Fig 2: TCF4 and IRF4 function downstream of p38a to regulate Blimp1 transcription.a Flow cytometry analysis of iPCs differentiated from cultured Cas9tg/+ B cells transduced with retroviruses encoding p38a or Tcf4 sgRNA (CD19+GFP+BFP+CD138+). Immunoblot analysis of BLIMP1, TCF4, and Actin expression in Tcf4 sgRNA expressing B cells at indicated time points after being transferred onto IL-21 feeder cells. b Schematic diagram of a putative TCF4-binding site in the Prdm1 locus. CUT&Tag analysis of TCF4 binding genomic fragments including the reported TCF4 binding site in Blimp1 enhancer region in cultured p38a KO and WT B cells at day 2 after transferred onto IL-21 feeder cells (n = 3 per group). c Flow cytometry analysis of iPCs among cultured p38a KO B cells transduced with retroviruses encoding TCF4 (CD19+GFP+CD138+) (n = 6 per group), protein levels of TCF4, IgG H-chain, BLIMP1, p38a and Actin were analyzed by immunoblot. d–f Same as described in (a–c) except that Irf4 sgRNAs were used or IRF4 was overexpressed in cultured p38a KO B cells. g Cultured p38afl/flVavcreCas9tg/+ and p38afl/flCas9tg/+ B cells were transduced with retroviruses encoding sgRNA targeting p38a, Tcf3, Tcf4 and Irf4 at day 2 of culture (n = 3 per group). Percentages of iPCs in sgRNA transduced B cells (CD19+GFP+BFP+CD138+) were analyzed by flow cytometry at day 8. NTC, non-targeting control. Data were representative of at least three independent experiments. Each symbol represents a representative sample. Small horizontal lines indicate the mean (±s.d.). Data were analyzed by two-tailed unpaired t-tests. Source data are provided as a Source Data file.

Fig 3: The activation of TCF3, TCF4 and IRF4 downstream of p38a in PC differentiation.a Cultured p38a-/- B cells were transduced with retroviruses encoding p38a or p38a kinase-dead mutations (K53M or T180A/Y182F) (GFP+) and luciferase reporter harboring TCF3, TCF4 or IRF4 binding sites in Prdm1 enhance region at day 2, and luciferase activity in retrovirus-infected B cells (CD19+GFP+) was analyzed at day 8 in iPC culture (n = 3 per group). b p38-/- 293 A cells were transfected with indicated plasmids, and the luciferase activity of IRF4 mutants was analyzed 24 h after transfection. Immunoblot analysis of p38a, p-p38a, IRF4 and Actin in p38-/- 293 A cells transfected with indicated plasmids (n = 3 per group). c Cultured Irf4-/- B cells were transduced with retroviruses encoding IRF4 or IRF4 mutants (T267A, T268A, T269A) at day 2, iPCs in retrovirally transduced B cells (CD19+GFP+CD138+) 6 days after transduction were analyzed by flow cytometry (n = 5 per group), and protein levels of IRF4, IgG H-chain, BLIMP1 and Actin were analyzed by immunoblot. Data were representative of at least three independent experiments. Each symbol represents a representative sample. Small horizontal lines indicate the mean (±s.d.). Data were analyzed by two-tailed unpaired t-tests. Source data are provided as a Source Data file.

Fig 4: Screen for Blimp1 retina-specific enhancers.(A) Schematic of the Blimp1 genomic locus on mouse chromosome 10 showing nine DNase hypersensitive sites (DHS) near the gene. (B) Schematic of the retinal explant enhancer assay. The nine DHS sequences (A-I) are inserted upstream of a TATA box minimal promoter driving nuclear-localized GFP (nGFP). A separate plasmid contains the ubiquitous EF1a regulatory element driving nuclear-localized Cherry expression. Constructs are co-electroporated into P0 retinal explants and collected for histological analysis after one day in vitro (DIV). (C-C”) Co-electroporation of TATA-box only (no enhancer) and cherry plasmids. GFP+ nuclei (green) are rarely observed and express GFP at low levels. In contrast, Cherry+ nuclei (red) are widely distributed. Most GFP+ cells do not co-express Blimp1 (grey, arrowheads, insets), but about 25% are co-labeled (arrows). A similar fraction of Cherry+ cells co-express Blimp1 (asterisks, insets). This represents the probability that electroporated plasmids will end up in Blimp1+ cells by chance after 1 DIV. (D-D”) Element F drives strong GFP expression in explants and most of these cells co-express Blimp1 (arrows, insets). Arrowheads mark GFP+ cells that do not co-express Blimp1. (E-E”) Element H drives little GFP expression and the few positive cells do not co-express Blimp1 (arrowheads, insets). Blimp1+ cells are electroporated, as Cherry+ cells co-express Blimp1 (asterisks). (F-F”) Element I showed robust GFP expression with nuclei localized to the middle of the retina. Some GFP+ cells co-express Blimp1 (arrows, insets), but about 80% are Blimp1 negative (arrowheads, insets). GCL, ganglion cell layer. Scale bar is 50 µm for panels and 10 µm for insets. (G) Plot of the average percentage of GFP+ or Cherry+ cells that co-express Blimp1 for the active elements. Error bars represent the standard deviation (S.D.). Only element F shows a significant enrichment of Blimp1 co-expression compared to Cherry and TATA box GFP controls (*, unpaired t-test, P <0.001). The high standard deviation in the TATA only condition is due to the paucity of GFP+ cells.

Fig 5: Control of colony size dramatically impacts the fraction of primordial germ cell-like cells (PGCLCs).(A–D) Different diameter colonies stained for TFAP2C, SOX17, EOMES at 42 hr and quantification, (B) SOX17 vs. TFAP2C scatterplot colored for colony size. (C) Same plot colored for EOMES expression. (D) SOX17+ subpopulations for each colony diameter. (E–G) 100 µm colonies differentiated with BMP only or with incipient mesoderm-like state (iMeLC) pre-differentiation stained for TFAP2C, NANOG, PRDM1 at 48 hr and quantification. C3 = 3 µM CHIR-99021, other notation is like in Figure 4. (I–K) Stainings and quantification of pSMAD1 and SMAD2/3 for different size colonies. Scale bars 50 µm.