HEB in the Spotlight: Transcriptional Regulation of T-Cell Specification, Commitment, and Developmental Plasticity

Figure 5

Developmental phenotype of HEB^−/−Rag-1^−/− T-cell progenitors expressing transgenic HEBAlt. HEB^+/− mice were bred with Rag-1^−/− mice to generate HEB^+/−Rag-1^−/− mice, which were timed mated to generate (a) HEB^+/+Rag-1^−/− and (b) HEB^−/−Rag-1^−/− embryos. Similarly, HEB^+/−Rag-1^−/− mice were bred with HEBAlt^Tg mice to generate HEBAlt^TgHEB^+/−Rag-1^−/− mice, which were timed mated to generate (c) HEBAlt^TgHEB^+/+Rag-1^−/− and (d) HEBAlt^TgHEB^−/−Rag-1^−/− embryos. Fetal livers were genotyped, lineage depleted (lineage positive fraction: B cells, myeloid cells, red blood cells). Fetal liver LSK (lineage negative, Sca1⁺, ckit⁺) cells were sorted and cultured on OP9-DL1 for 7 days to allow developmental progression to the DN3 stage. At day 7, lymphocytes were gated on the CD45⁺CD4⁻CD8⁻ fraction and sorted for the DN3 cells (CD44⁻CD25⁺), which were cultured on fresh OP9-DL1 stroma with 5 ng/mL IL7 and Flt3L. Four days later, whole cell cultures were analysed by flow cytometry. All plots were gated on the CD45⁺CD4⁻CD8⁻ fraction.

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