Figure 3. Esrrb promotes the expression of formative genes.

a: Schematic representation of E14 cells transfected with an inducible Esrrb-Ires-Venus vector (EIV) and differentiated for 96h with or without Doxycycline (DOX) treatment. Cells were sorted for presence or absence of Venus expression (EIV+ and EIV- respectively) and further characterised.

b: Left: Representative images of Alkaline phosphatase staining after clonal assay of cells expressing EIV or an inducible Empty vector (iEmpty), cultured in 2iL or without 2iL for 96h (96), in the presence of DOX. Right: Barplot showing number of AP positive colonies in cells expressing EIV or iEmpty, cultured in 2iL or without 2iL for 96h, in the presence or absence of DOX (+D or - D). Mean +/-SD of 4 biological replicates. Two-sided unpaired Student t-test.

c: Heatmaps showing mean-scaled normalised expression levels, measured by RNA-seq, of selected naive, formative and committed genes in E14 cells expressing EIV cultured in 2iL or differentiated for 96h in the presence or absence of DOX (96-DOX or 96+DOX respectively). Integration of n=2 independent experiments.

d: Expression levels of selected naive and formative genes measured by qPCR in cells treated as described in Fig. 3b. Mean of n=2 independent experiments. Expression of naive genes is normalised to iEmpty cells kept in 2iL -D. Formative genes are normalised to E14 cells differentiated for 48h.

e: Scatter plot showing transcriptome analysis of E14 cells expressing EIV cells differentiated for 96h in N2B27 with or without DOX. Down-regulated (Log2FC < -1 and p-value < 0.01) and Up-regulated (Log2FC > 1 and p-value < 0.01) genes are plotted on the left or right part of the panel respectively. The Y-axis indicates the mean expression on a log scale. Genes belonging to the 6 genes signatures described are represented by coloured dots. Selected genes are highlighted. Integration of n=2 independent experiments.