Innate Lymphoid Cells First Emerge from HSC-Independent Erythro-Myeloid Progenitors (EMP) during Mammalian Development
Authors: McGrath Kathleen E. , Myers Jacquelyn , Kingsley Paul D. , Fernandez Nestor , Fegan Katherine , Guilfoyle Emma , Schofield Taylor L. , Hidalgo Daniel , Socolovsky Merav , Sturgeon Christopher , Palis James 
Published: December 2024

Thousands of HSC-independent EMP originate from hemogenic endothelium in the yolk sac and engraft the fetal liver to serve as the major source of blood cells in the murine embryo. Lineage tracing and in vitro culture approaches have previously established that EMP generate not only erythroid and myeloid cells but also NK cells, a type of innate lymphoid cell (ILC). To shed further light on the differentiation and lineage potential of EMP we first performed single cell transcriptomic studies of primary E9.5 Kit+CD41+CD16/32+ EMP. Dimensionality reduction revealed cell clusters expressing marker genes associated with definitive erythroid, megakaryocyte, macrophage, myeloid, and lymphoid/ILC cells, as well as a cluster containing an endothelial gene signature consistent with the hemogenic endothelial (HE) origin of EMP in the yolk sac. Additionally, we identified presumptive multipotential and megakaryocyte/erythroid bipotential clusters bridging the HE- and unilineage-biased cell clusters. The identification of differentially expressed immunophenotypic markers allowed us to perform functional colony-forming assays, which confirmed the lineage enrichment of erythroid, myeloid, and multipotential EMP subpopulations. Integration of the E9.5 EMP scRNA-Seq dataset with E12.5 and E14.5 fetal liver scRNA-Seq datasets support the concept that most lineage-biased EMP continue their lineage-specific differentiation upon seeding the liver. Given the presence of a lymphoid/ILC cluster in our E9.5 scRNA-Seq dataset, we asked if EMP contain not only NK but also broader ILC potential. Primary E9.5 EMP generated populations of immunophenotypically defined ILC1, ILC2 and ILC3, including the LTi subset of ILC3 when cultured in vitro on OP9 or on OP9DL4 cells. Lineage-tracing studies with the tamoxifen-inducible CSF1RMerCreMerRosa26YFP mouse model confirmed the HSC-independent origin of ILC3 present in the E14.5 fetal liver. Intriguingly, lineage tracing studies using the Flk/switch mouse model revealed subpopulations of ILC3 in adult lung and spleen that are Tomato+, suggesting that they are derived independently of Flt3-expressing lymphoid progenitors. We also found that ILC3 potential was present in human induced pluripotent stem cell-derived HOXAneg (WNTi), yolk sac-like progenitors suggesting that an HSC-independent origin of ILCs is evolutionarily conserved. Taken together our findings support the concept that EMP emerge from hemogenic endothelium in the yolk sac, begin to differentiate prior to seeding the fetal liver, and provide definitive erythroid, megakaryocyte, innate myeloid, and broad innate lymphoid cell populations to meet the needs of the developing mammalian embryo prior to the establishment of HSC-derived blood cell production. Further, they support the concept that ILCs have at least two developmental origins; initially from myeloid-biased EMP and subsequently from HSC-derived lymphoid-biased progenitors.