Extended Data Fig. 1: Sustained signaling for PGCLC specification restricts subsequent development.
(a) RNA-sequencing data 6,14,24 of in vitro induced day 4 PGCLCs and in vivo PGCs for PGC genes. Bar plot represents mean values (PGC week5/7/9, n = 2 biological samples; migratory, 37 cells; mitotic, 332 cells; mitotic arrest, 309 cells). (b) Example of gating strategies for flowcytometry analysis with PGCLC d5 from Fig. 1b. Cell population is gated based on SSC-A/FSC-A, single cell population based on FSC-A/FSC-W and live cell population using DAPI. % gating populations/the parental populations. (c) Targeting strategy for reporter ES cells. Guide (g)RNA for CRISPR-Cas9 and genotyping primers (P1-P5) are indicated. (d) Genotyping PCR for reporter ES cells. +: wild type allele; KI: knock-in allele; NC: water control; WT: wildtype cells; −/+DRE: before (−) and after (+) puromycin resistant cassette excision. The experiment was repeated independently two times with similar results. (e) Flowcytometry and (f) RT-qPCR of PGCLC induction from NANOS3–tdTomato/DMRT1-mVenus double reporter male ES cells (WIS2) with inducible SOX17 = GR and TRE-PRDM1 cell line (clone 2) treated with DEX and dox (SOX17 + PRDM1) for five days in the presence or absence (no cytokine) of PGC specification cytokines, BMP2, SCF and EGF (BSE). Percentage for +/−DMRT1-mVenus in NANOS3-tdTomato positive cells are indicated. Values in (F) are normalized with the housekeeping gene GAPDH and relative changes against no tg/no Cy/DEXdox(-). A repeat experiment with independent clone with similar results is shown in Fig. 1e. (g) Quantitative PCR for copy number for genomic integration of the piggybac plasmids for SOX17 = GR and TRE-PRDM1 in clone 1 and 2. Biologically independent experiments, n = 2.(h) Western blot for expression of SOX17, PRDM1, LAMINB1 in the nuclear protein extract for the transgenic cell lines 1 and 2 in the presence of DEX and dox for 3 days, and PGCLCs for day 5 induced with cytokines, BMP2, SCF and EGF. The experiment was repeated independently two times with similar results. Dose response of DEX (μM) and dox (μg/ml) (i), DEX (j) and dox (k) for the expression of DMRT1 reporter in NANOS3+PGCLCs. Percentage for +/−DMRT1-mVenus in NANOS3-tdTomato positive cells are indicated. The graphs show the percentages from the two independent experiments.
