Single-Cell Transcriptomics Identifies the Hyaluronan Receptor Stabilin-2 As Crucial for Determining Yolk Sac Haematopoietic Output

Supplementary Information Shvartsman, Pavlovich, Oatley et al.

Single-cell transcriptomics identifies the hyaluronan receptor Stabilin-2 as crucial for determining yolk sac haematopoietic output

Maya Shvartsman1+, Polina V. Pavlovich1,2,3+, Morgan Oatley1+, Kerstin Ganter1, Rachel McKernan1, Radvile Prialgauskaite1, Artem Adamov1,3, Konstantin Chukreev1,3, Nicolas Descostes1, Andreas Buness1,4, Nina Cabezas-Wallscheid2 & Christophe Lancrin1,*

1 European Molecular Biology Laboratory, EMBL Rome - Epigenetics and Neurobiology Unit, via E. Ramarini 32, 00015 Monterotondo, Italy.

2 Max Planck Institute of Immunobiology and Epigenetics, Stübeweg 51, D-79108, Freiburg, Germany.

3 Moscow Institute of Physics and Technology, Institutskii Per. 9, Moscow Region, Dolgoprudny 141700, Russia.

4 Current address: Core Unit for Bioinformatics Data Analysis, Universitätsklinikum Bonn, Sigmund-Freud-Str. 25, 53127 Bonn, Germany

* Correspondence: [email protected]

+ Co-first authors

Supplementary Fig. 1 a E9.5 E11.5

Cells similarity matrix 1204 columns, 1204 rows Cells similarity matrix 1311 columns, 1311 rows

AGM VE-Cad+ AGM VE-Cad+ YS VE-Cad+ YS VE-Cad+ AGM VE-Cad- AGM VE-Cad- YS VE-Cad- YS VE-Cad- AGM_EC AGM_EC AGM_EC_2 AGM_Ery AGM_Neurons AGM_Neurons AGM_Mes AGM_Mes YS_EC EMP YS_Ery Mac YS_EMP YS_EC YS_Ery YS_Ery_2 YS_Liver_like YS_Mes

b E9.5 E11.5

Supplementary Fig. 1: CONCLUS cells similarity matrices and heatmaps with marker genes (a) Similarity matrices of cells for E9.5 (left) and E11.5 (right) datasets. (b) Heatmaps with top 35 positive marker genes per clusters. Mean-centred normalised data are shown. The legend for states and groups within an embryonic day is highlighted. Plots were generated with built-in functions of CONCLUS. Supplementary Fig. 2

a E9.5 E11.5

AGM VE-Cad+ AGM VE-Cad+ YS VE-Cad+ YS VE-Cad+ AGM VE-Cad- AGM VE-Cad- YS VE-Cad- YS VE-Cad-

AGM_EC AGM_Ery AGM_EC AGM_Neurons AGM_EC_2 AGM_Mes AGM_Neurons EMP AGM_Mes Mac YS_EC YS_EC YS_Ery YS_Ery YS_EMP YS_Ery_2 YS_Liver_like YS_Mes

Supplementary Fig. 2: Comparison of clustering of E9.5 and E11.5 datasets using Seurat and CONCLUS (a) t-SNE plots of E9.5 (left) and E11.5 (right) generated by Seurat and colored by clusters from Seurat. For E9.5, we chose scaling using negative binomial model and the resolution 0.6. Linear model regressed on the number of UMIs (nUMI) showed similar grouping. For E11.5, we show the result of the linear model regressed on the nUMI and the resolution 1.5. (b) t- SNE plots with the coordinates from (a) with highlighted states according to the design of the experiments. (c) t-SNE plots with the coordinates from (a) where colors show clusters of CONCLUS from the Figure 2a. The arrows indicate the clusters which were not found with Seurat but identified with CONCLUS. Supplementary Fig. 3 a 0 7 1 20 20 16 2 3 1 3 12 d 14 4 10 10 Arterial endothelial trajectory 5 2 Brain endothelial trajectory 15 6 13 10 Doublets 7 0 0 6 Endocardium trajectory 8 9 tSNE_2 tSNE_2 Liver endothelial trajectory 0 9 Lymphatic endothelial trajectory 10 −10 −10 5 Venous and capillary endothelial trajectory 11 12 13 −20 −20 14 8 4 11 15 16 −30 −30

