Day 11 mouse fetal thymus: phenotype and search for the point of commitment.

This phenotype study of uncommitted hematopoietic cells (UHC) and early T cell precursors (ETCP) in the thymus provides an understanding of the commitment process from UHC to ETCP. The study of genes that are involved in this process depends on the proper identification of these early cells. Nonetheless, most current phenotype studies of these cells are based on the observations from the late stages of fetal or adult thymocytes. Though conclusions drawn from these studies are insightful, cell maturation occurs so fast during thymus development that some important phenotypic nuances go unnoticed if one only looks at late-stage cells. Furthermore, even though early-stage thymocytes are phenotypically similar to those of late stage, they may have different properties. In order to study those thymus populations at the very beginning of their differentiation and commitment, 11-day mouse fetal thymuses were tested. This shows, using day-12 CFU-S as a measure, that at this stage of development there are UHC present. Thymocytes at this very initial stage of development are isolated for the first time, and their phenotypes as well as their differentiation potentials are analyzed. The result shows that UHC are detected only in the Mac-1- C-kit+ subset, which comprises 2% of the total thymus population. Surprisingly, 93% of the C-kit+ population is Mac-1+, which generates T cells with a frequency of 1/72. This indicates that the Mac-1 molecule is a differentiation marker rather than a lineage-specific marker. In addition, C-kit+ thymocytes at this stage do not express any T cell markers, such as Thy1.2, IL-2R alpha, CD2 and Mel-14. These thymocytes are very akin to UHC, since they express Sca-1, Wga, and CD34, and are likewise similar to ETCP, as they express Pgphigh, Mhc-Ihigh, Hsalow, and FcRlow. Moreover, they express a high level of adhesion molecules such as Lfa-1, Icam-1 and Lpam-1. As expected, these C-kit+ all contain the hematopoietic cell marker CD45. Low expression of CD4 (the typical marker associated to the earliest T cell precursor in adult thymuses) is also found in 10% of the C-kit+ population. While the C-kit+ population at this stage is more homogeneous than at any other stage of fetal thymus development, there are still markers (B220, CD5, Tsa, Mac-1, CD4, and Sca-1) that can split this population into other subsets. However, the majority of these markers are present in the Mac-1+ C-kit+ population, indicating that the C-kit+ population is essentially made of two populations (Mac-1+ and Mac-1- subsets). Interestingly, two major single-positive populations appear to emerge from this C-kit+ population one day later, namely Thy1.2+ IL-2R alpha-and Thy1.2- IL-2R alpha +. These two major single-positive populations seem to be derived directly from the Mac-1+ rather than from the Mac-1- subset of the C-kit+ population. Thus, these data suggest that important phenotypes are present during the early differentiation process. These phenotypes have never been shown before. Hopefully, this study will open up a new avenue for the study of very early stage T cell sublineages and their relationship to uncommitted thymic hematopoietic cells.