Fig 2: BAF-L deficiency causes defects in spermiogenesis. (A–D) Representative images of H&E staining and quantifications show decreased numbers of elongating germ cells in the BAF-L KO testis at the age of 4 weeks, 5 weeks, and 6 weeks. Error bars indicate SD. * p < 0.05, *** p < 0.001 by two-tailed Student’s t-test. Green arrows indicate elongating/elongated spermatids. (E) Illustration for the 16 developmental stages of mouse spermatids. (F) Representative images of ACRV-1 labeling in 4-week-old BAF-L KO testis. Red arrows indicate stage 1–4, green arrows indicate stage 5–8, blue arrows indicate stage 9–11, and purple arrows indicate 12–16. (G) Quantification shows comparable ACRV-1-positive cells in 4-week-old BAF-L KO testis. Error bars indicate SD. N.S by two-tailed Student’s t-test. n = 3 for WT and n = 4 for KO. (H) Quantification shows a delay in spermiogenic progress in 4-week-old BAF-L KO testis. Error bars indicate SD. * p < 0.05, ** p < 0.01 by two-tailed Student’s t-test. n = 3 for WT and n = 4 for KO. (I) Western blots show no difference in EpCAM protein level in 4-week-old and 8-week-old BAF-L KO testis. (J,K) Representative images of SCP3 and GATA-4 labeling in 4-week-old and 8-week-old BAF-L WT/KO testis.

Fig 3: The effects of doxorubicin (DOX) and 3i-1000 on GATA4 levels and caspase activation in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and neonatal rat ventricular myocytes (NRVMs) after short-term exposure. For high-content analysis, the cells were exposed simultaneously to 100 nM DOX and 3i-1000 for 24 h after which they were fixed and stained. Imaging and analysis was carried out using CellInsight High-Content Screening Platform. Representative images of GATA4 staining in a hiPSC-CMs and b primary NRVM cultures. c Average intensity of nuclear GATA4 staining. d Proportion of cardiomyocytes positive for fluorescent caspase-3/7 activity reporter. Adjustments of individual colour channels to enhance brightness and contrast were made identically to all representative images. The results are expressed as mean + SEM (n = 3–4). *P < 0.05 vs. control (randomized block ANOVA followed by Tukey’s HSD) (colour figure online)

Fig 4: GATA4-deficient HS architecture and gene expression are disrupted at E14.5. (A–H) GATA4 IHC of control and G4 cKO stomachs from E16.5 (n = 6 control, 6 mutant), E14.5 (n = 7 control, 11M) and E12.5 (n = 5 control, 6 mutant) embryos showed depleted GATA4 protein in mutants. Small regions of GATA4+ cells were evident in mutant HS. GATA4 protein loss became more evident in mutants between E12.5 to E14.5. (I–P) H&E staining of E16.5 (n = 6 control, 6 mutant), E14.5 (n = 5 control, 8 mutant), and E12.5 (n = 4 control, 4 mutant) control and G4 cKO HS showed disrupted epithelial structure at E16.5 and E14.5. E16.5 control HS epithelium was columnar and lined with primordial buds, whereas G4 cKO HS epithelium appeared stratified, containing few abnormal primordial buds. E14.5 control HS epithelium had a typical morphology containing intraepithelial spaces, which are associated with glandular morphogenesis, whereas mutant HS epithelium was thinner with sparse, smaller intraepithelial spaces. (Q) GATA4 protein expression and epithelial structure were normal in HS from Gata4+/+Shh Cre E14.5 embryos (n = 2). Boxed regions are shown at higher magnification in insets (A–Q). (R) Differentiated HS epithelial cell transcripts detected by qRT-PCR were decreased in E14.5 G4 cKO HS compared with control HS (n = 3 mutant, 3 control). (S) Expression of stratified squamous epithelial cell transcripts detected by qRT-PCR was increased in E14.5 G4 cKO HS compared with control HS. All were expressed in control FS (n = 13 control HS, 12 mutant HS, 3 control FS). All scale bars = 50 μm. All qRT-PCR used HS or FS epithelial cells. Error bars show SEM. P values determined by a Student’s t test or 1-way analysis of variance as appropriate. ∗P ≤ .05, ∗∗P ≤ .001, ∗∗∗∗P ≤ .0001.

Fig 5: Early Commitment to hESC Differentiation in Gastruloids and IPS Cells(A) Left: schematic of the duration of BMP4 pulses used to drive hESC differentiation. Right: representative images of hESCs growing on micropatterns showing active SMAD (pSMAD 1/5/8), GATA3, and tri-lineage specific markers (BRY, CDX2, SOX2) after 1 and 72 h of BMP4 (50 ng/mL) treatment, respectively. Micropattern diameter 500 µm.(B) Left: schematic of the duration of BMP4 pulses used to drive differentiation in KOLF2-C1 iPS cells. Center: representative images of pluripotency markers (NANOG and SOX2) and lineage-specific markers (BRY, CDX2, and GATA4) in response to 15 min and 60 min pulses of BMP4 stimulation (50 ng/mL). Cells cultured without BMP4 and with sustained (S) BMP4 were used as controls. Images are shown as the merge image between the gene of interest (red channel) and DAPI (blue channel). Scale bar represents 100 µm. Right: representative images of colony morphology for the different experimental conditions. Scale bar represents 50 µm.(C) Quantification of the fraction of positive cells showing the expression of pluripotency (NANOG and SOX2) and mesoderm-specific (BRY, CDX2, and GATA4) genes at day 3 following different pulses of BMP4. n > 500 cells were analyzed for each experimental condition.