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

转录因子GATA-3对T细胞的发育和功能至关重要，包括对T谱系的承诺。因此，造血祖细胞中Gata3的失活导致早期胸腺祖细胞（Early Thymic progenitor, ETPs）几乎完全缺失，目前认为这是T细胞发育的最早胸腺内阶段。长期造血干细胞（lt - hsc）中也有显著的Gata3表达，新出现的数据表明，GATA-3对lt - hsc的关键特征有复杂的影响。为了能够无创地监测Gata3的表达，我们通过将IRES-vYFP盒插入内源性Gata3基因位点的3- '未翻译区，产生了一种新的Gata3敲入小鼠品系，称为GATIR。正如我们的拨款申请所示，这是第一个没有内源性Gata3表达缺失或损伤的Gata3报告小鼠。我们的初步数据为GATIR小鼠在解决早期T细胞发育和造血干细胞（HSC）功能中一些长期存在的问题和争议提供了强有力的证据。在未来的实验中，我们希望利用GATIR小鼠(i)识别和全面表征胸腺内ETPs之前的T淋巴细胞发育阶段，（ii）研究在lt - hsc中二分Gata3表达的作用。为此，我们产生了第二种Gata3敲入小鼠，称为GATFU，作为额外的工具，它允许在蛋白质水平上对GATA-3表达进行非侵入性监测。然而，GATFU小鼠仍然需要更全面的表征来揭示它们的全部潜力，这是我们项目提案的另一个目标。最后，我们想评估我们在过去资助期的主要发现和我们在GATIR小鼠中获得的新的初步数据在多大程度上也适用于胚胎T淋巴生成。综上所述，本文提出的实验应有助于弥合长期以来在早期T淋巴生成和HSC功能方面的知识空白。

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.