Extended Data Fig. 2: Switching signaling in nascent PGCLCs for further development.

(a and b) Flowcytometry of NANOS3-tdTomato against DMRT1-mVenus. NANOS3-tdTomato/DMRT1-mVenus reporter 4i ES cells were induced PGCLCs with the cytokines indicated for 8 days. BSE: BMP2, SCF and EGF; Ra: retinoic acid, 20 μM; ActA: Activin A, 20 μg/ml; SE: SCF and EGF. Percentage populations of NANOS3 positive and DMRT1 positive or negative cells in live single cells are shown. (c) Flowcytometry of NANOS3-tdTomato against DMRT1-mVenus. 3-days induction of nascent PGCLCs with BSE followed by Ra 20 μM, SCF 100 μg/ml, EGF 50 μg/ml and, Activin A 100 μg/ml, TGFβ 100 μg/ml or Nodal 100 μg/ml. Percentage populations of NANOS3 and DMRT1 double positive cells in live cell population are shown. (d) Flowcytometry histogram of DMRT1-tdTomato. DMRT1-tdTomato female reporter line (Shef-6) was induced nascent PGCLCs with BSE for 3 days and followed by Ra 20 μM+Activin A 100 μg/ml+SCF 100 μg/ml+EGF 50 μg/ml for 6 days (d3 + 6). Parental Shef-6 ES cells without reporter was used as a negative control. (e) Flowcytometry and immunofluorescence for PGCLCs induced with BSE (d8 + 0) or BSE 3 days followed by RASE (d3 + 6) from conventional ES cells cultured in E8 via precursors of mesendoderm (E8-preME) or 4i ES cells (4i). Values in flowcytometry plots indicate percentage of DMRT1 positive or negative in NANOS3-tdTomato positive cell populations. Scale bar in immunofluorescence image shows 50 μm. The experiment was repeated independently two times with similar results. (f) Immunofluorescence of TFAP2C and 5-methylcytosine (5mC) for PGCLCs induced with BSE (d6 + 0) or BSE 3 days followed by RASE (d3 + 6). Scale bar =50 μm. The experiment was repeated independently two times with similar results.