−20 0 20 40 −20 0 20 40 tSNE_1 tSNE_1 b Cdh5 Stab2

20 20

10 ExpressionCdh5 10 ExpressionStab2 4 5 0 3 0 4 3 2 tSNE_2 tSNE_2 2 −10 1 −10 1 0 0 −20 −20

−30 −30 −20 0 20 40 −20 0 20 40 tSNE_1 tSNE_1

Lyve1

10 ExpressionLyve1 4 0 3 2 tSNE_2 −10 1 0 −20

−30 −20 0 20 40 tSNE_1

Cdh5 Cdh5 Stab2 Stab2 Lyve1 Lyve1 c 20 20 20 ● ● ●● ●● ● ●● ● ● ● ● ● ● ●●● ● ● ●●● ● ● ● ●● ● ● ●● ● ● ●●● ● ● ●● ● ● ● ●● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ●● ● ● ●● ● ●● ● ●● ●● ● ●● ● ● ● ● ●● ● ● ●● ● ● ●● ●● ● ●● ●● ● ● ●●● ● ●● ● ● ●●● ● ● ● ●● ●● ● ● ●● ● ●● ● ● ●● ● ●● ● ● ●●● ● ● ● ● ● ● ●●● ● ● ● ● ● ●●● ●● ● ● ●● ● ● ●●●● ● ●●●● ●● ● ● ● ● ●●● ● ● ●●● ● ● ● ●●● ● ● ●●●● ● ●● ● ● ● ●● ● ●● ● ● ● ●● ● ● ●●● ● ● ● ●● ● ● ● ● ● ● ●●● ● ● ●● ● ● ● ● ● ● ●●● ● ●● ● ● ● ●● ● ● ● ●●●●●● ● ● ● ● ●●●●●● ● ● ● ●● ● ● ● ● ● ● ●●●● ● ● ● ●● ● ● ● ● ●●● ● ● ● ● ●● ● ● ● ● ●●● ● ● ● ● ●●●●●● ● ●●● ● ●● ●●●●●● ●●● ●● ●●● ● ●● ●●●●●● ●●● ●● ● ● ● ●●●● ● ● ● ●●● ● ●● ● ● ● ● ● ●● ● ●● ● ● ● ● ● ●● ● ●●● ● ●● ●● ●●● ●●● ●● ● ●●● ●●● ●●●●● ●● ● ●●● ●●● ●●●●● ●● ●●● ● ● ● ● ● ●● ● ●●● ● ● ● ● ● ●●● ● ● ● ● ● ● ●●● ●●●● ●●●●● ●● ● ●● ● ● ● ● ●● ● ●● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● 10 ●●● ● ● ● ● 10 ●●● ● ● ● ● ● ●● ● ● ● ● ● ● ●●● ● ● ● ●●● ● ● ● 10 ● ●● ● ● ● ●● ●●● ●● ●● ● ●● ●● ●●● ●● ●● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ●● ●●●●●● ●● ● ● ●● ● ● ●●● ● ● ● ●●● ● ●● ● ● ● ● ● ●● ● ● ● ●●● ● ● ●●● ● ●●●● ● ● ●●●●● ● ● ●●●●● ● ●●● ●● ● ● ● ● ● ● ● ● ● ● ● ●●●●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ●● ● ● ●● ●● ● ● ● ●●● ● ● ●●● ● ● ● ●● ●● ● ● ●●● ●● ● ● ●● ●●● ●● ● ● ●● ●● ● ● ●●● ●●● ● ●● ●●● ●●● ●●● ● ●● ●●● ●●●● ●● ● ● ● ●● ●●● ● ●●●● ●●● ●●●●● ● ●●● ● ●●●● ●●● ●●●●● ● ●●● ●● ●●●● ●●●● ● ● ●●● ●●●●●● ● ● ●● ● ● ●●● ●●●●●● ● ● ●● ● ●●● ●● ●●● ●●● ●● ●● ● ●● ●●●●● ●● ●●●●● ● ●●● ●● ●●● ● ● ●● ● ●●● ● ●● ●●● ● ●●● ● ●● ●●● ● ● ●●●●● ● ●● ● ●● ● ●●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● expression ● expression ● ● ● ● ●● ●● ● ●● ●● ● ● expression ● ● ● ● ● ● ● ● ●● ●●●● ● ●●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ●● ●● ● ●● ●● ●● 12 ● ●● ●●● ● ● ● ●●● ● ● ●●● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ●●●● ●●●● ● ● 0 ● ● ● ●● 0 ● ● ● ●● ● ●●●● ●●● ●● ● ●●● ●● ● 0 ●● ●●●●● ● ●●● 9 ● ●●● ●● ● ● ●●●● ● ●●●● ● ● ●● 9 ● ● 9 ●●●●●

