GSE176306
Unveiling complexity and multipotentiality of early heart fields
Through high-resolution single cell and genetic lineage/clonal analyses, we show an unsuspected clonal relationship between extraembryonic mesoderm and cardiac lineages.  Single-cell transcriptomics and trajectory analyses uncovered two mesodermal progenitor sources contributing to left ventricle cardiomyocytes, one embryonic and the other with an extraembryonic gene expression signature.  Additional lineage-tracing studies revealed that the extraembryonic-related progenitors reside at the embryonic-extraembryonic interface in gastrulating embryos, and produce distinct cell types forming the pericardium, septum transversum, epicardium, dorsolateral regions of the left ventricle and atrioventricular canal myocardium, and extraembryonic mesoderm.  Clonal analyses demonstrated that these progenitors are multipotent, giving rise to not only cardiomyocytes and serosal mesothelial cell types but also, remarkably, extraembryonic mesoderm. Overall design: To prepare single cells for scRNA-seq, Mesp1-Cre; Rosa26-tdT genetically-labeled embryos at E7.25, E7.5, E7.75 and E8.25 were dissected in cold sterile 1 X PBS without Ca2+, Mg2+ under a stereo microscope.  Embryos were staged based on their morphology.  The Reichert's membrane and ectoplacental cone were removed, and Mesp1-Cre; Rosa26-tdT genetically-labeled embryos were selected and imaged.  The yolk sac was removed from one of the three E8.25 samples in order to enrich for cardiac cells.  Individual embryos were placed into a 1.5 ml microfuge tube and incubated in 0.25% Trypsin-EDTA (Gibco, Catalog # 25200056) at 37°C with inversion every two minutes for 30 min until no visible tissue remained.  The solution was pipetted once with a p1000 and neutralized by adding 0.75 ml DMEM containing 10% FBS (Gibco).  Cells were then passed through a 100-µm cell strainer (BD Biosciences, Catalog # 352360) and single tdT+ cells were obtained by fluorescence-activated cell sorting (FACS) on a BD Influx Cell sorter (BD Biosciences).  Living cells were gated on FSC, SSC, DAPI- and tdT+.  After sorting, cells were centrifuged at 300g for 4 minutes and pooled or kept as individual embryos.  E7.25 embryos were pooled due to the low number of Mesp1-lineage positive cells.  Libraries were prepared using the Chromium Single Cell 3' Library and Gel Bead Kit v2 (PN-120237) and Chromium i7 Multiplex Kit (PN-120262) according to instructions from 10X Genomics (https://www.10xgenomics.com/resources/user-guides/).  Prior to sequencing cDNA, libraries were verified by the D1000 ScreenTape system (Agilent) and quantified via Qubit™ Flex Fluorometer (Thermofisher, Catalog # Q33327).  All libraries were sequenced twice on the HiSeq 4000 (Illumina) at the UCSD IGM genomics core.  An initial shallow sequencing run was done for quality control and to determine the number of cells captured.  An individual sample was excluded from further analysis due to a low number of reads per cell (60%) as analyzed by Cell Ranger (10X genomics).  A second deeper sequencing run was subsequently performed ensuring an approximate equal read depth per cell across the samples, resulting in an average of 60,450 UMIs (unique molecular identifiers) per cell and an average sequence saturation of 65.7%