Figure 6. The XaXa and XiXIST+Xa states are stabilized during ESC derivation.
A) (i) Quantification of RNA FISH patterns for XIST and X-linked gene HUWE1 and co-immunostaining for H3K27me3, in cells of male blastocysts at 48hrs and 96hrs after plating in ESC culture media. No enrichment of H3K27me3 was observed. “Male” classification was based on the predominant mono-allelic HUWE1 and/or XIST pattern. (ii) Representative images of the XIST/HUWE1 RNA FISH (left) and the H3K27me3 co-staining (right) for the two main patterns observed in (i). The XIST signal was typically weak.
B) As in (A), except for seven female pre-implantation blastocysts, classified based on two distinct nuclear regions of XIST and/or HUWE1 RNA FISH signal.
C) Quantification of the XCI patterns and representative RNA FISH images of XIST, HUWE1 and ATRX, in the blastocyst outgrowths and throughout early passages (p) for the XaXa ESC lines UCLA17 (i), UCLA18 (ii), and UCLA9 (iii) without any freeze/thaw cycle.
D) As in (C), but for the early passages of UCLA14 (i) and UCLA8 (ii), which were XiXIST+Xa at the earliest passage. Representative RNA FISH images capture the XIST-positive and XIST-negative states, while mono-allelic ATRX expression is maintained.
See also Figure S6.