6 6 6

3 3 3 ●●● ●●● ●● ● ●● ● ● ●●●● ● ●● ● ● ●●●● ● ● ● ●●● ● ●●●●●● ●●●● ● ●●●●●● ●●●● ● ●●●●●● ● ●● tSNECoords[, 2] ● ● ● ● tSNECoords[, 2] ● ● ● ● ●● ● ●● ● ● ●● ●● ●● ● ● ● ●● ●● ●● ● tSNECoords[, 2] ● ●●● ●● −10 ●● ● ●● ● ● ● −10 ●● ● ●● ● ● ● −10 ●● ●●● ●● ● ● ● ● ● ● ●●● ● ● ● ● ●●● ●● ● ● ● ● ●●● ●●●●● ● ● ●●●●● ● ● ●●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ●●●● ● ● ● ● ●● ● ● ● ● ●●●●● ● 0 ● ● ● ● ●●●●● ● 0 ● ● ●●●● ● ●● ●●●● ●● ●●●● ● ● ● ●●● 0 ● ●● ●● ● ● ●● ●● ● ● ●● ● ●● ●●●● ● ● ● ●●●●●● ●●●● ● ● ● ●●●●●● ●●●● ●●●● ●● ● ●●● ● ● ● ●●● ● ● ● ●● ●●● ● ● ●●● ● ●● ● ● ● ● ●● ● ●●● ●● ● ● ● ● ●● ● ●●● ● ● ● ● ● ●● ● ●●● ●●● ●● ● ● ●●● ●● ● ● ●●● ●● ● ●●● ●●● ●● ● ● ●●● ●●● ●● ● ● ●●● ●●●● ●● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●● ●● ●● ●● ●● ● ● ●

●● ●● ● ●● ●● ●● −20 −20 −20 ● ●●●● ●●●● ●●●● ●●● ● ●●● ● ●● ● ● ●●● ● ●●● ●● ●●● ●●● ●●● ●●● ●● ● ● ●● ● ● ● ● ● ●●● ● ●● ●●● ● ●● ●●●● ● ●● ●●● ●● ●●● ●● ●●● ● ● ●●●● ● ●●●● ● ●●●● ●● ● ●● ●● ● ●● ●● ● ●● ●● ●●● ●● ●●● ●● ●●● ● ●●●●●●● ● ● ●●●●●●● ● ● ●●●●●●● ● ●●●● ●●●● ●●●● ● ●● ● ●● ● ●●

−20 −10 0 10 20 −20 −10 0 10 20 −20 −10 0 10 20 tSNECoords[, 1] tSNECoords[, 1] tSNECoords[, 1] Supplementary Fig. 3: Top YS endothelial marker genes are highly expressed by the liver endothelium in embryonic and adult stages (a) The t-SNE plots show the 26,107 endothelial cells from the Cao et al. mouse organogenesis atlas according to specific trajectories (left panel) and based on their belonging to distinct clusters (right panel). (b) The t-SNE plots show the expression of the indicated genes. The black ellipse highlights the cells with liver endothelial features: co-expression of Stab2 and Lyve1. The scale of gene expression is indicated next to each plot. (c) The t-SNE plots show the liver single cells from the Tabula Muris dataset. The black ellipse highlights the cells with liver endothelial features: co-expression of Stab2 and Lyve1. The scale of gene expression is indicated next to each plot.

SupplementarySupplementary Fig. Fig. 4 4: Single-cell q-RT-PCR analysis of the different YS endothelial cell populations a a

GroupID GroupID YS_SC1 Adgrg1 YS_EC Ikzf1 Genes I Haematopoietic YS_HEC Ptpn6 15 YS_SC2 Dock2 YS_PreHPC_I Sfpi1 Was YS_PreHPC_IIYS_SC3 Dok2 YS_ProHPC Runx1 YS_SC4 Sla Laptm5 10 Alox5ap YS_SC5 Coro1a Pla2g4a GroupID GroupIDexpression Log Dapp1 Adgrg1 YS_EC Ikzf1 Cxcl12 15 YS_HEC Col3a1 Ptpn6 15 2 Dock25 YS_PreHPC_I Gene Gpr126 Sfpi1 Was YS_PreHPC_II Fbn1 Dok2 YS_ProHPC Acta2 Runx1 Sla Pde3a Laptm5 10 Alox5ap

Sox6 Coro1a Itgam Pla2g4a Lrp1 Dapp10 Gata2 Cxcl12 Col3a1 5 Gata1 Gpr126 5 Fbn1 Epor Acta2 Notch1 Pde3a Sox6 Cdh2 Itgam Serpinf1 Lrp1 0 Gata2 Gfi1 Gata1 Mycn Epor Notch1 Gfi1b Cdh2 Hbb−bh1 Serpinf1 Gfi1 Col4a5 Mycn Bmp4 Gfi1b Hbb−bh1 Snai1 Col4a5 Kit Bmp4 Snai1 Pde2a Kit Erg Pde2a Erg Lmo2 Lmo2 Cbfa2t3 Cbfa2t3 Nmt2 Nmt2 Lgals9 Lgals9 Tal1 Tal1 Smad2 Hmmr Smad2 Csf1r Hmmr Ptprc Smad6 Csf1r Smad7 Ptprc Rbm38 Smad3 Smad6 Ptk2b Smad7 Plcg2 Atp2a3 Rbm38 Pbx1 Smad3 Bcl11a Nfe2 Ptk2b Angpt1 Plcg2 Ikzf2

