/- heart. (F) Higher magnification of Pdgfd+/- heart, strong staining in blood vessel bifurcation (arrow). (G) Pdgfd+/- counterstained with hematoxylin and eosin. (H-K) Representative images of Pdgfd+/- heart sections from 21 weeks old mice, showing podocalyxin (H, J) staining as a marker of endothelial cells (green) and X-gal staining (blue). (H) Artery showing X-gal co-staining with podocalyxin (upper arrow) and X-gal staining outside of podocalyxin staining (lower arrow) and (I) in the same section, artery showing X-gal without podocalyxin. (J) A vein showing limited X-gal staining (arrow), and (K) the same section, without podocalyxin staining. (L-O) Representative confocal images of a Pdgfd+/- heart section from 16 weeks old mice showing immunofluorescent alpha-smooth muscle actin (αSMA) and enzymatic X-gal staining visualized by transmitted light. (L) Arteries showing αSMA (red) and X-gal staining (black). (M-O) Magnifications from (L), arrows pointing at black X-gal staining. (M) Arteries showing αSMA (red) and X-gal staining (black). (N) αSMA (white), no transmitted light. (O) X-gal staining (black). Scale bars 100 μm.
Fig 4.
Pdgfd is predominantly expressed in arterial endothelial cells, but vSMCs can also express Pdgfd.
(A) Overview picture showing X-gal stained (black arrows) vessels in whole mount stained postnatal (day P4) mesenteric tissue. (B-J) Whole mount co-stained with X-gal (black arrows) visualized by transmitted light and αSMA to differentiate between arterial (A, red arrows) and venous (V, blue arrows) vessels. Dashed lines showing veins. (B-C) Representative pictures showing prominent PdgfdLacZ expression in postnatal (day P4) mesenteric arteries, but not in veins. (B) αSMA (red) and X-gal (black). (C) αSMA (white), no transmitted light. (D) X-gal (black). (E-G) Representative pictures showing strong PdgfdLacZ expression in arteries and distinct PdgfdLacZ expressing cells in veins of mesenteric tissue from 16 weeks old mice. (E) αSMA (red) and X-gal (black). (F) αSMA (white), no transmitted light. (G) X-gal (black). (H-J) Representative pictures showing PdgfdLacZ expression in arteries of postnatal (day P4) skin tissue, while veins are negative. (H) αSMA (red) and X-gal (black). (I) αSMA (white), no transmitted light. (J) X-gal (black). (*) Indicates hair follicles that appear false positive, because of their structural characteristics. Scale bars 100 μm or 200 μm.
Fig 5.
NG2-expressing pericytes are disorganized in the cardiac vasculature of Pdgfd-/- mice.
Representative images from wildtype and Pdgfd-/- hearts showing tissue morphology and vascular stainings. (A-D) Hematoxylin and eosin staining showing normal morphology in Pdgfd-/- hearts compared to Pdgfd+/+ hearts. (E-G, I-K and M-O) Representative images of PECAM and NG2 staining showing decreased expression of NG2 and altered morphology of NG2-expressing pericytes in Pdgfd-/- (n = 4) hearts compared with wildtype hearts (n = 4). (E) PECAM and NG2 stained wildtype heart. (F) Magnification of right box in (E), arrow indicating a wildtype pericyte with processes spread out along the vessel. (G) Magnification of the left box in (E), arrows indicating NG2-stained pericyte processes covering wildtype vessels. (H) Quantification of vascular density, PECAM (pixels)/total area. (I) PECAM and NG2 stained Pdgfd-/- heart. (J) Magnification of the left box in (I), arrow indicating a Pdgfd-/- pericyte, with a rounder morphology than the corresponding wildtype pericyte in (F). (K) Magnification of the right box in (I), arrows indicating vessel segments not covered by NG2 stained pericyte processes. (L) Quantification of NG2 staining displayed as pericyte coverage, NG2/PECAM pixel ratio. (M-O) Magnifications NG2-positive pericyte processes between vessels, showing abnormal pericyte morphology in Pdgfd-/-. (M) Typical thin wildtype processing. (N) Pdgfd-/- pericyte with rough body morphology, located between vessels, with thick processes connected to vessels. (O) Pdgfd-/- pericyte with thick, rough, processes ending abruptly. (A-E) age 12 weeks and (E-O) age 15–17 weeks. Scale bars 20 or 50 μm. Error bars indicating standard deviation. **, p < 0.01.
Fig 6.
Pdgfd-/- mice display mild circulatory defects.
(A) qPCR analysis on RNA from Pdgfd+/+ (n = 3) and Pdgfd-/- (n = 3) mice (aged 18–20 weeks), of endothelial marker Pecam1, mural markers Cspg4 and Desmin and (B) developmental markers Notch1 and Gata4. (C-D) Wet and dry weight of hearts from 20–22 weeks old Pdgfd+/+ (n = 15) and Pdgfd-/- (n = 16) mice, normalized to body weight. (E) Tail-cuff blood pressure measurements showing systolic and diastolic blood pressure in Pdgfd+/+ and Pdgfd-/- male mice, aged 17–18 weeks. (Representative experiment, Pdgfd+/+ n = 5 and Pdgfd-/- n = 6). In (A-B), error bars indicate standard deviation. In (C-E), boxes indicate 2nd and 3rd quartile, middle bar indicates median and whiskers show min and max, *, p < 0.05 **, p < 0.01.
Table 2.
Clinical chemistry markers in serum.
Fig 7.
Pdgfd-/- mice show normal glucose homeostasis.
(A-D) Representative images of Pdgfd+/- pancreas sections from 21 weeks old mice, showing podocalyxin (green) staining as a marker of endothelial cells and X-gal (blue) staining. (A) Exocrine pancreas, X-gal stained vessel (arrow) and (B) same section without podocalyxin staining. (C) Endocrine pancreas, X-gal staining co-expressed with podocalyxin (arrow) and (D) same section without podocalyxin staining. Dashed lines denotes the pancreatic islet. Representative images from wildtype and Pdgfd-/- pancreas showing tissue morphology and different vascular stainings. (E-F) Hematoxylin and eosin staining showing normal morphology in Pdgfd-/- pancreas compared with wildtype pancreas, from 18 weeks old mice. (G-H) Representative images of wildtype (n = 6) and Pdgfd-/- (n = 6) pancreatic islets from 18 weeks old mice, stained for insulin and glucagon. Scale bars, 50μm. (I-M) Quantifications of stainings in (G-H), (I) number of islets per section area, (J) mean islet area, (K) total insulin area per section area, (L) insulin area per islet area, (M) insulin to glucagon ratio. (N-O) Glucose tolerance test in mice aged 17–20 weeks, and insulin tolerance tests in mice aged 19–23 weeks, Pdgfd+/+ (n ≥12) and Pdgfd-/- (n≥15) mice. Error bars indicating standard deviation. In (J), boxes indicate 2nd and 3rd quartile, middle bar indicates the median and whiskers show min and max. *, p < 0.05. ***, p < 0.001.