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. 2021 Aug 26;12:5136. doi: 10.1038/s41467-021-25370-4

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a Schematics of the in vitro protocol used to differentiate EpiSCs towards anterior primitive streak (APS) and pre-somitic mesoderm (PSM). b ScRNA-seq of in vitro PSM differentiation. Cells were harvested at different time-points, as indicated in a. Unsupervised clustering and UMAP visualisation reveal temporally distinct populations of NMPs (orange-red), MPCs (light pink) and PSM (light blue). Individual cells are coloured according to annotated cluster identities. Reconstructed developmental trajectories are indicated with black dashed lines. c Heatmap showing expression of key genes along the path towards PSM. Cells acquire a NMP signature at 24 h, and subsequently undergo progressive maturation towards PSM. NMPs present at 36–48 h express higher levels of posterior Hox genes (Hox6-9), consistent with the acquisition of more posterior axial identity. Colour bar indicates the intensity associated with normalised expression values. d Western blot analysis confirms absence of PBX1 and PBX2 proteins in the Pbx-DKO cell lines obtained by inactivating Pbx1 and Pbx2 in 129 EpiSCs with a CRISPR/Cas9 approach. e Schematics of wild-type (WT) and Pbx-DKO EpiSCs differentiation towards PSM. The time-points selected for scRNA-seq analyses are highlighted in black. f Monocle’s pseudotemporal ordering of WT (black) and Pbx-DKO cells (yellow) spanning the NMP-to-PSM transition (24–48 h), showing inability to generate PSM and increased number of NMPs/MPCs caused by loss of PBX. g Pseudotime analysis of PSM differentiation showing distribution of WT (black) and Pbx-DKO cells (yellow) expressing the NMP genes Sox2 and T-Bra and the PSM marker Tbx6. h Representative immunofluorescence staining for T-BRA (white), SOX2 (red) and TBX6 (green) at 48 h of differentiation reveals an accumulation of NMPs (T-BRA^pos;SOX2^pos, dashed white lines) in Pbx-DKO cells. Scale bar: 50 µm. i Histograms representing the percentage of progenitors in WT (black) and Pbx-DKO cells (yellow) at 48 h of differentiation. Upper panel: percentage of MPCs (T-BRA^pos;TBX6^pos) relative to DAPI^pos cells (total cells) or to TBX6^pos cells (PSM). Lower panel: percentage of NMPs (T-BRA^pos;SOX2^pos) and PSM (TBX6^pos) cells relative to DAPI^pos cells. Data are mean ± s.e.m. P-values indicated in the figure were calculated by two-tailed unpaired t-test (n = 3 biological replicates).