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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. 1 — a Gross morphology of E9.0 Pbx1^−/−;Pbx2^−/− (Pbx-DKO) embryos showing enlarged tailbud (tb), shortened trunk and rudimentary somites (s). Scale bar: 200 µm. b Dorsal views of E8.5 Pbx1^−/−;Pbx2^+/− (Pbx-com) and Pbx-DKO mouse embryos stained with FOXC2 (green) and UNCX4.1 (red) antibodies display abnormal somitogenesis. The cartoon on the left is a schematic representation of the E8.5 mouse tailbud. CLE caudal lateral epiblast, NSB node-streak border, PS primitive streak, PSM pre-somitic mesoderm, Som somites, NT neural tube, A anterior, P posterior. Scale bars: 70 µm. In a and b, asterisks denote the newly generated somites. c Schematics illustrating paraxial mesoderm differentiation from bipotent NMPs to somites, showing intermediate states and associated lineage markers. EMT epithelial-to-mesenchymal transition. d Strategy used to perform single-cell RNA-seq (scRNA-seq) of mouse embryonic tailbuds. e ScRNA-seq of E8.5/E9.0 control, Pbx-com and Pbx-DKO embryonic tailbuds. Unsupervised clustering and UMAP visualisation reveal reduced aPSM (dark blue arrowhead) and expanded MPCs/pPSM (light blue arrowhead) cell populations in Pbx mutants. Individual cells are coloured according to annotated cluster identities: NMPs neuromesodermal progenitors, pNT pre-neural tube, MPCs/pPSM mesodermal progenitor cells/posterior pre-somitic mesoderm, aPSM anterior pre-somitic mesoderm, IM intermediate mesoderm, LPM lateral plate mesoderm, Alnt allantois, Endth endothelial cells, Bld blood, Endo endoderm, Spp splanchnopleura, Noto notochord, str stressed cells. f Percentage of MPCs and aPSM cells in control (black), Pbx-com (light pink) and Pbx-DKO (yellow) tailbuds at E8.5 and E9.0. P-values indicated in the figure were calculated by two-sided Fisher’s exact test. g Expression analysis of selected lineage markers within the MPCs/pPSM cluster isolated from control and Pbx mutant tailbuds. Dot plot shows the relative expression of the NMP markers T-Bra, Sox2, Wnt3a, Fgf8, and the mesodermal genes Tbx6, Cited1, Dll1, Msgn1, indicating co-expression of NMP and MPC markers in Pbx-com and Pbx-DKO embryos. Colour bar indicates the intensity associated with normalised expression values. h Monocle’s pseudotemporal ordering of paraxial mesoderm-related clusters displaying the progressive maturation of control cells (black) from NMPs to aPSM and the defective NMP-to-PSM transition of Pbx-DKO cells (yellow).