Notch2Genes II Haematopoietic Atp2a3 Cbfb Pbx1 Pag1 Stk26 Bcl11a Rab38 Nfe2 Med12l Mctp1 Angpt1 Myb Ikzf2 Dnmt3b Itgb3 Notch2 Itga2b Cbfb Ppia Acvrl1 Pag1 Ptprb Stk26 Ramp2

Col4a2 Rab38 Cdh5 Med12l Kdr Tek Mctp1 Pecam1 Myb Sox17 Meis2 Dnmt3b Esam Itgb3 Emcn Pcdh12 Itga2b Cd44 Ppia Cd47 Tgfb1 Acvrl1 Fli1 Ptprb Emb Ramp2 Mdk Ly6e Col4a2 Gpx1 Cdh5 Dock8 Stxbp2 Kdr Pfas P1_CD44low_Stab2neg_KITpos_01_04 P3_CD44low_Stab2neg_KITpos_01_10 P3_CD44low_Stab2neg_KITpos_01_12 P2_CD44low_Stab2neg_KITpos_01_14 P2_CD44low_Stab2neg_KITpos_01_06 P2_CD44high_01_12 P2_CD44low_Stab2neg_KITpos_01_03 P2_CD44low_Stab2neg_KITpos_01_10 P3_CD44low_Stab2neg_KITpos_01_07 P3_CD44low_Stab2neg_KITpos_01_01 P2_CD44low_Stab2neg_KITpos_01_12 P2_CD44high_01_06 P1_CD44high_01_08 P1_CD44low_Stab2neg_KITpos_01_07 P2_CD44low_Stab2neg_KITpos_01_16 P1_CD44low_Stab2neg_KITpos_01_08 P2_CD44low_Stab2neg_KITpos_01_15 P1_CD44low_Stab2neg_KITpos_01_13 P3_CD44low_Stab2pos_KITpos_01_14 P1_CD44low_Stab2neg_KITpos_01_03 P1_CD44low_Stab2neg_KITpos_01_15 P1_CD44low_Stab2neg_KITpos_01_16 P2_CD44low_Stab2neg_KITpos_01_11 P2_CD44low_Stab2neg_KITpos_01_07 P1_CD44low_Stab2neg_KITpos_01_11 P1_CD44low_Stab2neg_KITpos_01_10 P1_CD44low_Stab2neg_KITpos_01_12 P1_CD44low_Stab2neg_KITpos_01_06 P1_CD44low_Stab2neg_KITpos_01_02 P1_CD44low_Stab2neg_KITpos_01_05 P1_CD44high_01_10 P2_CD44neg_Stab2neg_01_02 P2_CD44low_Stab2neg_KITneg_01_11 P2_CD44low_Stab2neg_KITpos_01_05 P1_CD44low_Stab2pos_KITpos_01_16 P1_CD44high_01_04 P1_CD44high_01_07 P1_CD44high_01_06 P1_CD44high_01_12 P1_CD44high_01_05 P1_CD44low_Stab2pos_KITneg_01_02 P3_CD44low_Stab2neg_KITneg_01_20 P3_CD44low_Stab2neg_KITpos_01_11 P2_CD44low_Stab2neg_KITneg_01_13 P2_CD44neg_Stab2pos_01_01 P2_CD44high_01_14 P1_CD44high_01_01 P2_CD44high_01_04 P2_CD44high_01_03 P2_CD44high_01_15 P2_CD44high_01_10 P2_CD44high_01_13 P2_CD44low_Stab2neg_KITpos_01_13 P2_CD44high_01_01 P2_CD44high_01_02 P1_CD44high_01_03 P1_CD44high_01_02 P2_CD44high_01_11 P2_CD44high_01_07 P2_CD44high_01_09 P3_CD44high_01_04 P3_CD44high_01_07 P3_CD44high_01_05 P3_CD44high_01_03 P3_CD44high_01_08 P3_CD44high_01_01 P3_CD44high_01_13 P3_CD44high_01_06 P3_CD44low_Stab2pos_KITneg_01_09 P1_CD44low_Stab2pos_KITpos_01_12 P2_CD44neg_Stab2pos_01_05 P1_CD44low_Stab2pos_KITpos_01_14 P1_CD44low_Stab2pos_KITpos_01_09 P1_CD44low_Stab2pos_KITpos_01_13 P1_CD44neg_Stab2pos_01_05 P1_CD44low_Stab2pos_KITneg_01_06 P1_CD44neg_Stab2neg_01_03 P1_CD44low_Stab2pos_KITpos_01_07 P1_CD44low_Stab2pos_KITneg_01_09 P1_CD44low_Stab2pos_KITneg_01_05 P1_CD44low_Stab2pos_KITpos_01_11 P1_CD44low_Stab2pos_KITpos_01_02 P1_CD44low_Stab2pos_KITpos_01_04 