Fig. 5: GATA factors are required for initial Xist upregulation in vivo.
a,b, Expression of GATA TFs during early development assessed by scRNA-seq42,57. C, cell; PrE, primitive endoderm; VE, visceral endoderm. c–g, Zygotic TKO of Gata1, Gata4 and Gata6. c, Schematic depiction of the experimental workflow, where zygotes, generated by IVF were electroporated with Alt-R CRISPR/Cas9 ribonucleoprotein complex pre-assembled with three crRNAs targeting the Gata1, Gata4 and Gata6 coding sequences. Embryos were allowed to develop to the eight-cell stage. d, Schematic depiction of Gata1, Gata4 and Gata6 genomic loci with regions targeted by crRNAs shown as blue lines. e, Staining of the indicated GATA TFs. Dashed lines represent the nuclei as detected by DAPI staining. For the numbers indicated, two biological replicates were merged. f,g, RNA-FISH for Xist and the X-linked Huwe1 gene (nascent transcript) at the eight-cell stage. Only female embryos (two Huwe1 signals) were included in the analysis. In g, the summed fluorescence intensity within the automatically detected Xist clouds is shown for individual cells. Embryos from two biological replicates were pooled (individual replicates are shown in Extended Data Fig. 7b). Statistical comparison was performed with a two-sided Wilcoxon ranksum test. The central mark indicates the median, and the bottom and top edges of the box indicate the first and third quartiles, respectively. The top and bottom whiskers extend the boxes to a maximum of 1.5 times the interquartile range; cell (embryo) numbers are indicated on top. The scale bars in e and f represent 10 μm. Source numerical data are available as source data.
