Mice

All mice were housed and maintained on a 12/12 h light/dark cycle at the Animal Facility of Zhongshan Ophthalmic Center, Sun Yat-Sen University. Six-week old nude mice were purchased from the GemPharmatech Company (Nanjing, China). Pdgfd deficient mice were generated by the Cyagen Company (Nanjing, China), and bred on a C57BL/6J background for more than six generations. Briefly, two gRNAs targeting Pdgfd exon 2 together with Cas9 mRNA were injected into mouse zygotes, which were subsequently transplanted into pseudopregnant mice. The neonatal mutant mice were genotyped by PCR and sequencing. The positive founder (F0) was bred to establish Pdgfd+/− mouse line. The primers used for genotyping the Pdgfd deficient mice by PCR are 5′-ATAACATAGTAAAGCGAAAACATGAACTG-3′ and 5′-GGCCACTCTTGTGGAAGATAATCTC-3′. A 928 bp PCR product represents the wild-type mice, and a 457 bp band represents the deletion of the Pdgfd gene. The absence of Pdgfd exon 2 mRNA in Pdgfd−/− mouse hearts (Fig. S5c) was confirmed by qRT-PCR. The primers are 5′-TTCCCGAACAGCTACCCAAG-3′ and 5′-TCCTTGGAGGGATCTCCTTGT-3′.

Cell culture and treatment

Mouse ESCs E14 (Darwin Core Facility, Baylor College of Medicine) were maintained in culture dish coated with 0.1% gelatin (Sigma-Aldrich, St. Louis, USA) in ESC medium containing KO DMEM medium (Thermo Fisher Scientific, Gibco, Waltham, MA, USA) and supplemented with 15% (v/v) fetal bovine serum (FBS; Gibco, Waltham, MA, USA), β-mercaptoethanol (0.1 µM), GlutaMax-I supplement (2 mM), MEM non-essential amino acids (0.1 mM), 1% (v/v) Penicillin–Streptomycin, LIF (1000 U/mL, Millipore, Billerica, MA, USA), and 2 inhibitors (CHIR99021, PD325901 at 3 µM and 1 µM, respectively) (Hejian Technology Co., Suzhou, China). HEK293T cells (COBIOER, Nanjing, China) were maintained in DMEM medium (Sigma-Aldrich, St. Louis, USA) with 10% (v/v) FBS, 1% Penicillin–Streptomycin. Mouse embryonic fibroblasts (MEFs, Cyagen, Santa Clara, CA, USA) serves as feeder cells and were maintained in DMEM medium with 10% FBS and 1% Penicillin–Streptomycin. The MEFs were treated with Mytomycin C (10 µg/mL, Sigma-Aldrich, St. Louis, USA) for 3 h before culturing in ESC medium. All cells were cultured at 37 °C in humidified air with 5% CO2.

For PDGFD or PDGFB protein stimulation, ESCs were starved in serum-free medium overnight and then stimulated with 50 ng/mL PDGFD protein (R&D, Minneapolis, MN, USA) or PDGFB protein (PeproTech, Cranbury, NJ, USA) for indicated timepoints. For ERK inhibition, the MEK inhibitor PD0325901 (1 μM) was applied for 2 days in ESC medium without 2i. For TPA treatment, cells were starved in serum-free medium overnight followed by treatment with 200 nM TPA (New England Biolabs, Ipswich, MA, USA) for 15 or 30 min. For Crenolanib treatment, cells were starved in serum-free medium overnight, followed by treatment with 50 ng/mL PDGFD protein together with 100 nM Crenolanib (Cayman Chemical, Ann Arbor, Michigan, USA) or control goat IgG (R&D, Minneapolis, MN, USA) for 30 min. The cells were then collected for Western blot.

ESC differentiation

For LIF withdrawal-induced ESC differentiation, ESCs were cultured in ESC medium in the absence of LIF for 3 days [38]. For RA-induced ESC differentiation, ESCs were cultured in ESCs medium containing 5 µM RA (Sigma-Aldrich, St. Louis, USA) for three days [39]. For embryoid body (EB)-induced ESC differentiation, EB formation was performed for 6 days (see detailed description below). For endothelium differentiation, 5 × 105 ESCs were cultured in a 100 mm petri dish in the absence of LIF for 2 days and cultured further with 30 ng/mL recombinant human VEGFA protein (PeproTech, Cranbury, NJ, USA) for 7 days [40].