P1_CD44low_Stab2neg_KITneg_01_13 P1_CD44low_Stab2pos_KITneg_01_14 P1_CD44low_Stab2pos_KITneg_01_07 P1_CD44low_Stab2pos_KITpos_01_08 P2_CD44low_Stab2pos_KITpos_01_05 P3_CD44low_Stab2neg_KITneg_01_11 P3_CD44low_Stab2neg_KITneg_01_16 P1_CD44low_Stab2pos_KITpos_01_01 P3_CD44low_Stab2neg_KITneg_01_12 P3_CD44low_Stab2neg_KITneg_01_02 P3_CD44low_Stab2neg_KITneg_01_09 P3_CD44low_Stab2neg_KITneg_01_05 P3_CD44low_Stab2neg_KITneg_01_03 P3_CD44low_Stab2neg_KITneg_01_08 P3_CD44low_Stab2neg_KITneg_01_15 P2_CD44low_Stab2neg_KITneg_01_07 P1_CD44low_Stab2pos_KITpos_01_03 P1_CD44low_Stab2pos_KITneg_01_01 P3_CD44low_Stab2pos_KITneg_01_01 P2_CD44low_Stab2neg_KITpos_01_09 P1_CD44high_01_09 P2_CD44low_Stab2neg_KITneg_01_05 P1_CD44low_Stab2neg_KITneg_01_15 P3_CD44low_Stab2pos_KITneg_01_06 P3_CD44low_Stab2neg_KITpos_01_04 P3_CD44low_Stab2neg_KITneg_01_10 P2_CD44neg_Stab2neg_01_08 P3_CD44low_Stab2neg_KITpos_01_03 P3_CD44low_Stab2pos_KITpos_01_12 P3_CD44neg_Stab2pos_01_01 P2_CD44low_Stab2pos_KITneg_01_07 P3_CD44low_Stab2pos_KITpos_01_01 P3_CD44neg_Stab2pos_01_05 P3_CD44neg_Stab2pos_01_06 P3_CD44low_Stab2pos_KITneg_01_08 P2_CD44neg_Stab2pos_01_08 P2_CD44neg_Stab2neg_01_04 P2_CD44low_Stab2neg_KITneg_01_14 P3_CD44neg_Stab2neg_01_01 P3_CD44low_Stab2pos_KITneg_01_02 P3_CD44low_Stab2pos_KITpos_01_08 P3_CD44low_Stab2neg_KITneg_01_14 P3_CD44neg_Stab2neg_01_03 P3_CD44low_Stab2pos_KITneg_01_05 P2_CD44low_Stab2neg_KITneg_01_15 P3_CD44neg_Stab2pos_01_08 P2_CD44low_Stab2pos_KITneg_01_08 P3_CD44neg_Stab2neg_01_04 P3_CD44neg_Stab2neg_01_08 P2_CD44low_Stab2neg_KITneg_01_03 P2_CD44low_Stab2pos_KITneg_01_03 P1_CD44low_Stab2pos_KITneg_01_04 P2_CD44low_Stab2pos_KITpos_01_03 P3_CD44neg_Stab2pos_01_03 P2_CD44neg_Stab2neg_01_05 P1_CD44low_Stab2pos_KITneg_01_16 P2_CD44low_Stab2pos_KITpos_01_12 P2_CD44neg_Stab2pos_01_07 P3_CD44neg_Stab2pos_01_07 P1_CD44low_Stab2pos_KITneg_01_11 P1_CD44low_Stab2pos_KITneg_01_03 P1_CD44neg_Stab2pos_01_02 P1_CD44low_Stab2neg_KITneg_01_11 P1_CD44low_Stab2pos_KITneg_01_08 P1_CD44low_Stab2neg_KITneg_01_03 P1_CD44low_Stab2neg_KITpos_01_09 P1_CD44low_Stab2neg_KITneg_01_02 P1_CD44low_Stab2pos_KITpos_01_06 P1_CD44neg_Stab2neg_01_07 P1_CD44low_Stab2pos_KITneg_01_13 P2_CD44low_Stab2pos_KITpos_01_09 P1_CD44neg_Stab2pos_01_04 P2_CD44low_Stab2pos_KITpos_01_07 P1_CD44low_Stab2neg_KITneg_01_14 P3_CD44low_Stab2pos_KITpos_01_15 P1_CD44low_Stab2pos_KITpos_01_10 P2_CD44low_Stab2pos_KITpos_01_01 P1_CD44neg_Stab2pos_01_07 P1_CD44low_Stab2pos_KITneg_01_15 P2_CD44low_Stab2neg_KITneg_01_02 P3_CD44low_Stab2pos_KITneg_01_11 P2_CD44low_Stab2pos_KITneg_01_01 P3_CD44low_Stab2pos_KITpos_01_11 P2_CD44low_Stab2pos_KITneg_01_10 