Fig 2: Low Concentrations of BMP9 Modulate the Calcineurin/NFATc1 Signaling Pathway(A) qRT-PCR analysis of BMP9 effects on the mRNA levels of transcription factors known to be essential for lymphatic differentiation. FLK-1+ vascular precursors at day 5 were plated on CellTracker CMFDA-stained OP9 stromal cells. BMP9 (0.3 ng/mL) was added at day 6. After 24 hr of treatment, the CMFDA-negative cell population was sorted and collected for gene expression analysis. Data are the mean ± SEM of five experiments.(B) NFATc1 protein expression analysis after BMP9 stimulation. FLK-1+ vascular precursors at day 5 were stimulated in suspension in serum-free medium with or without 0.3 ng/mL BMP9 for 20 min. Immunoblotting was performed using an antibody against NFATc1 or actin proteins as a loading control. Results of a representative experiment, out of three, are illustrated. Sizes of the molecular weight markers are indicated on the left.(C) Inhibition of NFATc1 signaling by cyclosporin A prevents the BMP9-induced increase in LYVE-1+ cell formation. LYVE-1-positive cell formation was measured by flow cytometry 4 days after addition of BMP9 in the absence or presence of cyclosporin A. Data are the mean ± SEM of three experiments.*p < 0.05, significantly different from control untreated cells (A) and significantly different from treatment with BMP9 alone (C) using unpaired Student's t test. See also Figure S3.

Fig 3: Postnatal phenotype of Clp1K/K newborns on a c57Bl/6-NMRI mixed genetic background. (A) Overall skeletal muscle activity score of newborn Clp1+/+ and Clp1K/K littermates on a c57Bl/6-NMRI mixed genetic background. Quantitative data are represented as mean and SEM from 9 to 12 newborns in each group [*P = 0.0374 (two-tailed t-test)]. (B) Viability score of newborn Clp1+/+ and Clp1K/K littermates on a c57Bl/6-NMRI mixed genetic background. Quantitative data are represented as mean and SEM from 9 to 12 newborns in each group [P = 0.0684 (two-tailed t-test)]. (C) Lung morphology of newborn Clp1+/+ and Clp1K/K littermates on c57Bl/6 and c57Bl/6-NMRI mixed genetic backgrounds shown by HE histology. Representative images are shown from seven newborns from three litters. Bars, 50 µm. (D) Visualization of pulmonary lymphatics in Clp1+/+ and Clp1K/K newborns on c57Bl/6 and c57Bl/6-NMRI mixed genetic backgrounds. Representative images are shown for LYVE1 immunostaining and DAPI from five newborns per group in two litters. Bars, 50 µm.

Fig 4: High fat diet results in obesity and subcutaneous tissue inflammation. A. Body weight of obese and control mice. B. Gross photograph of subcutaneous tissue adipose deposition in control and obese mice (top). Representative photomicrograph of full-thickness back punch biopsy specimens in control and obese mice (20× magnification). Brackets indicate subcutaneous fat deposition. C. Mean subcutaneous fat thickness in back punch biopsy of control and obese mice. D. Serum cholesterol, triglycerides, and glucose levels in 12 hour fasting blood samples from control and obese mice. E., F. Representative low (E; 10x) and high (F; 40x) power photomicrographs of control and obese mice back punch (upper) and tail (lower) histological sections stained for CD45 (brown) and LYVE-1 (red). Black box in figure E is magnified in F. G. Mean number of CD45+ cells/hpf (40x) in control and obese mice. H. Mean number of peri-lymphatic CD45+ cells/hpf (40x) in control and obese mice.

Fig 5: LEC-specific deletion of Ilk confirms ILK-controlled VEGFR3 signalling A–DLSM images of stained cross-sections through the jls/pTD of an E13.5 Prox1-Cre ERT2 ;Ilk +/+ embryo (referred to as “control”) and an E13.5 Prox1-Cre ERT2 ;Ilk ?/? embryo (referred to as “induced ILK K.O.”). Red dots indicate sites of tyrosine phosphorylation (p-Tyr) in proximity to VEGFR3. Arrowheads point to PLA dots within the Lyve1-stained area. Scale bars: 10 µm.EQuantification of the PLA dots indicating VEGFR3 with phosphorylated tyrosine (p-Tyr) per LEC in E13.5 control or induced ILK K.O. embryos (n = 7 control and n = 8 induced ILK K.O. embryos), *P = 0.035.Data information: Data are presented as means ± SEM, shown as percentage of control embryos, unpaired two-tailed Student's t-test.