It is widely believed that hematopoiesis after birth is established by hematopoietic stem cells (HSCs) in the bone marrow and that HSC-independent hematopoiesis is limited only to primitive erythro-myeloid cells and tissue-resident innate immune cells arising in the embryo. Here, surprisingly, we find that significant percentages of lymphocytes are not derived from HSCs, even in 1-year-old mice. Instead, multiple waves of hematopoiesis occur from embryonic day 7.5 (E7.5) to E11.5 endothelial cells, which simultaneously produce HSCs and lymphoid progenitors that constitute many layers of adaptive T and B lymphocytes in adult mice. Additionally, HSC lineage tracing reveals that the contribution of fetal liver HSCs to peritoneal B-1a cells is minimal and that the majority of B-1a cells are HSC independent. Our discovery of extensive HSC-independent lymphocytes in adult mice attests to the complex blood developmental dynamics spanning the embryo-to-adult transition and challenges the paradigm of HSCs exclusively underpinning the postnatal immune system.

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

Recent advances in developmental immunology have revealed a hematopoietic stem cell (HSC)-independent origin for various innate immune lineages, including mast cells (MCs). It is now established that adult bone marrow (BM) long-term HSCs do not regenerate MCs but, instead, the physiological production of MCs starts before the emergence of HSCs in the aorta-gonad-mesonephros (AGM) region and is mostly completed before birth. However, while the AGM region represents a major site of MC generation during ontogeny, whether the first emerging HSCs in the AGM or fetal liver (FL) possess the potential to regenerate MCs is unknown. Here, we combined three fate-mapping mouse models with detailed HSC transplantation assays to determine the potential of AGM and FL HSCs to produce MCs. We show that HSCs from E11.5 AGM and E12.5 FL efficiently repopulated MCs in recipients. In stark contrast, HSCs from ≥E14.5 FL failed to reconstitute MCs. An Endothelial (EC) fate-mapping study confirmed the EC origin of the majority of MCs. Additionally, our HSC-labeling showed that HSCs do not produce MCs in a physiological setting. Hence, although most MCs are generated and maintained via an HSC-independent pathway, the earliest HSCs to emerge in the AGM and seed the early FL can produce MCs, but only during a minimal time window. Our results challenge the stem cell theory in hematology and EC-derived mast cells may contribute to the pathogenesis of postnatal mast cell disorders.

Abstract 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.