P3_CD44neg_Stab2neg_01_06 P2_CD44low_Stab2pos_KITpos_01_15 P1_CD44neg_Stab2pos_01_03 P2_CD44low_Stab2pos_KITpos_01_04 P1_CD44low_Stab2neg Pecam1 Sox17 Meis2 Genes Endothelial Esam Emcn Pcdh12 Cd44 Cd47 Tgfb1 Fli1 Emb Mdk Ly6e Gpx1 Dock8 Stxbp2 Pfas P1_CD44low_Stab2neg_KITpos_01_04 P3_CD44low_Stab2neg_KITpos_01_10 P3_CD44low_Stab2neg_KITpos_01_12 P2_CD44low_Stab2neg_KITpos_01_14 P2_CD44low_Stab2neg_KITpos_01_06 P2_CD44high_01_12 P2_CD44low_Stab2neg_KITpos_01_03 P2_CD44low_Stab2neg_KITpos_01_10 P3_CD44low_Stab2neg_KITpos_01_07 P3_CD44low_Stab2neg_KITpos_01_01 P2_CD44low_Stab2neg_KITpos_01_12 P2_CD44high_01_06 P1_CD44high_01_08 P1_CD44low_Stab2neg_KITpos_01_07 P2_CD44low_Stab2neg_KITpos_01_16 P1_CD44low_Stab2neg_KITpos_01_08 P2_CD44low_Stab2neg_KITpos_01_15 P1_CD44low_Stab2neg_KITpos_01_13 P3_CD44low_Stab2pos_KITpos_01_14 P1_CD44low_Stab2neg_KITpos_01_03 P1_CD44low_Stab2neg_KITpos_01_15 P1_CD44low_Stab2neg_KITpos_01_16 P2_CD44low_Stab2neg_KITpos_01_11 P2_CD44low_Stab2neg_KITpos_01_07 P1_CD44low_Stab2neg_KITpos_01_11 P1_CD44low_Stab2neg_KITpos_01_10 P1_CD44low_Stab2neg_KITpos_01_12 P1_CD44low_Stab2neg_KITpos_01_06 P1_CD44low_Stab2neg_KITpos_01_02 P1_CD44low_Stab2neg_KITpos_01_05 P1_CD44high_01_10 P2_CD44neg_Stab2neg_01_02 P2_CD44low_Stab2neg_KITneg_01_11 P2_CD44low_Stab2neg_KITpos_01_05 P1_CD44low_Stab2pos_KITpos_01_16 P1_CD44high_01_04 P1_CD44high_01_07 P1_CD44high_01_06 P1_CD44high_01_12 P1_CD44high_01_05 P1_CD44low_Stab2pos_KITneg_01_02 P3_CD44low_Stab2neg_KITneg_01_20 P3_CD44low_Stab2neg_KITpos_01_11 P2_CD44low_Stab2neg_KITneg_01_13 P2_CD44neg_Stab2pos_01_01 P2_CD44high_01_14 P1_CD44high_01_01 P2_CD44high_01_04 P2_CD44high_01_03 P2_CD44high_01_15 P2_CD44high_01_10 P2_CD44high_01_13 P2_CD44low_Stab2neg_KITpos_01_13 P2_CD44high_01_01 P2_CD44high_01_02 P1_CD44high_01_03 P1_CD44high_01_02 P2_CD44high_01_11 P2_CD44high_01_07 P2_CD44high_01_09 P3_CD44high_01_04 P3_CD44high_01_07 P3_CD44high_01_05 P3_CD44high_01_03 P3_CD44high_01_08 P3_CD44high_01_01 P3_CD44high_01_13 P3_CD44high_01_06 P3_CD44low_Stab2pos_KITneg_01_09 P1_CD44low_Stab2pos_KITpos_01_12 P2_CD44neg_Stab2pos_01_05 P1_CD44low_Stab2pos_KITpos_01_14 P1_CD44low_Stab2pos_KITpos_01_09 P1_CD44low_Stab2pos_KITpos_01_13 P1_CD44neg_Stab2pos_01_05 P1_CD44low_Stab2pos_KITneg_01_06 P1_CD44neg_Stab2neg_01_03 P1_CD44low_Stab2pos_KITpos_01_07 P1_CD44low_Stab2pos_KITneg_01_09 P1_CD44low_Stab2pos_KITneg_01_05 P1_CD44low_Stab2pos_KITpos_01_11 P1_CD44low_Stab2pos_KITpos_01_02 P1_CD44low_Stab2pos_KITpos_01_04 P1_CD44low_Stab2neg_KITneg_01_13 P1_CD44low_Stab2pos_KITneg_01_14 P1_CD44low_Stab2pos_KITneg_01_07 P1_CD44low_Stab2pos_KITpos_01_08 