Fig 2: PDGFD promotes ESC vascular differentiation and represses ESC self-renewal by activating ERK signaling via PDGFR-β. Higher levels of PDGFD expression in ESCs activates PDGFR-β and ERK, resulting in increased expression of EC markers and decreased SOX2 expression, thus increased ESCs differentiation into endothelial cells and decreased ESCs self-renewal

Fig 3: PDGFR‐β activation aggravates the course of renal disease models, whereas blocking partially reverses the fibrotic phenotype A, BFive days after the induction of unilateral ureteral obstruction (UUO), a model of obstructive nephropathy and interstitial fibrosis, we assessed fibrosis (collagen I, α‐SMA) and inflammation (F4/80), the classical readout parameters in this model. Compared to wt mice with UUO, Foxd1Cre::Pdgfrb +/J mice developed significantly stronger fibrosis and inflammation (A). Representative pictures of the histological stainings are shown in (B). Scale bar = 50 µm. All data in (A) and (B) show means ± SD; wt n = 4, Foxd1Cre::Pdgfrb +/J n = 3. Statistical analysis was performed by unpaired two‐tailed Student's t‐test. *P < 0.05. Exact P‐values are provided in Appendix Table S4.C–HAngiotensin II infusion via an osmotic pump induced hypertension similarly in wt and Foxd1Cre::Pdgfrb +/J mice (C). In this model of hypertensive injury, the kidney function and even the histopathology were largely unaffected in wt mice, whereas the Foxd1Cre::Pdgfrb +/J mice showed significantly increased BUN values and prominent glomerulopathy after 28 days compared to the starting values (day 0), to wt animals after 28 days, and to age‐matched Foxd1Cre::Pdgfrb +/J mice without angiotensin II infusion (D). Consistently, the creatinine clearance per g bodyweight decreased in the hypertensive Foxd1Cre::Pdgfrb +/J mice (E). (F) PAS staining showed more prominent mesangial expansion and intratubular protein casts indicative of proteinuria only in hypertensive Foxd1Cre::Pdgfrb +/J mice. (G) Hypertension did not induce mesangial expansion and activation in wt mice, but led to a significant increase in hypertensive Foxd1Cre::Pdgfrb +/J mice as visualized by α‐SMA staining. (H) Histomorphometric analysis of α‐SMA‐positive percentage of the glomerular tuft area. All data in (C–H) show means ± SD, wt n = 7, Foxd1Cre::Pdgfrb +/J n = 5. Statistical analysis was performed by unpaired two‐tailed Student's t‐test. *P ≤ 0.05 compared to wt of the same time point, § P ≤ 0.05 compared to d0, # P ≤ 0.05 compared to same‐aged non‐hypertensive mice. Exact P‐values are provided in Appendix Table S4.I–PTwenty‐two‐week‐old Foxd1Cre::Pdgfrb +/J mice were treated with imatinib (daily gavage, 50 mg/kg bodyweight for 21 days; Foxd1Cre::Pdgfrb +/J + imatinib) and were compared to control Foxd1Cre::Pdgfrb +/J mice receiving vehicle (water; Foxd1Cre::Pdgfrb +/J + water). Age‐matched wt mice are indicated as dashed lines. (I) Histomorphometric quantifications showed a significant reduction of α‐SMA abundance in the glomerular tufts of imatinib‐treated Foxd1Cre::Pdgfrb +/J mice. (J) Glomerular cellularity evaluated by counting the total number of cells per glomerular tuft, normalized to the area, was significantly reduced in imatinib‐treated Foxd1Cre::Pdgfrb +/J mice compared to control mice. (K, L) Histomorphometric quantifications of collagen I showed no effect of imatinib treatment on mesangial sclerosis in the glomeruli (K), whereas reduced interstitial fibrosis in imatinib‐treated Foxd1Cre::Pdgfrb +/J mice compared to controls was found (L). (M) Hemoglobin serum levels were significantly improved in imatinib‐treated mice. (N) Representative pictures of the histological stainings of α‐SMA (left) and collagen I (middle: glomeruli; right: cortical interstitium). Scale bar = 50 µm. (O, P) Total PDGFR‐β protein levels were decreased in Foxd1Cre::Pdgfrb +/J mice after imatinib treatment, as shown using Western blot (O) and its densitometric evaluation (P). Bar graphs show means ± SD, n = 5 per group. Statistical analysis was performed by unpaired two‐tailed Student's t‐test. *P ≤ 0.05 Foxd1Cre::Pdgfrb +/J + water versus Foxd1Cre::Pdgfrb +/J + imatinib. Source data are available online for this figure.

Fig 4: Characterization of blood vessels in the pre-implantation murine uterus. H&E, IHC, double staining IF and fluorescently labeled dextran in E3.5 transverse uterine sections. Ovals indicate areas of the uteri magnified (B1 – F1). The orientation is the same for all panels; anti-mesometrial (am) is at the bottom and mesometrial (m) is at the top. (A) H&E at E3.5 highlighting the luminal epithelium (arrows), inner circular and outer longitudinal myometrium [dashed bracket (myo)], and endometrial stroma (st). (B) CD31+ ECs are detected throughout the endometrial stroma and myometrium. (C) CD31 and NG2 staining. NG2+ mural cells are associated with CD31+ ECs in the stroma, myometrium and serosa (s). (D) CD31 and PDGFR-β staining. PDGFR-β+ murals cells are associated with CD31+ ECs in the stroma, myometrium and serosa. (E) CD31 and SMA staining. SMA labels glandular epithelium (g) and smooth muscle cells in the myometrium and serosa and is not associated with CD31+ ECs in the stroma. (F) FITC-dextran is detected throughout the endometrial stroma, in both small and large capillaries (F1, arrowheads), and myometrium. DAPI identifies all nuclei in IF images. Bar in A – E =100 μm. Bar in F = 500 μm. Bar in B1 – F1 = 50 μm.

Fig 5: Location of Notch3 (N3) in relation to endothelial cells and mural cells in the peri-implantation uterus. H&E and double staining IF of E3.5 (A – D) and E6.5 (E – H) uterine sections. Ovals indicate areas of the uteri magnified (B1 – D1, F1 – H1). (A) H&E at E3.5 highlighting the luminal epithelium (arrows), inner circular and outer longitudinal myometrium [dashed bracket (myo)], endometrial stroma (st) and serosa (s). (B – D) Notch3+ cells are observed throughout the myometrium and serosa and in endometrial glands (g). (B) Notch3 and CD31. Notch3 is not expressed in CD31+ ECs. (C) Notch3 and PDGFR-β. Notch3 is expressed in PDGFR-β+ mural cells in the endometrial stroma (arrowheads in C1). (D) Notch3 and SMA. Notch3 and SMA staining overlaps in the myometrium and serosa. (E) H&E at E6.5 highlighting the embryo (e), inter-implantation sites (i), and myometrium (myo). (F – H) Notch3+ cells are observed throughout the myometrium. (F) Notch3 and CD31. Notch3 is not expressed in CD31+ ECs. (G) Notch3 and PDGFR-β. Notch3 is not associated with PDGFR-β in the decidua or myometrium. (H) Notch3 and SMA. Notch3 and SMA staining overlaps in the myometrium. DAPI identifies all nuclei in IF images. am, anti-mesometrial; m, mesometrial. Bar in A and E =100 μm. Bar in B – D and F – H =500 μm. Bar in B1 – D1 and F1 – H1 = 50 μm.