Fig 1: Distinct temporal combinations of RA and BMP4 direct different mESC identities(A) Schematic of the source of patterning signals in the neural tube.(B) Schematic of three RA ± BMP4 protocols used to direct mESCs toward dorsal spinal cord identities through an NMP intermediate.(C–E) Protocols 2 and 3, but not protocol 1, induce phosphorylated Smad1/5/8 (p-Smad1/5/8) by day 5 showing active BMP signaling.(F–K) Rosettes expressing Sox2 (NPCs, red, I, K), Pax3 (dPs, red, F, H), Pax6 (dPs, green, F, H), and Tuj1 (neurites, green, I, K) are observed in protocols 1 and 3, but not in protocol 2 (G, J), by day 9.(L) Timeline for bulk RNA-seq sample acquisition (n = 3).(M) Principal-component analysis (PCA) identified similar trajectories for protocols 1 (red, R-branch) and 3 (blue, B-branch), which are distinct from the trajectory of protocol 2 (green, C-branch).(N) Pearson correlation analysis showing the cells from protocols 1 (R9) and 3 (B9) are transcriptionally similar and distinct from those in protocol 2 (C9) at day 9.(O) Weighted gene co-expression network analysis (WGCNA) identified the successive downregulation of a rRNA biogenesis module implicated in the loss of pluripotency (Watanabe-Susaki et al., 2014; Woolnough et al., 2016). Conversely, neural modules are sequentially upregulated in the R- and B-branches, while a cardiac module is sequentially upregulated in the C-branch.See Figure S1E for other modules. Scale bars: 100 µm (C–K).