P2_CD44low_Stab2pos_KITpos_01_05 P3_CD44low_Stab2neg_KITneg_01_11 P3_CD44low_Stab2neg_KITneg_01_16 P1_CD44low_Stab2pos_KITpos_01_01 P3_CD44low_Stab2neg_KITneg_01_12 P3_CD44low_Stab2neg_KITneg_01_02 P3_CD44low_Stab2neg_KITneg_01_09 P3_CD44low_Stab2neg_KITneg_01_05 P3_CD44low_Stab2neg_KITneg_01_03 P3_CD44low_Stab2neg_KITneg_01_08 P3_CD44low_Stab2neg_KITneg_01_15 P2_CD44low_Stab2neg_KITneg_01_07 P1_CD44low_Stab2pos_KITpos_01_03 P1_CD44low_Stab2pos_KITneg_01_01 P3_CD44low_Stab2pos_KITneg_01_01 P2_CD44low_Stab2neg_KITpos_01_09 P1_CD44high_01_09 P2_CD44low_Stab2neg_KITneg_01_05 P1_CD44low_Stab2neg_KITneg_01_15 P3_CD44low_Stab2pos_KITneg_01_06 P3_CD44low_Stab2neg_KITpos_01_04 P3_CD44low_Stab2neg_KITneg_01_10 P2_CD44neg_Stab2neg_01_08 P3_CD44low_Stab2neg_KITpos_01_03 P3_CD44low_Stab2pos_KITpos_01_12 P3_CD44neg_Stab2pos_01_01 P2_CD44low_Stab2pos_KITneg_01_07 P3_CD44low_Stab2pos_KITpos_01_01 P3_CD44neg_Stab2pos_01_05 P3_CD44neg_Stab2pos_01_06 P3_CD44low_Stab2pos_KITneg_01_08 P2_CD44neg_Stab2pos_01_08 P2_CD44neg_Stab2neg_01_04 P2_CD44low_Stab2neg_KITneg_01_14 P3_CD44neg_Stab2neg_01_01 P3_CD44low_Stab2pos_KITneg_01_02 P3_CD44low_Stab2pos_KITpos_01_08 P3_CD44low_Stab2neg_KITneg_01_14 P3_CD44neg_Stab2neg_01_03 P3_CD44low_Stab2pos_KITneg_01_05 P2_CD44low_Stab2neg_KITneg_01_15 P3_CD44neg_Stab2pos_01_08 P2_CD44low_Stab2pos_KITneg_01_08 P3_CD44neg_Stab2neg_01_04 P3_CD44neg_Stab2neg_01_08 P2_CD44low_Stab2neg_KITneg_01_03 P2_CD44low_Stab2pos_KITneg_01_03 P1_CD44low_Stab2pos_KITneg_01_04 P2_CD44low_Stab2pos_KITpos_01_03 P3_CD44neg_Stab2pos_01_03 P2_CD44neg_Stab2neg_01_05 P1_CD44low_Stab2pos_KITneg_01_16 P2_CD44low_Stab2pos_KITpos_01_12 P2_CD44neg_Stab2pos_01_07 P3_CD44neg_Stab2pos_01_07 P1_CD44low_Stab2pos_KITneg_01_11 P1_CD44low_Stab2pos_KITneg_01_03 P1_CD44neg_Stab2pos_01_02 P1_CD44low_Stab2neg_KITneg_01_11 P1_CD44low_Stab2pos_KITneg_01_08 P1_CD44low_Stab2neg_KITneg_01_03 P1_CD44low_Stab2neg_KITpos_01_09 P1_CD44low_Stab2neg_KITneg_01_02 P1_CD44low_Stab2pos_KITpos_01_06 P1_CD44neg_Stab2neg_01_07 P1_CD44low_Stab2pos_KITneg_01_13 P2_CD44low_Stab2pos_KITpos_01_09 P1_CD44neg_Stab2pos_01_04 P2_CD44low_Stab2pos_KITpos_01_07 P1_CD44low_Stab2neg_KITneg_01_14 P3_CD44low_Stab2pos_KITpos_01_15 P1_CD44low_Stab2pos_KITpos_01_10 P2_CD44low_Stab2pos_KITpos_01_01 P1_CD44neg_Stab2pos_01_07 P1_CD44low_Stab2pos_KITneg_01_15 P2_CD44low_Stab2neg_KITneg_01_02 P3_CD44low_Stab2pos_KITneg_01_11 P2_CD44low_Stab2pos_KITneg_01_01 P3_CD44low_Stab2pos_KITpos_01_11 P2_CD44low_Stab2pos_KITneg_01_10 P3_CD44neg_Stab2neg_01_06 P2_CD44low_Stab2pos_KITpos_01_15 P1_CD44neg_Stab2pos_01_03 P2_C b b YS_SC1 YS_SC2 YS_SC3 YS_SC4 YS_SC5

