(AiCAii and BiCBii) are z-maximum projection, (AiiiCAiv and Biii) are single optical sections. resulting from the administration of tamoxifen at E6+8h (i) and E7+7h (ii, iii) in immunostained with cTnnT to reveal the cardiomyocytes (blue).(TIF) pbio.3001200.s002.tif (5.3M) GUID:?47F43319-111D-4ED1-BD63-05509028A617 S3 Fig: CreErt2 nuclear localisation 2 hours after tamoxifen administration. (A) Representative embryos resulting from a 2-hour pulse of tamoxifen via oral gavage (0.08 mg/bw) immunostained with oestrogen receptor. Embryos have been immunostained simultaneously and image under the same conditions. Maximum z-projection (iCiii) and single optical sections (ivCvi) are shown.(TIF) pbio.3001200.s003.tif (3.9M) GUID:?F29B0538-F179-4B53-B71D-A058A9D46CF5 S4 Fig: Recombination of the R26RtdTomato reporter is occurring 2.5 hours after tamoxifen administration by oral gavage. (A, B) PCR amplicons generated from the genomic region in which Cre-mediated recombination occurs from s embryos (A), resolved on an agarose gel (B). Before recombination, the PCR product is 1,145 bp (white rectangle); after recombination, it is 274 bp (black rectangle). Template gDNA was extracted from either an ear clip of an adult mouse (untreated) or embryos (i, ii) following oral gavage with Tamoxifen, as labelled. An increase in the proportion of the recombined band PTC-028 can be seen over time following Tamoxifen administration. The data can PTC-028 be found in S2 Raw image.(TIF) pbio.3001200.s004.tif (12M) GUID:?9D3EC4E5-37FC-4894-BE17-36F7747A024B S5 Fig: mouse and immunostained for Foxa2 (green). tdTomato-positive cells are localised in the pericardium, cardiomyocytes and endoderm but not in the endocardium. cardio, cardiomyocyte; CC, cardiac crescent; cTnnT, cardiac troponinin T; endo, endoderm; LA, left atria; LV, left ventricle; OFT, outflow tract; RA, right atria; RV, right ventricle. Scale bars: 200 m in (A) and 100 m in (B).(TIF) pbio.3001200.s005.tif (6.6M) GUID:?B94D7969-8154-43E7-BB1D-987300102226 S6 Fig: T and Foxa2 colocalise in primitive streak cells. (ACD) Single optical sections from same embryos as shown in Fig 4. E6+21h MS (ACA) and LS (BCB) FLJ14936 and E7+7h EB (E, F) embryos are immunostained for T (red) and Foxa2 (green). Views are lateral/slightly posterior. Insets in Ai, Aii, Bi, and Di show magnified views (ACC). White arrows point to T+/Foxa2+ double positive cells in the definitive endoderm (Ai, Aii) at MS position in MS-LS embryos (Bi and Ci). Scale bar: 100 m. EB, early bud stage; LS, late-streak; MS, mid-streak; PS, primitive streak.(TIF) pbio.3001200.s006.tif (21M) GUID:?F7207AFE-A319-418F-ABFE-E1F5D29D8907 S7 Fig: Segmentation of the proximal and distal primitive streak cells. (A) Example of a segmented images based on T signal for PTC-028 the proximal cells (i) and Foxa2 signal for the distal cells (ii). Segmentation for only the tdTomato-positive cells is shown in (iii). Merge of the 2 segmented images (i and ii) is shown in (iv).(TIF) pbio.3001200.s007.tif (8.6M) GUID:?E22BCFC7-2569-4714-B61F-31C71A16FE00 S8 Fig: Characterisation of the lineage-positive mesodermal cells. (A) Representative embryo at about E7.5. (B, C) Representative embryos resulting from the administration of tamoxifen at E6+21h in immunostained for Foxa2 (blue) and Cer1 (blue) (B) or Foxa2 (blue) and Flk1 (green) (C). Inset in BiCCi show magnified view (B, C) in single optical section. (D) Representative E7.5 embryo immunostained for DKK1 (red) and Foxa2 (green). (E) Representative TCFdsred embryo (red) at E7.5 immunostained for Foxa2 (green). Ant, Anterior; post., posterior; PS, primitive streak. Scale bar: 100 m.(TIF) pbio.3001200.s008.tif (12M) GUID:?E72F5F9E-2353-41B9-B535-4EAFB23A52BB S9 PTC-028 Fig: Bre-cerulean line report BMP signalling activity in the mesoderm. (ACA) Colocalisation of the Cerulean signal and P-Smad1/5/8 in Bre-cerulean embryos at the cardiac crescent stage. (A) z-max proj. (A) Single optical projection. (A) Magnified view form insets in A. cc., cardiac crescent; e, endoderm; p, pericardium. Scale PTC-028 bar: 100 m.(TIF) pbio.3001200.s009.tif (14M) GUID:?CFDF7FB2-3889-4609-A4D8-CDF365D07916 S10 Fig: Outflow tract and atrial progenitors are located away from regions with high BMP signalling activity. (A) tdTomato localisation in embryos immunostained against P-Smad1/5/8 following tamoxifen administration at E7+7h. cc, cardiac crescent; pm, pharyngeal mesoderm. Yellow arrow points to a Phospho-Smad1/5/8+/tdTomato+ cell, red arrows point to Smad1/5/8?/tdTomato+ cells. Scale bar: 100 m.(TIF) pbio.3001200.s010.tif (9.0M) GUID:?DC7A167B-AD1E-4FE6-8115-040E092666C9 S11 Fig: Assignment of cluster identities in scRNA-seq E7+7h dataset. (A) UMAP plot of the Pijuan and colleagues.