Analysis of the functional divergence of the Notch ligands Delta1 and Delta4 in vitro and in vivo

进化保守的Notch信号通路介导相邻细胞之间的直接交流，在线虫、昆虫和脊椎动物等多种生物的多种发育过程中起着关键作用。DLL1和DLL4是两种哺乳动物DSL蛋白，它们的氨基酸序列相似（47%相同，14%相似的氨基酸）和结构域结构相似（两者都含有相同数量的egf样重复序列和c端PDZ结合结构域），并且都具有激活Notch配体的功能。这些配体在发育和成人组织中以独特和重叠的模式表达。在肠上皮中，DLL1和DLL4在隐窝细胞中共表达，并在维持隐窝祖细胞池中起冗余作用。在胸腺中，DLL1和DLL4均在胸腺上皮细胞中表达，在基质细胞上表达的两种配体均能诱导造血祖细胞的T细胞体外发育。然而，在体内，DLL4是t淋巴生成所必需的Notch配体。DLL4能更好地与胸腺细胞上的NOTCH受体结合，在体外诱导t细胞发育需要较低的稳态细胞表面水平。与这些观察结果一致的是，对纯化的DLL1、DLL4和NOTCH1片段进行的生化研究表明，DLL4与NOTCH1结合的亲和力提高了约10倍。出乎意料的是，我们使用两个小鼠模型的初步数据显示，尽管DLL4具有更强的Notch激活电位，但在胚胎早期发育过程中，DLL4不能取代dll1在躯体发生和肌肉分化过程中的功能，这表明这些密切相关的DSL蛋白在生化和功能特性上存在深刻差异。在本研究中，我们提出测试假设，即DLL1和DLL4的特定蛋白质结构域赋予这些配体不同的生化特性，从而根据环境导致非冗余和冗余功能。这将通过分析嵌合DLL1/4配体蛋白来实现，其中这些蛋白的结构域已经交换。嵌合配体将在体外使用已建立的转基因策略分析其Notch激活电位和生化特性，以及在体内胚胎发育过程中。这些实验将阐明Notch配体DLL1和DLL4的不同生化特性，这些特性是两种密切相关的DSL蛋白激活Notch的不同结果的基础，并将提供这一至关重要的信号通路如何在体内根据环境被激活和微调的新见解。

The evolutionary conserved Notch signaling pathway mediates the direct communication between adjacent cells, and is pivotal for the regulation of multiple developmental processes in diverse organisms such as nematodes, insects and vertebrates. DLL1 and DLL4 are two mammalian DSL proteins that are similar in their amino acid sequence (47% identical plus 14% similar amino acids) and domain structure (both contain the same number of EGF-like repeats, and a C-terminal PDZ binding domain) and both function as activating Notch ligands. These ligands are expressed both in unique and overlapping patterns during development and in adult tissues. In the intestinal epithelium DLL1 and DLL4 are coexpressed in crypt cells and act redundantly in maintaining the crypt progenitor cell pool. In the thymus DLL1 and DLL4 are both expressed in thymic epithelial cells, and both ligands expressed on stromal cells can induce T cell development of hematopoietic progenitors in vitro. However, in vivo DLL4 is the essential Notch ligand required for T-lymphopoiesis. DLL4 binds better to NOTCH receptors on thymocytes and a lower steady-state cell surface levels is required to induce T-cell development in vitro. Consistent with these observations biochemical studies with purified fragments of DLL1 and DLL4 and NOTCH1 have shown that DLL4 binds to NOTCH1 with approximately 10-fold higher affinity.Unexpectedly, our preliminary data using two mouse models show that -despite the stronger Notch activation potential of DLL4- during early embryonic development DLL4 cannot replace DLL1s function during somitogenesis and muscle differentiation, suggesting profound differences in the biochemical and functional properties of these closely related DSL proteins. In this study we propose to test the hypothesis that specific protein domains of DLL1 and DLL4 confer different biochemical properties to these ligands and thereby lead to non-redundant and redundant functions depending on the context. This will be achieved by the analysis of chimeric DLL1/4 ligand proteins in which domains of these proteins have been swapped. Chimeric ligands will be analyzed in vitro for their Notch activation potential and biochemical properties as well as during embryonic development in vivo using an established transgenic strategy. These experiments should elucidate the divergent biochemical properties of the Notch ligands DLL1 and DLL4 that are the basis for the different outcomes of NOTCH activation by two closely related DSL proteins and should provide novel insights how this critically important signaling pathway is activated and fine-tuned in vivo depending on the context.