CD44high CD44high CD44high CD44high CD44high

CD44low_Stab2neg_KITne CD44low_Stab2neg_KITne CD44low_Stab2neg_KITne CD44low_Stab2neg_KITne CD44low_Stab2neg_KITne g g g g g

CD44low_Stab2neg_KITpo CD44low_Stab2neg_KITpo CD44low_Stab2neg_KITpo CD44low_Stab2neg_KITpo CD44low_Stab2neg_KITpo s s s s s

CD44low_Stab2pos_KITne CD44low_Stab2pos_KITne CD44low_Stab2pos_KITne CD44low_Stab2pos_KITne CD44low_Stab2pos_KITne g g g g g

CD44low_Stab2pos_KITpo CD44low_Stab2pos_KITpo CD44low_Stab2pos_KITpo CD44low_Stab2pos_KITpo CD44low_Stab2pos_KITpo s s s s s

CD44neg_Stab2neg CD44neg_Stab2neg CD44neg_Stab2neg CD44neg_Stab2neg CD44neg_Stab2neg

CD44High Stab2Neg KitPos CD44Low Stab2Neg KitPos CD44Low Stab2Pos KitPos CD44Neg Stab2Pos CD44Low Stab2Neg KitNeg CD44Low Stab2Pos KitNeg CD44Neg Stab2Neg

Supplementary Fig. 4: Single-cell q-RT-PCR analysis of the different YS endothelial cell populations (a) Heatmap of gene expression showing the different clusters from FACS isolated YS single- cells of the indicated phenotypes at E10 and E11.5 following sc-q-RT-PCR of 96 genes. (b) Pie-charts indicating the phenotype of the cells composing the five different clusters defined in (a). Supplementary Fig. 5 a day 1 day 2 day 3

5 5 5 10 0.18% 10 0.14% 10 0.21%

4 4 4 10 10 10

3 3 3 10 10 10

0 0 0

VE-Cad-efluor660 3 3 3 -10 -10 -10

3 3 4 5 3 3 4 5 3 3 4 5 -10 0 10 10 10 -10 0 10 10 10 -10 0 10 10 10 Stab2-AF488 b c

0.16 T2A TRE dCas9-VPR 5’ LTR rtTA HygroR 0.12

Stab2 T2A U6 Pr. EF1a PuroR BFP gRNA 0.08

Inducible Stab2 ES cells 0.04 Relative RNA quantity RelativeRNA

0 Control no dox Control with dox d iStab2 no dox iStab2 with dox 30 ** p = 0.0061

25 (%)

Neg 20

15 VE-Cad

Pos 10

CD41 5

0 Control No Dox iStab2 No Dox

Supplementary Fig. 5: Ectopic expression of Stab2 disrupts in vitro EHT (a) FACS plots showing the expression of VE-Cad and Stab2 in an in vitro haemangioblast differentiation time course. (b) Scheme describing the inducible Stab2 ESC line. (c) RT-q-PCR results for dCAS9-VPR at day 3 of haemangioblast culture for the indicated samples. A representative experiment is shown. (d) Tukey’s box plots showing the frequency of CD41Pos VE-CadNeg for the indicated conditions. N=7 (** indicate p-value < 0.01, two tailed t-test). Description of the supplementary files

Supplementary File 1: Marker Gene Lists of E9.5 clusters Each worksheet contains a list of top 100 marker genes for a given cluster. The genes are classified based on the statistical analysis performed by CONCLUS (see method section).

Supplementary File 2: Marker Gene Lists of E11.5 clusters Each worksheet contains a list of top 100 marker genes for a given cluster (apart from the AGM_Ery cluster which had only 75 significantly up-regulated genes). The genes are classified based on the statistical analysis performed by CONCLUS (see method section).

Supplementary File 3: Results of GO term analysis The worksheet contains the results of GO term analysis performed with DAVID Bioinformatics Functional Annotation tool on the marker gene lists generated by CONCLUS.