Nagaokaet al.,Science 367 , eaaw4115 (2020) 6 March 2020 6of9
ACFGHDEBFig. 4. Regulatory coverages of ZGLP1 and STRA8 for the oogenic program.
(A) Overlap of genes up-regulated by BMP2 between the wild-type andZglp1–/–
mPGCLC-derived cells. The numbers of the genes up-regulated [log 2 (RPM + 1) > 4,
log 2 (fold change) > 2 from the control at c4, c5, c7, or c9] in the respective
clones are shown. Key GO enrichments are shown. (B) Expression kinetics of the
457 up-regulated genes (A) in the wild-type andZglp1–/–mPGCLC-derived
cells at c4, c5, c7, and c9. Fold changes from the control culture are shown for
the respective clones. Box plots represent values for the 25th,50th, and 75th
percentiles. (C) PCA of the transcriptomes of germ cells in vivo (E9.5 to E11.5
PGCs and E12.5 to E15.5 female or male germ cells) and wild-type andZglp1–/–
mPGCLC-derived cells (G10) cultured with BMP2 and RA from c3. Each plot
represents one transcriptome. Transcriptomes from two replicates (for E9.5 PGCs
and E14.5 to E15.5 oocytes, three replicates) are plotted. The color code is as
indicated. (D) Expression dynamics ofZglp1andStra8in the wild-type,Zglp1−/−,
andStra8−/−mPGCLC-derived cells cultured with BMP2 and RA from c3 onward
(relative to the wild-type mPGCLC-derived cells at c9). (E) Left: Western blot
analysis of the STRA8 expression at c7 in the wild-type andZglp1–/–mPGCLC-
derived cells cultured with BMP2 and RA from c3 onward. Right: STRA8 levels
(normalized byb-ACTIN) from two biological replicates. The mean value for the
wild-type mPGCLC-derived cells is set as 1.0. Error bars indicate SDs. (F)Ex-
pression of key oogenic and meiotic genes at c7 in the wild-type andZglp1−/−
mPGCLC-derived cells in the cultures as indicated. The expression in SC+cells
sorted by FACS was analyzed by quantitative polymerase chain reaction. TheDCt
from the average Ctvalues of two housekeeping genes,ArbpandPpia, is shown
for each gene. The mean values from two independent experiments, each with two
technical replicates, are shown. Error bars indicate SDs. N.D., not detected. The
color code is as indicated. (GandH) Overlap of differentially expressed genes
(DEGs) [log 2 (RPM + 1) > 4, log 2 (fold change) > 2 at c4, c5, c7, or c9] between the
wild-type andZglp1–/–PGCLC-derived cells (common DEGs in clone E5 and
G10; 530 up-regulated and 281 down-regulated) and DEGs between the wild-type
andStra8–/–mPGCLC-derived cells (common DEGs in clone B6 and F6; 224
up-regulated and 152 down-regulated). Key GO enrichments are shown.RESEARCH | RESEARCH ARTICLE