GSE167588
Adult-repopulating lymphoid potential is not confined to the arterial endothelial cells in yolk sac
During embryogenesis, hematopoietic stem progenitor cells (HSPCs) are believed to  be derived from hemogenic endothelial cells (HECs). Moreover, arterial feature is  proposed to be a prerequisite for the HECs to generate HSPCs with lymphoid potential.  Whereas the molecular basis of the hematopoietic stem cell-competent HECs has been  delicately elucidated within the embryo proper, the functional and molecular  heterogeneity of HECs in the extra-embryonic yolk sac (YS) remains largely  unresolved. In this study, we firstly identified six molecularly different endothelial  populations in the mid-gestational YS through integrated analysis of several single-cell  RNA sequencing (scRNA-seq) datasets, and validated the arterial vasculature  distribution of Gja5+ ECs by using a Gja5-EGFP reporter mouse model. We further  explored the hemogenic potential of different EC populations based on their Gja5-  EGFP and CD44 expression. The lineage differentiation potentials of ECs were  spatiotemporally different and the B lymphoid potential was detected in the YS ECs as  early as embryonic day (E) 8.5 regardless of their arterial feature. Unexpectedly,  capacity of generating hematopoietic progenitors with in vivo lymphoid potential was  found in non-arterial in addition to arterial YS ECs at E10.0-E10.5. Importantly, the  distinct identities of E10.0-E10.5 ECs with a hemogenic potential between those from  YS and from intra-embryonic caudal region were revealed by further scRNA-seq  analysis. Taken together, these findings extend our knowledge about the hemogenic  potential of ECs from anatomically and molecularly different vascular beds, providing  a theoretical basis for better understanding the sources of HSPCs during mammalian  development. Overall design: Sequencing libraries were prepared following a modified single-cell tagged reverse  transcription (STRT) protocol as previously reported (Li et al., 2017; Picelli et al.,  2013). The cells were first judged by morphology to assess their state, with cells in  good condition being picked and directly placed into lysis buffer. The reverse  transcription reaction was performed using a sample-specific 25-nucleotide (nt) oligo  dT primer containing an 8-nt barcode (TCAGACGTGTGCTCTTCCGATCTXXXXXXXX-  DDDDDDDD-T25, where X represents the sample-specific barcode,  and D stands for the unique molecular identifier (UMI)). After reverse transcription and  second-strand cDNA synthesis, the cDNAs were amplified by 16 cycles of PCR. The barcoded DNAs were then pooled together and purified using AMPure XP beads.  Biotinylated pre-indexed primers were used to further amplify the PCR product by an  additional four cycles of PCR to introduce biotin tags to the 3' ends of the amplified  cDNAs. Approximately 300 ng cDNA was sonicated into 300-bp fragments using the  Covaris S2 system and enriched with Dynabeads MyOneTM Streptavidin C1 beads.  Libraries were constructed using a Kapa Hyper Prep Kit (Kapa Biosystems) and were  then submitted to150-bp paired-end sequencing on an Illumina Hiseq X Ten  platform (Novogene).