Extended Data Fig. 9: Heterogeneities in BAF155 and CARM1 within the early embryo trigger differential expression of keratins at the eight-cell stage.
From: Keratins are asymmetrically inherited fate determinants in the mammalian embryo
a, Live-imaging of an embryo expressing K8-Emerald, H2B-RFP and RFP-Utrophin confirms that the first cells to assemble keratin filaments are sister cells. The microtubule bridge connecting sister cells can be identified by RFP-Utrophin accumulation (white arrowheads)41. Data are from three independent experiments. b, Scheme shows the stereotypical 3D organization of a tetrahedral four-cell embryo. The vegetal blastomere is located distal from the polar body. c, Selective photoactivation of the vegetal blastomere. The vegetal blastomere is identified based on its distal position from the polar body. The vegetal cell nucleus is then targeted with a two-photon laser (820 nm light) to photoactivate H2B-paGFP. 2D confocal planes show efficient photoactivation immediately after 820 nm light illumination. Data are from three independent experiments. d, The first cells to form keratin filaments are unrelated to the order of cell divisions during the 4- to 8-cell stage transition. χ2 test. e, BAF155 knockdown reduces BAF155 immunofluorescence levels relative to control blastomeres, while BAF155 overexpression increases them. Embryos were microinjected with BAF155 siRNAs or high levels of BAF155 RNA respectively at the one-cell stage. ***P < 0.0001; ANOVA test. f, Embryos treated with trichostatin A (TSA) display extensive keratin filament formation, while embryos treated with actinomycin D (Act D) do not form filaments. *P = 0.0489; ***P < 0.0001; ANOVA test. g, Microinjection of K8 and K18 mRNA into the one-cell embryo causes premature assembly of an extensive keratin filament network throughout early blastomeres before the eight-cell stage. ***P < 0.0001; two-sided Fisher’s exact test. h, BAF155-overexpressing embryos treated with actinomycin D do not form keratin filaments at the eight-cell stage. Data are from three independent experiments. i, Dimethyl-BAF155 is lowest in the vegetal blastomere. **P = 0.004; unpaired, two-tailed Student’s t-test. j, CARM1 overexpression increases CARM1 immunofluorescence levels relative to control blastomeres. Embryos were microinjected with high levels of Carm1 RNA at the 1-cell stage. ***P < 0.0001; unpaired, two-tailed Mann–Whitney U-test. k, CARM1 overexpression reduces keratin filament assembly. ***P = 0.0007; unpaired, two-tailed Student’s t-test. l, Overexpression of BAF155 or mutant BAF155(R1064K) causes premature keratin filament assembly at the four-cell stage. **P = 0.009 for BAF155 overexpression; **P = 0.005 for BAF155(R1064K); two-sided Fisher’s exact test. m, BAF155-knockdown blastomeres (white arrowheads) display lower levels of CDX2 than control cells (orange arrowheads) at the same stage. BAF155 siRNAs were microinjected into only one cell of the two-cell embryo. ***P < 0.0001; ANOVA test. n, CARM1-overexpression blastomeres (white arrowheads) display lower levels of CDX2 than control blastomeres (orange arrowheads) at the same stage. High levels of Carm1 RNA were microinjected into only one cell at the two-cell stage. **P = 0.005 for control inner cells; *P = 0.04 for CARM1 overexpression outer cells; **P = 0.006 for CARM1-overexpression inner cells; ANOVA test. Scale bars, 10 μm.
