Fig. 1: Temporal resolution and morphological effects of inhibiting p38-MAPK signaling in developing blastocysts. | Communications Biology

Fig. 1: Temporal resolution and morphological effects of inhibiting p38-MAPK signaling in developing blastocysts.

From: p38-MAPK-mediated translation regulation during early blastocyst development is required for primitive endoderm differentiation in mice

Fig. 1

a Scheme illustrating the experimental protocol used to resolve the temporal nature of p38-MAPKi in the PrE deficit phenotype. The lineage markers NANOG and GATA4 denote EPI and PrE cells, respectively. b Quantification of the ratio of GATA4-positive PrE cells to total ICM cells in control (n = 21) and p38-MAPKi (n = 22) conditions from confocal images of E4.5 embryos transiently cultured with DMSO or SB220025 between E3.5 + 4 h and E3.5 + 7 h (right); the means and standard deviations are highlighted. Representative z-section blastocyst confocal microscopy projections are shown on the left (scale bars = 20 µm). c Quantification of the ratio of GATA4-positive PrE cells to total ICM cells in control (DMSO) (n = 42) and p38-MAPKi (SB220025) (n = 38) conditions for treatment between E3.5 and E4.5 (recapitulation of previously published observations36,37); the means and standard deviations are highlighted. d Quantification of the equatorial cavity areas (µm2) in fixed blastocysts cultured (E3.5-E4.5) in control (DMSO; n = 20) and p38-MAPKi (SB220025; n = 17) conditions. Representative confocal equatorial z-sections of each embryo group are shown, with the cavity circumference indicated (dashed yellow line) (upper panel scale bars = 20 µm). e Equatorial areas of control (DMSO; n = 32) and p38-MAPKi (SB220025; n = 32) blastocysts imaged in live culture from E3.5-E4.5. The sub-panels show segments of the recordings between 3 and 10 h, 10 and 15 h, and 15 and 22 h. The data are presented as box-and-whisker plots, with lines connecting the median points (blue for DMSO and red for SB220025). The numbers within the sub-panels are the mean equatorial areas. The bar graph denotes the percentages of hatching blastocysts observed at the end of the imaging period (E4.5). f qRT-PCR-derived expression levels (normalized to Tbp) of the ICM lineage markers Nanog and Gata4 (upper) and the p38-MAPK paralogous genes Mapk11-14 (lower) during early blastocyst maturation (at E3.5 + 0 h, E3.5 + 3 h, E3.5 + 6 h, E3.5 + 9 h, and E3.5 + 10 h). Data presented as box plots with the mean expression levels across the timeline expressed as line graphs.

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