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. 2020 Mar 20;30(5):376–392. doi: 10.1038/s41422-020-0300-2

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© The Author(s) 2020

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 Representative FACS plots for sorting of the T1 pre-HSCs (CD31⁺CD45⁻CD41^lowKit⁺CD201^high) from E11.0 AGM region of mouse embryos for the subsequent scRNA-seq. b Violin plots showing the expression levels of indicated genes in tif-HEC (including clusters HEC and PK44), T1 pre-HSC and lateAEC. c t-SNE plot of the cells included in the filtered initial dataset, PK44 dataset and T1 pre-HSC dataset, with clusters mapped on it. Clusters HEC and PK44 are combined as tif-HEC. d PCA plot of tif-HEC and T1 pre-HSC populations. e. Enriched terms of PC2-positive and -negative genes are shown, corresponding to the properties distinguishing tif-HEC and T1 pre-HSC, respectively. f Heatmap showing top 20 positive and negative genes of PC2. Genes are ordered by their contributions to PC2. g Trajectory of AEC clusters, tif-HEC and T1 pre-HSC inferred by Mpath. Arrows indicate the development directions predicted by sampling stages. h Representative FACS plots for sorting of the PK44 cells from E10.0 AGM region (left) and analysis of the immunophenotypic T1 pre-HSCs (right) after cultured in vitro for 4 days. i Representative CD31 immunostaining on the cultures of single T1 pre-HSCs from E11.0 AGM region, showing typical morphologies regarding distinct differentiation capacities. Cell frequencies of each type are also shown. Data are from 7 independent experiments with totally 89 embryos used. Scale bars, 400 μm.