Embryoid body (EB) formation

ESCs were seeded at a density of 4 × 104 cells/mL using a hanging drop (25 µL) method and cultured without LIF (−LIF) in ESC medium for six days in 150 mm dishes. At day 6, all the EBs were harvested, photographed and analyzed [41].

Detection of secreted PDGFD

Mouse ESCs were plated in two 10 cm dishes (1 × 106 each) and cultured in ESC medium. The next day, one dish was refreshed with normal ESC medium while the other switched to ESC medium without LIF (−LIF). After 48 h, ESCs were rinsed with PBS and maintained in 5 mL serum-free ESC medium with or without LIF for 2 days. The medium were then concentrated using Amicon Ultra-4 centrifugal filter units (UFC801024) and analyzed by Western blot. Ponceau S staining was used as a loading control.

PDGFB knockdown by siRNA

ESCs were transfected with siRNA oligos (GenePharma) targeting mouse PDGFB or scrambled siRNA as a control using Lipofectamine 2000 (Invitrogen). ESCs were trypsinized and 1 × 106 cells were seeded in a 60 mm culture dish with 4 mL medium/well. For each well, 200 pmol of siRNA was mixed with 500 µL of Opti-MEM (Thermo), and 10 µL Lipofectamine 2000 (Invitrogen) was mixed with another 500 µL of Opti-MEM, and incubated for 5 min. The two different mixtures were subsequently combined and incubated for 15 min, and added into each well. After 6 h, the culture medium was changed with new medium. Forty-eight hours after transfection, the ESCs were harvested for analysis. The sequences of the PDGFB siRNA oligos are: sense, 5′-GCCUGCAAGUGUGAGACAGUA-3′, and antisense, 5′-UACUGUCUCACACUUGCAGGC-3′.

PDGFR-β neutralization

ESCs were starved in serum-free medium overnight. The next day, ESCs were refreshed with serum-free medium and supplemented with 50 ng/mL PDGFD protein together with 0.6 μg/mL PDGFR-β receptor neutralizing antibody (Thermo Fisher Scientific, Invitrogen, Waltham, MA, USA) or control goat IgG (R&D, Minneapolis, MN, USA) for 30 min. The cells were then collected for Western blot analysis.

Generation of stable Pdgfd knockdown or overexpression ESCs

To generate stable Pdgfd knockdown ESCs, 1 μg of pLent-Pdgfd-4in1-shRNA plasmids (Vigene Biosciences, Rockville, MD, USA) expressing shRNAs targeting four different mouse Pdgfd sequences were transfected into HEK293T cells together with 2 μg of lentivirus packing plasmids pSPAX2 and pMD2G (Addgene, Watertown, MA, USA) using Lipofectamine 2000 (Life Technologies, Waltham, MA, USA). The supernatants containing the viral particles was collected after 48 h and added to the ESCs in the presence of 5 μg/mL of polybrene (Santa Cruz, Dallas, TX, USA). After 6 h, new ESC medium was added. The ESCs were selected using puromycin (2 μg/mL). The pLent-4in1-shRNA-GFP-Puro plasmid (Vigene Biosciences, Rockville, MD, USA) was used as a negative control. The stable pdgfd-overexpressing ESCs were constructed following the same procedure. Mouse pdgfd gene sequence was inserted into the plent-EF1a-FH-CMV-GFP-P2A-puro plasmid (Vigene Biosciences, Rockville, MD, USA). PCR primers used are 5′-CG GGATCCCGATGCAACGGCTCGTTTTAGT-3′ and 5′-ATAGCGGCCGCTCGAGGTGGTCTTGAGCTG CAGAT-3′. The shRNA sequences used are listed in Table S3.

Generation of Pdgfd −/− and Pdgfd +/+ ESCs

Pdgfd−/− and Pdgfd+/+ ESCs were derived from 3.5 day-old blastocyst-stage embryos obtained from Pdgfd+/− heterozygous breeding. Blastocysts were collected by flushing of oviducts and uteri using M2 media (Sigma-Aldrich, St. Louis, USA). Embryos were collected to four-well plates (Thermo Fisher Scientific, Waltham, MA, USA) with feeder cells in ESC medium. After 6–12 days, the ICM outgrowth was re-plated after trypsinization using 0.25% trypsin (Invitrogen Corp., Grand Island, NY, USA) on four-well plates with feeder cells. When the ESCs were sub-confluent, they were plated into larger gelatin-coated culture dishes. The ESCs were passaged every 2–4 days and genotyped by PCR.

