Lymphoid Precursors Arise Independently at Multiple Sites During Development.

Abstract 699

Primitive hematopoietic cells arise on embryonic day 7.0 (E7.0) within the murine extra-embryonic yolk sac (YS) followed by formation of definitive hematopoietic cells on E8.25. The first cells capable of long term multi-lineage repopulation in an adult irradiated mouse arise in the embryo proper on E10.5. The temporal and spatial emergence of lymphoid precursors capable of forming mature T and B lymphocytes remains controversial. Cardiac contractions begin on E8.25, leading to the circulation of cells between various sites of hematopoiesis thus making it difficult to identify definitively the site where lymphoid precursors emerge. We have previously utilized a transgenic knock out murine mutant in which no heartbeat develops to examine hematopoietic cell emergence. NCX1 is a protein required for the initiation of cardiac contractions. The ncx1 null embryos develop normally until about E10.5, and then succumb to the lack of a systemic circulation. Single cell suspensions from within the para-aortic splanchnopleure (P-Sp) of the embryo proper and from YS at E8.5 and E9.5 were isolated and plated on the OP9-Dl1 stromal cell line that is known to support T lymphopoiesis. We observed that both YS and P-Sp independently displayed the potential to produce mature T cells including CD4 and CD8 DN, DP, and SP cells. We observed that both sites can give rise to both αaβ and γδ T cells independently at a frequency approaching 1:165 hemogenic endothelial cells plated. Cells derived from both YS and P-Sp demonstrated lymphoid specific VDJ rearrangement. Adoptive transfer of the lymphoid cells derived from these sites into NOD/SCID/IL2Rg null recipient mice revealed engraftment within multiple lymphohematopoietic organs including thymus, spleen and liver in the recipient mouse and the presence of circulating donor T cells. Stimulation of splenocytes from transplanted animals with anti-CD3 antibodies induced proliferation of these cells suggesting intact antibody responses in YS and P-Sp derived T cells. Development of mature functional T lymphocytes from precursors isolated from YS and P-Sp before the presence of a functional thymus brings forward the hypothesis for an extra-thymic multi-site origin of T cells in the early embryo. The presence of prospective lymphoid precursors more than a day earlier than the adult reconstituting hematopoietic stem cell (HSC) first observed at E10.5 during development suggests that either the HSC may be present at earlier points in development than is believed or that there might be a HSC independent emergence of lymphoid precursors in the developing embryo.