Extended Data Fig. 7: Experimental manipulations of keratin levels show that keratins regulate CDX2 to specify the first trophectoderm cells of the embryo.
From: Keratins are asymmetrically inherited fate determinants in the mammalian embryo
a, Immunofluorescence for K8 in embryos microinjected with siRNAs for K8 and K18 at the one-cell stage, or into only one cell at the two-cell stage. This double-knockdown approach extensively eliminates keratin filament assembly. Data are from five independent experiments. b, Knockdown of K8 and K18 in half of the embryo also eliminates filament formation by K19. White arrowheads show knockdown cells. Data are from three independent experiments. c, Keratin overexpression causes a premature and widespread assembly of a keratin network within the 8- to 16-cell stage embryo. Images show examples of embryos microinjected with high levels of K8 and K18 RNA at the 1-cell stage, or into one cell of the 2-cell embryo. Data are from three independent experiments. d, Keratin overexpression causes some filaments to be inherited by inner cells of the 16-cell stage embryo (yellow segmented cell indicated by arrow in left panel). 2D view shows keratin filament organization within outer and inner cells of keratin overexpressing embryos (right). Data are from three independent experiments. e, Inner cells in keratin overexpressing embryos express lower levels of nuclear YAP and CDX2 than outer cells. *P = 0.04; ***P < 0.0001; unpaired, two-tailed Mann–Whitney U-test. f, Knockdown of YAP using siRNAs microinjected into one cell of the two-cell embryo reduces CDX2 levels, in both keratin-positive and keratin-negative cells. H2B-RFP was co-injected with the siRNAs to identify the knockdown cells (white arrowheads). ***P < 0.0001; ANOVA test for CDX2. Right graph shows that our knockdown approach using YAP siRNAs effectively reduced YAP levels. *P = 0.03, unpaired, two-tailed Mann–Whitney U-test for YAP. g, Scheme depicting cloning strategy to generate rescue constructs for K8 and K18. The coding regions of K8 and K18 are indicated by thick yellow arrows, and the targeted sequence locations for the keratin siRNAs used in this study are indicated by the red arrows. The specific siRNA target sequences are highlighted in yellow, corresponding to the keratin wild-type (WT) sequence (top rows). The rescue construct sequences are indicated (bottom rows). Note the conservation of amino acid sequence despite the scrambling of DNA bases throughout the siRNA target sequence. In each experiment, H2B-RFP was co-injected with the siRNAs and/or mRNAs to label the injected half of the embryo, and 100% of H2B-positive cells displayed keratin filaments when injected with the rescue construct. K8/K18-knockdown cells express lower levels of CDX2 than control cells with keratins, but this phenotype is rescued when the keratin rescue constructs are co-injected with keratin siRNAs. ***P < 0.0001; ANOVA test. Scale bars, 10 μm.
