GATA-3 is required for early T lineage progenitor development

Abstract

Most T lymphocytes appear to arise from very rare early T lineage progenitors (ETPs) in the thymus, but the transcriptional programs that specify ETP generation are not completely known. The transcription factor GATA-3 is required for the development of T lymphocytes at multiple late differentiation steps as well as for the development of thymic natural killer cells. However, a role for GATA-3 before the double-negative (DN) 3 stage of T cell development has to date been obscured both by the developmental heterogeneity of DN1 thymocytes and the paucity of ETPs. We provide multiple lines of in vivo evidence through the analysis of T cell development in Gata3 hypomorphic mutant embryos, in irradiated mice reconstituted with Gata3 mutant hematopoietic cells, and in mice conditionally ablated for the Gata3 gene to show that GATA-3 is required for ETP generation. We further show that Gata3 loss does not affect hematopoietic stem cells or multipotent hematopoietic progenitors. Finally, we demonstrate that Gata3 mutant lymphoid progenitors exhibit neither increased apoptosis nor diminished cell-cycle progression. Thus, GATA-3 is required for the cell-autonomous development of the earliest characterized thymic T cell progenitors.

GATA3 Abundance Is a Critical Determinant of T Cell Receptor β Allelic Exclusion

“…Proplatelet formation is inhibited after either diminished or excessive expression of the transcription factor MafG in megakaryocytes (67), and T cell development in the thymus is blocked when there is either too little or too much GATA3 (32,33,40,68,69). Of note in this regard, the inactivation of one Gata3 allele often results in a mild reduction in T cell development in humans (HDR [hypoparathyroidism, deafness, and renal dysplasia] syndrome) (70)(71)(72) and in mice (49), while a 2-fold increase in the GATA3 protein abundance has been shown to induce T cell lymphoma (73).…”

“…Development from DN3 to DN4 stage thymocytes was blocked in adult mice that were conditionally ablated for Gata3 at the DN3 stage using an Lck-Cre transgene to inactivate Gata3 flox/flox (34) as well as in adoptively transferred animals that were reconstituted with homozygous hypomorphic mutant Gata3 g/g fetal liver HSC and progenitors (32). Here we show that an elevated GATA3 abundance leads to a forfeiture of Tcrb allelic exclusion by analyzing the recombination status of both Tcrb alleles in single thymocytes recovered from both Tg Cd2-GATA3 and wild-type mice.…”

“…3 in reference 49) (Gata3 g/g mutants could not be examined directly since the homozygous mutation leads to perinatal lethality [50]). In initially curious contrast, GFP fluorescence was never observed at the ETP, DN2, or DN3 stage in Gata3 g/ϩ heterozygous mutants (32,49). We then hypothesized that this apparent contradiction might be explained by the facts that the Gata3 g allele is a hypomorph and that its reduced activity, if expressed in ETPs (where Gata3 is monoallelic), would not be adequate to allow these cells to survive, while ETPs that initially express the wild-type (Gata3 ϩ ) allele should be able to developmentally progress in a normal fashion.…”

“…Gata3 g (32,50), GATA3 Tg Cd2-GATA3 transgenic line 720 (36), TCR␣␤ transgenic OT-II.2 (53) (purchased from Jackson Laboratory), and TCR␤ transgenic (52) (generously provided by Karen Hathcock at National Institutes of Health, Baltimore, MD) mice were described previously and were maintained on a congenic C57BL/6J background. C57BL/6J inbred mice used for backcrossing and FVB/NJ mice were purchased from Jackson Laboratory.…”

“…Since the Gata3 monoallelic-to-biallelic transcriptional switch (49) is first observed at around the same developmental stage (DN2/DN3) as when the Tcrb loci initiate VDJ genomic DNA rearrangement, we hypothesized that Gata3 monoallelic expression might be functionally associated with allelic exclusion at the Tcrb locus. We previously generated a GATA3-enhanced green fluorescent protein (eGFP) fusion protein knock-in allele (Gata3 g ) by embryonic stem (ES) cell gene targeting (32,50), and by both RNA-fluorescence in situ hybridization (FISH) and RT-PCR single nucleotide variant (SNV) sequencing of single thymocytes, we recently demonstrated that a Gata3 monoallelic-to-biallelic transcriptional switch initiates at around the DN2 stage (49). In concert with those observations, hypomorphic Gata3 g/g homozygous thymocytes were shown to exhibit a second peak of GFP fluorescence starting at the DN2 stage in thymocytes that were generated after adoptive transfer of fetal liver hematopoietic stem cells (HSC) and progenitors (see Fig.…”