Zombie red dye

A randomly-selected subset of pregnant females was euthanized 13 days (E13.5) after vaginal plug detection. E13.5 embryos were isolated from euthanized pregnant dams, and E13.5 gonads containing primordial germ cells (PGCs) were isolated using Leica MZ9.5 Binocular Stereo Microscope. Gonads were identified and sexed by their characteristic morphology at E13.5 (Extended data Fig. 13a, b) and sex was verified by PCR^{26 (link)}. Primer sequences are given in Extended data Table 1. Gonads from same-gender embryos in each litter were pooled prior to tissue dissociation. Gonads were enzymatically digested using Embryoid Body Dissociation Kit (Miltenyi Biotec). Next, total dissociated gonad cells were stained with Zombie Red Dye (BioLegend), PE Mouse anti-SSEA-1 (BD Pharmingen), and Alexa-Fluor 647 Mouse anti-CD61 (BD Pharmingen). Primordial germ cells were purified based on the expression of Zombie Red⁻/SSEA-1⁺/CD61⁺ using BD FACS Aria II Cell Sorter (BD Bioscience). Somatic gonad cells were purified based on the expression of Zombie Red⁻/SSEA-1⁻/CD61. The gating strategy and purity of the isolated cells is shown in Extended data Fig. 14.

TBT, dexamethasone, isobutylmethylxanthine, insulin were purchased from Sigma-Aldrich (St. Louis, MO). Rosiglitazone (ROSI) was purchased from Cayman Chemical (Ann Arbor, MI). Embryoid Body Dissociation Kit (#130–096-348) was purchased from Miltenyi Biotec (North Rhine-Westphalia, Germany). Zombie Red Dye (#77475) was purchased from BioLegend (San Diego, CA). PE Mouse anti-SSEA-1 (#560142) and Alexa-Fluor 647 Mouse anti-CD61 (#563523) were purchased from BD Biosciences (Franklin Lakes, NJ). Blood glucose meter kits (BG1000) were purchased from Clarity Diagnostics (Boca Raton, FL). Mouse Leptin ELISA Kit (#90030), Mouse C-peptide ELISA kit (#80954) and mouse insulin ELISA kit were purchased from Crystal Chem (Elk Grove Village, IL, USA). Arima-HiC Kit (A510008) was purchased from Arima Genomics (San Diego, CA). Ultra-pure formaldehyde (#18508) was purchased from Ted Pella Inc (Redding, CA). MinElute Reaction Cleanup Kit (#28206) was purchased from Qiagen (Hilden, Germany).

EBs were harvested at day 10 of the cardiovascular differentiation protocol to conduct flow cytometry analyses using a standard staining protocol.^[56] Briefly, cells were incubated with Zombie Red dye (BioLegend) to exclude dead cells and CD29‐PE (MCA2298PE, AbD Serotec, UK) for 25 min at 4 °C protected from light. After washing, the Foxp3 Staining Buffer Kit (421403, BioLegend) was used to fix the cells for 30 min on ice, followed by washing and permeabilization for 15 min at 4 °C. After blocking with BSA for 10 min, cells were incubated with CTNT primary antibody (ab8295, Abcam) for 35 min at 4 °C, washed and blocked with 5% BSA for 10 min at room temperature to avoid unspecific binding. The secondary anitbody (rat antimouse IgG1‐AF647, 406617, BioLegend) was added for 25 min at 4 °C, protected from light. For the analysis using the ImageStreamx mkII (Amnis Corporation, USA) with the INSPIRE instrument controller software with 40× magnification, 1 × 10⁴ single cells were acquired per sample. Data were analyzed with IDEAS Image analysis software. All samples were gated on single cells that were Zombie Red‐negative. The percentage of CTNT+ cells was determined and a control sample (stained with the same antibodies except the primary antibody) was used to set the background fluorescence.