Endothelial protease activity, organogenesis and birth defects

项目描述（申请人提供）：本项目旨在阐明血管内皮在胚胎发育和器官发生中的作用。微血管内皮细胞（mEC）排列在毛细血管上，具有众所周知的血管生成调节功能，但对其在器官发生中的作用知之甚少。我们的初步数据表明，进化上保守的、分泌的金属蛋白酶ADAMTS9是微血管内皮的产物，在正常器官发生中起重要作用。Adamts9-/-小鼠在妊娠7.5天（E7.5）死亡，在血管生成和器官生成开始之前。然而，使用缺乏Adamts9等位基因的小鼠，我们已经证明Adamts9影响血管和非血管发育过程。其中，mEC表达的Adamts9与颅面间质表达的Adamts20协同作用于小鼠次腭闭合，并影响心肌的发育和血管生成。此外，对桃聚糖（一种对发育至关重要的大型蛋白多糖）的ADAMTS9蛋白水解对上颚和心脏都至关重要。由于ADAMTS9在器官发生过程中由mEC在全球表达，而versican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，我们假设通过对分泌蛋白的蛋白水解修饰，mEC产生的ADAMTS9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对发育的影响，并阐明其潜在机制，需要一种条件靶向方法。本RO3提案的具体目的是在mEC中实现ADAMTS9的条件缺失，并对由此产生的小鼠表型进行形态学分析，从而解决对血管生成和器官发生的总体影响。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菌株在早期发育阶段（E8.5）的mEC中特异性删除Adamts9。此外，我们将利用他莫昔芬诱导的Cre表达（通过VE-cadherin-Cre ERT2小鼠），在发育后期（E10.5及更晚）的mEC中进行条节性条件缺失。由此产生的小鼠表型的形态学分析将确定对血管生成和器官发生的影响。意义：除了对血管生成、腭发育和心肌发育的预期影响外，预计该研究将阐明mec表达的ADAMTS9在器官发生中的广泛作用。这项工作对腭裂、心脏畸形等常见的人类出生缺陷具有直接意义，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.