Endothelial protease activity, organogenesis and birth defects

描述(申请人提供)：本项目的目标是阐明血管内皮细胞在胚胎发育和器官发生中的作用。微血管内皮细胞(MEC)排列在毛细血管内，在调节血管生成方面具有众所周知的功能，但对其在器官形成中的作用知之甚少。我们的初步数据表明，进化上保守的、分泌的金属蛋白酶ADAMTS9是微血管内皮细胞的产物，在正常器官发生中具有重要作用。Adamts9-/-小鼠在血管生成和器官生成开始之前，在妊娠7.5天(e7.5)时死亡。然而，在缺乏ADAMTS9一个等位基因的小鼠身上，我们发现ADAMTS9影响血管和非血管发育过程。具体地说，MEC表达的Adamts9与头面部间质表达的ADAMTS20在小鼠次级腭部闭合中起协同作用，并影响心肌发育和血管生成。此外，VERSICAN是一种对发育至关重要的大蛋白多糖，ADAMTS9对味觉和心脏都是至关重要的。由于ADAMTS9在器官发生过程中由MEC全局表达，并且Verscan在胚胎发生过程中广泛表达，我们有趣的观察表明MEC产生的ADAMTS9在发育过程中具有更广泛的影响。基于这些发现，我们推测MEC产生的ADAMTS9通过对分泌蛋白的蛋白水解性修饰，在器官发生过程中影响周围细胞。为了全面了解MEC衍生的ADAMTS9的发育影响，并阐明其潜在机制，需要一种有条件的靶向方法。这个R03方案的具体目的是在MEC中实现ADAMTS9的条件删除，并对所产生的小鼠表型进行形态分析，从而解决对血管生成和器官生成的整体影响。这将为继续研究已确定的发育缺陷，包括其潜在机制奠定基础。方法：我们将使用Tie2-Cre菌株在最早发育阶段(E8.5)的MEC中特异性地删除Adamts9。此外，我们将在发育后期(E10.5及以后)利用他莫昔芬诱导的Cre表达(通过VE-cadherin-Cre ERT2小鼠)在MEC中进行受调控的条件缺失。对所产生的小鼠表型的形态分析将确定对血管生成和器官生成的影响。意义：除了对血管生成、腭裂形成和心肌发育的预期影响外，可以预见的是，拟议的研究将阐明MEC表达的ADAMTS9在器官形成中的广泛作用。这项拟议的工作对常见的人类出生缺陷，如腭裂和心脏畸形具有直接意义，并可能为其他发育缺陷提供新的小鼠模型。

DESCRIPTION (provided by applicant): The goal of this project is to elucidate the role of vascular endothelium in embryonic development and organogenesis. Microvascular endothelial cells (mEC), which line capillaries, have a well-known function in regulation of angiogenesis, but less is known about their role in organogenesis. Our preliminary data suggests that the evolutionarily conserved, secreted metalloprotease ADAMTS9, a product of microvascular endothelium, has an important role in normal organogenesis. Adamts9-/- mice die by 7.5 days of gestation (E7.5), prior to the onset of angiogenesis and organogenesis. Nevertheless, using mice lacking one allele of Adamts9, we have shown that ADAMTS9 influences both vascular and non-vascular developmental processes. Specifically, Adamts9 expressed by mEC worked cooperatively with Adamts20 expressed by craniofacial mesenchyme in closure of the mouse secondary palate, and also influenced development of the myocardium and angiogenesis. Moreover, ADAMTS9 proteolysis of versican, a large, developmentally critical proteoglycan, was crucial in both the palate and the heart. Since ADAMTS9 is expressed globally by mEC during organogenesis and because versican is widely expressed during embryogenesis, our intriguing observations suggest a broader impact of mEC-produced ADAMTS9 during development. Based on these findings, we hypothesize that through the proteolytic modification of secreted proteins, ADAMTS9 produced by mEC influences surrounding cells during organogenesis. To gain a complete understanding of the developmental impact of mEC-derived ADAMTS9, and to elucidate the underlying mechanisms, a conditional targeting approach is required. The specific aim of this RO3 proposal is to achieve conditional deletion of ADAMTS9 in mEC, and perform morphologic analysis of the resulting mouse phenotype, thereby resolving the overall impact on angiogenesis and organogenesis. This will lay the groundwork for continued work on the identified developmental defects, including their underlying mechanisms. Approach: We will delete Adamts9 specifically in mEC at the earliest developmental stages (E8.5) using a Tie2-Cre strain. In addition, we will undertake regulated conditional deletion in mEC at later developmental stages (E10.5 and later) using tamoxifen-induced Cre expression (via a VE-cadherin-Cre ERT2 mouse). Morphologic analysis of the resulting mouse phenotypes will identify effects on angiogenesis and organogenesis. Significance: In addition to the expected impact on angiogenesis, palatogenesis and myocardial development, it is foreseen that the proposed study will elucidate a widespread role for mEC-expressed ADAMTS9 in organogenesis. The proposed work has immediate significance for common human birth defects, such as cleft palate and cardiac anomalies, and may provide new mouse models for other developmental defects.