Analysis of Pdgfd deficient embryos and neonatal mice

Pdgfd mutant embryos and neonatal mice were obtained from Pdgfd+/−  × Pdgfd+/− breeding. Briefly, 6–8 weeks old female mice were superovulated by intraperitoneal injection of 5 IU pregnant mare’s serum gonadotropin (PMSG; Solarbio, Beijing, China), and after 48 h, followed by the injection of 5 IU human chorionic gonadotrophin (hCG; Millipore, Billerica, MA, USA). The female mice were then caged with male mice at a one-to-one ratio. The presence of a vaginal plug in a female mouse about 12–20 h after hCG injection marks 0.5 days post copulation.

For mouse blastocyst collection, embryos were flushed from oviducts and uteri using M2 media (Sigma-Aldrich, St. Louis, USA) at E3.5. The blastocysts were subjected to whole transcriptome amplification using the PEPLI-g WTA single cell kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The product was 10-fold diluted, and 1 µL of the diluted product was used for qRT-PCR.

For E10.5 or E12.5 embryo collection, the embryos were dissected from uterus, washed with phosphate buffered saline (PBS) and then grinded in RIPA (Solarbio, Beijing, China) or Trizol Reagent (Invitrogen, Waltham, MA, USA) and subjected to Western blot or qRT-PCR.

Colony formation assay and alkaline phosphatase (AP) staining

ESC colony formation assay was performed by plating the ESCs onto 0.1% gelatin-coated 6-well plates at different densities (200, 400, 800 cells per well) and culturing for seven days. Colony formation was analyzed by AP staining prior to imaging. AP staining was performed using the AP detection kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s instructions.

Cell proliferation assay

To analyze ESC proliferation, 1 × 104 ESCs per well were plated in 12-well plates and cultured for 4 days. Cell numbers were quantified at 72 and 96 h using an automated cell counter (Inno-Alliance Biotech, Wilmington, DE, USA). Triplicate samples were used for each group at each time point.

RNA isolation, cDNA synthesis and quantitative real-time PCR (qRT-PCR)

Total RNA was isolated using the Trizol reagent (Invitrogen, Waltham, MA, USA) following the manufacturer’s instructions. A total of 2 µg RNA was reverse transcribed to cDNA using the FastQuant RT Kit (Tiangen Biotech, Beijing, China), and then amplified with qRT-PCR via SYBR Green PCR Master Mix (Vazyme Biotech, Nanjing, China) using an ABI QuantStudio 6 Flex device (Life Technologies, Waltham, MA, USA). When the expressions of multiple genes were investigated, to show the overall expression levels, the expression of each gene was normalized against GAPDH using a ∆Ct method [∆Ct = Ct of the gene − Ct of GAPDH. Relative gene expression = \(2^}} )}}\)]. When the expression of only one gene was investigated, a ∆∆Ct method was used [∆∆Ct = ∆Ct of the gene − ∆Ct of control. Relative gene expression = \(2^}} )}}\)]. The sequences of the primers used are listed in Table S4.

Cellular fractionation

To obtain different cellular fractionations, ESCs were lysed using cytoskeletal buffer (50 mM NaCl, 300 mM sucrose, 10 mM Pipes, pH 6.8, 3 mM MgCl2, 0.5% Triton X-100, protease inhibitor (Beyotime Biotechnology, Shanghai, China), 1 mM DTT, 1 mM PMSF) for 30 min on ice. After centrifugation for 10 min at 4 °C, the cytoplasmic fraction in the supernatant was collected. The nuclear fraction in the pellet was washed, lysed in RIPA buffer (Solarbio, Beijing, China) and collected.

