GATA3 was first discovered in a screen that sought to identify factors that bind to the human T-cell receptor-α (TCRa
. It is among the earliest genes that are expressed by progenitor cells following commitment to the T-cell lineage. As germline deletion of Gata3
results in embryonic lethality16
, analysis of the role of GATA3 in T-cell development required the generation of chimeric mice from immunodeficient (recombination-activating gene 2 (Rag2
. In these chimeric mice, T cells were not detected in the spleen or thymus, and there was little or negligible contribution of Gata3-/-
embryonic stem cells to the rudimentary Rag2-/-
thymus. B-cell and NK-cell development were unaffected. These data led to the conclusion that GATA3 expression is a hallmark of T cells, and that GATA3 is necessary for T-cell development.
But, is GATA3 expression sufficient for T-cell development? Gata3
mRNA is present in progenitors, such as fetal and adult haematopoietic stem cells (HSCs) in both mice and humans, before they commit to the T-cell lineage, which suggests that expression of GATA3 is not strictly T-cell specific18-22
. Physiological expression of GATA3 is not sufficient to abrogate the development of progenitors into non-T-cell lineages, as indicated by the finding that common lymphoid progenitors (CLPs; which have a LIN-
phenotype) express GATA3 while retaining B-cell and NK-cell developmental potential23
. However, might forced overexpression of GATA3 in non-T-lineage cells be sufficient to divert their development to the T-cell lineage? This issue has been a difficult to address for two reasons. First, from a technical standpoint, overexpression of GATA3 in mouse HSCs or fetal human CD3-
thymocytes in fetal thymic organ cultures (FTOCs) inhibits cell growth and/or induces apoptosis, which makes the analysis of developmental outcomes difficult24, 25
. Second, forced expression of GATA3 in non-T-lineage cells may induce developmental programmes that are normally controlled by other GATA-family proteins (such as GATA1 and GATA2). For example, overexpression of GATA3 in mouse HSCs diverted these cells to develop into erythroid and megakaryocytic lineages, which are fates usually specified by GATA124
. So, although GATA3 is clearly required for T-cell lineage development, its function prior to T-cell commitment remains unclear. The physiological role of GATA3 in early progenitor cell populations, such as HSCs, which co-express GATA3 and GATA219
, must be defined in the future by conditional knockout approaches, to discriminate the non-redundant functions of related GATA proteins.
During early T-lymphoid development, Gata3
mRNA is detectable in HSCs and the levels increase as development proceeds through the lymphoid progenitor, double negative 1 (DN1), DN2 and DN3 stages26, 27
. This expression pattern led to the suggestion that GATA3 expression occurs downstream of the Notch signalling pathway, which is known to be crucial for T-cell development. Indeed, the ligation of Notch on uncommitted precursors by culturing them with OP9 stromal cells that express the Notch ligand Delta-like ligand 1 (DLL1; the OP9-DLL1 culture system)28
forces their development into T cells and upregulates their expression of GATA329-31
, an effect that is reversed when Notch ligands are removed31
. As the Notch signalling pathway is both necessary and sufficient for T-cell commitment in vivo32, 33
, a simple model would predict that Notch signals in the thymus promote T-cell commitment by direct or indirect upregulation of GATA3 expression and suppress B-cell commitment by inactivation of B-cell-specifying factors.
If this model were correct, forced expression of GATA3 in the absence of Notch signalling would be expected to promote T-cell development. However, a recent study from the Rothenberg laboratory34
shows that the relationship between Notch and GATA3 is more complex and that constraining GATA3 expression is important to avoid cell death and alternative lineage specification. Using the OP9-DLL1 culture system, these investigators showed that the growth of purified HSCs (LIN-
), lymphoid progenitors (LIN-
) or DN thymocytes was inhibited by GATA3 overexpression. Moreover, the development of fetal (embryonic day 14.5) thymocytes, which comprise DN cells, that overexpress GATA3 was arrested at the DN1 and DN2 stages when cultured under T-cell-promoting conditions (that is, in the presence of Notch ligands)34
, an effect that is similar to the toxic effects of GATA3 observed in human FTOCs25
. Under B-cell-promoting conditions (that is, in the absence of Notch ligands), GATA3 overexpression failed to induce the differentiation of lymphoid progenitors (or fetal liver cells) into T cells34, 35
. In addition, too little GATA3 expression was detrimental to progenitor survival, as GATA3-deficient fetal liver cells were profoundly depleted from OP9 co-cultures despite expression of the active, intracellular domain of Notch135
. So, in the absence of Notch signalling, GATA3 overexpression is insufficient to drive T-cell differentiation, and in the presence of Notch signalling, GATA3 overexpression or lack of expression is toxic, which suggests that the levels of GATA3 must be kept ‘just right’ for thymocyte survival and development.
Unexpectedly, GATA3 overexpression by DN1 or DN2 thymocytes that were cultured in the absence of Notch ligands resulted in their differentiation into mast cells, as indicated by the upregulation of canonical mast-cell genes and a characteristic mast-cell morphology34
. Overexpression of GATA3 by cells at the later, DN3 stage of development did not lead to induction of the mast-cell programme, indicating that the developmental window for diversion of progenitors to the mast-cell lineage by GATA3 expression is narrow. As GATA1 and GATA2, but not GATA3, are normally expressed by mast cells, the forced overexpression of GATA3 used in this study does not necessarily indicate a physiological role for GATA3 during the terminal stages of mast-cell development. The finding that GATA proteins induce their own expression36, 37
may be relevant in this setting, as GATA3 overexpression probably induces the mast-cell programme indirectly through auto-activation of GATA1 and/or GATA2 expression. These studies show that keeping the expression of GATA3 (or perhaps any GATA protein) at the appropriate level in DN1 and DN2 cells is essential to suppress their development into alternative cell lineages. Together, these data suggest that Notch signals regulate thymocyte development by maintaining the expression of T-cell-specific genes, preventing the induction of non-T-cell-specific transcriptional programmes and, if necessary, leading to the demise of cells that express GATA3 at high levels. Although it is clear that Notch signalling precedes the upregulation of GATA3 expression by DN thymocytes, there is no clear evidence for direct regulation of GATA3 expression by Notch. So, in the presence of Notch signals, the developmental outcome of regulated GATA3 expression seems to be influenced by other cell-type and stage-specific factors.