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

转录因子加塔-3对T细胞发育和功能至关重要，包括对T谱系的定型。因此，造血祖细胞中Gata 3的失活导致几乎完全不存在早期胸腺祖细胞（ETP），目前认为ETP是T细胞发育的最早胸腺内阶段。在长期造血干细胞（LT-HSC）中也存在显著的Gata 3表达，并且新出现的数据表明加塔-3对LT-HSC的关键特征的复杂影响。为了能够非侵入性地监测Gata 3表达，我们通过将IRES-vYFP盒插入内源性Gata 3基因座的3-prime非翻译区中，产生了一种新的Gata 3敲入小鼠品系，称为GATIR。如我们的资助申请中所示，这是第一只没有遭受内源性Gata 3表达的伴随损失或损害的Gata 3报告小鼠。我们的初步数据为GATIR小鼠解决早期T细胞发育和造血干细胞（HSC）功能中一些长期存在的探索和争议问题的有用性提供了强有力的证据。在本授权申请中描述的未来实验中，我们希望利用GATIR小鼠（i）鉴定和全面表征胸腺内ETP之前的T淋巴发育阶段，以及（ii）研究LT-HSC中二分Gata 3表达的作用。为此，我们已经产生了第二系Gata 3敲入小鼠，称为GATFU，作为额外的工具，其允许在蛋白质水平上也非侵入性地监测加塔-3表达。然而，GATFU小鼠仍然需要更全面的表征来揭示其全部潜力，这是我们项目提案的另一个目标。最后，我们想评估在多大程度上我们的关键发现从过去的资助期和我们的新的初步数据获得的GATIR小鼠也适用于胚胎T淋巴细胞生成。总之，这里提出的实验应该有助于缩小长期存在的知识差距，在早期T淋巴细胞生成和HSC功能的最高生理相关性。

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.