Fig. 6: Functional interrogation of NOTCH signalling in human peri-implantation.
From: Distinct pathways drive anterior hypoblast specification in the implanting human embryo
a, Immunofluorescence images of embryos cultured from day 5 to day 7 in control conditions (N = 44 embryos), 20 µM DAPT (N = 8 embryos), 10 µM Compound-E (N = 14 embryos) or 20 µM MK-0752 (N = 9 embryos). b–d, Quantification of the number of epiblast (b), hypoblast (d) and CER1-positive hypoblast cells (d) at day 7. e, Immunofluorescence images of embryos cultured from day 7 to day 9 in control conditions (N = 39 embryos), DAPT (N = 9 embryos), Compound-E (N = 12 embryos) or MK-0752 (N = 17 embryos). f–h, Quantification of the number of epiblast (f), total hypoblast (g) and CER1-positive hypoblast cells (h) at day 9. i–k, Immunofluorescence images and quantification of 2D naive PXGL and primed human (h)ES cells treated for 48 h in control conditions (PXGL n = 2,336, primed n = 7,796) or 20 µM DAPT (PXGL n = 2,386, primed n = 6,136; N = 2 experiments) and 3D spheroids derived from primed hES cells cultured in control conditions (n = 364) or DAPT (n = 643; N = 2 experiments). Cells were stained for SOX2 and cleaved caspase 3 (Ccasp3) with spheroids additionally stained for PODLX. l, Immunofluorescence images of the differentiation of YSLCs that were treated with DAPT for 48 h during YSLC specification or maturation. m, Percentage of GATA6-positive cells that are CER1 positive. D2–D4: control n = 2,502; DAPT n = 2,690. D4–D6: control n = 2,500; DAPT n = 2,701; N = 2 experiments. n, Immunofluorescence images of pSMAD1.5.9 expression in hES cell-derived spheroids cultured in mTeSR+ medium (mTeSR+ n = 1,060 cells) or mTeSR+ medium conditioned on either control YSLC (YSLC CM n = 1,836 cells) or on YSLC differentiated with 48 h of DAPT treatment (YSLC + DAPT CM n = 501 cells). N = 3 experiments. o, Quantification of normalized pSMAD1.5.9 fluorescence from n. For box plots, box encompasses 25th and 75th quartiles with median marked by central line. Minimum and maximum and denoted by whiskers. Plus symbol marks the mean. Error bars denote standard error (j, k and m–o). Statistics: two-sided Mann–Whitney test (b–d and f–h); two-sided unpaired t-test (j and m); two-way analysis of variance (ANOVA) with Šidák’s multiple comparison’s test (k); one way ANOVA with Tukey–Kramer post-hoc (o). P < 0.06 is noted. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. Unmarked comparisons to control are not significant (NS). Exact P values presented in Supplementary Table 8. Scale bars: 100 µm (a and e) and 50 µm (i, l and n).