Western blot

Western blots were performed by separating proteins on SDS-PAGE gels followed by protein transfer to PVDF membranes (Bio-Rad). The antibodies used were: anti-PDGFD (sc-137030, Santa Cruz), anti-SOX2 (AF-2018, R&D), anti-BRACHYURY (sc-166962, Santa Cruz), anti-ERKs (4696, Cell Signaling Technology), anti-Phospho ERKs (4370, Cell Signaling Technology), anti-PDGFR-β (3169, Cell Signaling Technology), anti-Phospho PDGFR-β (Tyr1021) (2227, Cell Signaling Technology), anti-PECAM1 (222783, Abcom), anti-Phospho STAT3 (9145, Cell Signaling Technology), anti-STAT3 (9139, Cell Signaling Technology), anti-KDR (9698, Cell Signaling Technology), anti-HSP90 (7411, Cell Signaling Technology), anti-TUBULIN (RM2007, Ray Antibody), anti-β-ACTIN (RM2001, Ray Antibody), anti-GAPDH (70-Mab5465-040, MultiSciences), anti-HISTONE 3 (GB13102-1, Servicebio), Goat anti-mouse IgG (GAM0072, MultiSciences), Goat anti-rabbit IgG (GAR0072, MultiSciences), Rabbit anti-goat IgG (RAG0072, MultiSciences), anti-PDGFB (PA1-27394, Invitrogen), anti-uPA (17968-1-AP, Proteintech) and anti-PLASMIN (66399-1-Ig, Proteintech). The bands were visualized using a GBOX-CHEMI-XX8 device (SYNGENE).

Teratoma formation and immunohistochemistry analysis

The shControl or shPdgfd ESCs (1 × 106 cells/100 µL PBS) were injected subcutaneously into the dorsal flank regions of nude mice. Four weeks after injection, the teratomas were surgically dissected and weighed. Parts of the tumors were used for RNA extraction, and the rest were fixed in 4% paraformaldehyde, embedded in paraffin and sectioned (5 µm) for immunohistochemistry (IHC) staining.

For IHC staining, sections were placed at 60 °C for 1 h followed by deparaffinized with xylene and ethanol. Subsequently, the sections were processed in 10 mM citrate buffer (pH 6.0) and boiled for 10 min for antigen retrieval. After cooling in room temperature, the sections were incubated in 3% hydrogen peroxide for 10 min to block the endogenous peroxidase activity. The sections were stained with anti-Collagen IV (BioRad, Hercules, California, USA) overnight at 4 °C and then incubated using the Elivision kit (Maxim Biotech, Fuzhou, China) for 30 min at room temperature. The DAB color reagent (Maxim Biotech, Fuzhou, China) was used. Finally, hematoxylin staining was performed for nuclear visualization. Sections were visualized using a fluorescence microscope-Axio Imager Z2 (ZEISS, Oberkochen, Germany). ImageJ software was used to analyze the sections. Collagen IV density was calculated as Collagen IV+ pixels/total field pixels.

Immunofluorescence analysis

E12.5 mouse embryos or the hearts from P1 pups were fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, USA) overnight at 4 °C, and then transferred into 30% sucrose overnight at 4 °C. The tissues were then embedded in Tissue-Tek O.C.T. compound (Sakura Finetek, Tokyo, Japan) and sectioned. The frozen sections were placed in room temperature for 30 min, blocked with 5% donkey serum, 0.5% Triton X-100 in 1 × PBS for one hour at room temperature. The sections were stained with anti-PECAM1 (BD Biosciences, New Jersey, USA) overnight at 4 °C and then stained with fluorescein-conjugated secondary antibodies (Life Technologies, Waltham, MA, USA) for 1 h at room temperature. Sections were visualized using a fluorescence microscope-Axio Imager Z2 (ZEISS, Oberkochen, Germany). All sections were imaged using a × 20 objective. The ImageJ software was used for image analysis. The ventricle areas of the hearts were imaged and analyzed. Vascular density was calculated as PECAM1+ pixels/total field pixels.

RNA sequencing (RNA-seq)

Total RNA was extracted from shControl or shPdgfd ESCs (triplicates for each group) using Trizol reagent (Invitrogen, Waltham, MA, USA). RNA-seq was performed using an Illumina Nova Seq 6000 sequenator (Illumina, San Diego, CA, USA) by Kangcheng Bio-tech, Inc. (Shanghai, China). The differentially expressed genes (DEGs) were defined at p < 0.05 and fold change > 1.5. The DEGs were subjected to GO analysis using the topGO package for enriched biological processes, and the KEGG pathway analysis using Fisher’s exact test for enriched pathways (p < 0.05). GSEA enrichment plots were generated using the GSEA software [42]. Default parameters were used for GSEA analysis. The RNA sequencing data were accessible in GEO database (GSE172117).

Statistical analysis

GraphPad Prism (GraphPad Software, Inc.) was used for statistical analyses except for the RNA-seq data. All data are presented as mean ± SD. For comparisons between two groups, two-tailed Student’s t tests were used. For comparison among more than two groups, one-way ANOVA analysis was used. N numbers, p values and other detailed information are provided in the corresponding figure legends.