A transwell system was used to study the X-chromosome reactivation in E9.5 and E11.5 *X*^{GFP} PGCs cultured for a period of 48 hours.

(A) The percentage of X-linked GFP-expressing XX E9.5 PGCs after culture. Hindgut and the surrounding mesentery were dissociated and cultured in the top compartment with the lower compartment containing: medium only (control), one dissociated XX E11.5 UGR pair (XX), or one dissociated XY E11.5 UGR pair (XY). Red bars depict the median, *n* is the total number of transwell filter membranes (each containing 1.5× embryo) analysed. *, *p* < 0.05.

(B) The percentage of X-linked GFP-expressing XX E11.5 PGCs after culture. FACS-sorted PGCs (SSEA1-positive) were cultured in the top compartment with FACS-sorted XX (XX) or XY (XY) E11.5 UGR somatic tissue (SSEA1-negative). Red bars depict the median, *n* is the total number of embryos analysed.

(C,D) GFP-positive (white arrow) and GFP-negative (asterisk) *Xp-X*^{GFP} E9.5 PGCs (SSEA1-positive) after culture.

(E–J) Representative dot-plots showing FACS-analysis of E13.5 genital ridges from individual *XX* (E,F), *X*^{GFP}X (G), *X*^{GFP}X Sry (H), *X*^{GFP}Y (I), and *X*^{GFP}Y Sry (J) embryos.

(K,L) The percentage of FACS-analysed GFP-expressing PGCs (K) and surrounding somatic tissue (L) in the genital ridges of individual *X*^{GFP}X, *X*^{GFP}X Sry, *X*^{GFP}Y, and *X*^{GFP}Y Sry E13.5 embryos. PGCs and somatic cells were respectively positive and negative for PECAM1. Red bars depict the median, *n* is the total number of embryos analysed.

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