Genetic labeling of lymphoid precursor populations: In vivo fate mapping with pTa/iCre and Gata3/vYFP knock-in mice

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The transcription factor GATA-3 is crucial for T cell development and function, including commitment to the T lineage. Accordingly, inactivation of Gata3 in hematopoietic progenitor cells results in virtually complete absence of Early Thymic Progenitors (ETPs), currently considered the earliest intra-thymic stage of T cell development. There is also significant Gata3 expression in long-term hematopoietic stem cells (LT-HSCs), and emerging data suggest intricate effects of GATA-3 on key features of LT-HSCs. To be able to monitor Gata3 expression non-invasively, we have generated a novel Gata3 knock-in mouse strain, termed GATIR, by inserting an IRES-vYFP cassette into the 3-prime untranslated region of the endogenous Gata3 gene locus. As shown in our grant application, this is the first Gata3 reporter mouse not suffering from concomitant loss or impairment of endogenous Gata3 expression. Our preliminary data provide strong evidence for the usefulness of GATIR mice to address a number of long-standing questing and disputed issues in early T cell development and hematopoietic stem cell (HSC) function. In future experiments, described in this grant application, we wish to exploit GATIR mice (i) to identify and comprehensively characterize stages of T lymphoid development preceding intra-thymic ETPs and (ii) to investigate the role of dichotomous Gata3 expression in LT-HSCs. To this end, we have generated a second line of Gata3 knock-in mice, termed GATFU, as additional tool, which allow non-invasive monitoring of GATA-3 expression also at the protein level. However, GATFU mice still require a more comprehensive characterization to unveil their full potential, another goal of our project proposal. Finally, we would like to assess to what extent our key findings from the past funding period and our new preliminary data obtained with GATIR mice also apply for embryonic T lymphopoiesis. Taken together, the experiments proposed here should help to close long-standing knowledge gaps in early T lymphopoiesis and HSC function of highest physiological relevance.