Extreme GATA-3 dose dependence in early T cell development (36.28)

Abstract

Abstract GATA-3 expression first reaches peak levels during T-lineage commitment, but a specific requirement for GATA-3 at each of the early DN stages in T development has not been proven. Using retroviral expression of shRNA to reduce GATA-3 protein at each of the earliest stages of T-cell differentiation (DN1, DN2, DN3), we identify two dose-dependent effects: a defect in survival or expansion at DN1, and a separable developmental arrest of cells at DN2, which was cell-autonomous, and not relieved by a Bcl2 transgene. The DN2 arrest phenotype was extremely robust; we were able to document this developmental block even in T development assay cultures initiated with a single fetal liver derived precursor. The DN2-stage developmental block was confirmed using transduced Cre to induce deletion of a floxed Gata3 allele. Reduction of GATA-3 also increased PU.1 levels in pro-T cells, despite relatively normal T-lineage gene induction. To test roles of GATA-3 in exclusion of non-T fates, we forced expression of wild type, obligate repressor, and tamoxifen-inducible GATA-3 forms in precursors undergoing B and myeloid development. GATA-3 blocked B cell development via direct repression under conditions which remained permissive for myeloid development. Thus GATA-3 is required for at least two steps of T-cell progression to commitment, and restricts two different alternative fates by distinct mechanisms.