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SummaryThe stem cell theory that all blood cells are derived from hematopoietic stem cell (HSC) is a central dogma in hematology. However, various types of blood cells are already produced from hemogenic endothelial cells (HECs) before the first HSCs appear at embryonic day (E)11 in the mouse embryo. This early blood cell production from HECs, called HSC-independent hematopoiesis, includes primitive and definitive erythromyeloid progenitors that transiently support fetal blood homeostasis until HSC-derived hematopoiesis is established. Lymphoid potential has traditionally been detected in the extra-embryonic yolk sac (YS) and/or embryos before HSC emergence, but the actual presence of lymphoid progenitors at this stage remains unknown. In addition, whether HSCs in the fetal liver are the main source of innate-like B-1a cells has been controversial. Here, using complementary lineage tracing mouse models, we show that HSC-independent multipotent progenitors (MPPs) and HSC-independent adoptive B-lymphoid progenitors persist into adult life. Furthermore, HSCs minimally contribute to the peritoneal B-1a cell pool; most B-1a cells are originated directly from ECs in the YS and embryo and HSC-independent for life. Our discovery of extensive HSC-independent MPP and B-lymphoid progenitors in adults attests to the complex blood developmental dynamics through embryo to adult that underpin the immune system and challenges the paradigm of HSC theory in hematology.

Mast cell (MC) is an unique immune cell displaying wide variety of functions. Recent break through concluded MC generation is mostly provided by embryonic Yolk Sac (YS) and AGM, and post-natal BM HSC does not supply MC (HSC-independent) as our last presentation. Because YS erythro-myeloid progenitor (EMP) provide earliest tissue resident macrophage (brain microglia) and MC together as a common progenitor, and one report shows part of tissue resident macrophages are marked by IL-7 receptor (IL-7R) as an exception of lymphoid essential lineage marking, we hypothesized that MC could be also marked by IL-7R. We tested by utilizing IL-7Rcre/flox-dTom model and found that newborn to young peritoneal cavity (PerC) and skin MCs, surprisingly, exhibited more than 90% dTom positivity (90.8 ± 3.1%). IL-7R protein was not expressed on the mouse PerC, skin MCs and E12.5 Fetal Liver MC progenitors. To confirm functional IL-7R involvement for the MC development, we measured MCs in IL-7Rcre/+(Het) and IL-7Rcre/cre (KO) mice. Despite marked reductions of T/B cell counts, PerC MC count was comparable (WT: 2.4 ± 1.6 vs KO: 4.1 ± 2.3 × 10e4), suggesting IL-7R is temporarily expressed in early EMP stage in an only short period. We also tested MC differentiation from the adult BM dTom-negative Lin−Sca+Kit+ (LSK) cells. MC production was comparable between Het and KO and interestingly enough, both WT/KO LSK derived MCs showed least dTom positivity (3.4± 2.5 vs 4.7 ± 3.1 %) whereas more than 70% of macrophages turned dTom positive, suggesting embryonic and adult MC differentiation used different program respectively. Consistently, %dTom in PerC MC was reduced (79.2 ± 8.1 %) in old mice (> 1year), indicating that post-natal de novo MC production is minimum without IL-7R use. Supported by R01AI121197-01A1 NIAID R01AI147685-01 NIAID

Innate-immune like B-1a cells are unique in its functions and developmental origin compared to conventional adaptive immune B-cells (B-2 cells). Especially, their origin and developmental pathway have been argued over four decades between two proposed models: the lineage model and the selection model. Although many transplantation studies support the lineage model in which B-1a cells are derived from fetal progenitors, contradictory results of fetal liver transplantation studies raise a question as to whether fetal liver hematopoietic stem cells (HSCs) are the primary source of the peritoneal B-1a cells. In order to address this question, we took two approaches using transplantation assays and lineage tracing mouse models that enable us to trace Tomato+ progeny of endothelial cells (ECs, Cdh5CreERT mice) and HSCs (Fgd5CreERT2 mice) at a time of Tamoxifen (Tam) injection, respectively. Results: First, we transplanted HSC-precursors (pre-HSCs) from the aorta-gonad-mesonephros (AGM) region of the mouse embryo at embryonic (E) 11.5 into irradiated NSG neonates. Interestingly, we found more B-1a repopulating capacity than multi-lineage in the pre-HSC population. The lineage tracing of hemogenic ECs revealed that B-1a progenitors and HSCs were simultaneously produced from ECs during E9.5 to 10.5, but B-1a production is diminished after E11.5 while HSC were still labeled at E11.5. The HSC-tracing model demonstrated the minimal contribution of FL HSCs to the postnatal B-1a cell pool. Conclusion: Our data addressed the long-lasting controversy and showed HSC-independent B-1a cell development in the mouse embryo.