PROCOLLAGEN C-PROTEINASE ENHANCERS: IN VIVO ROLES

说明(申请人提供)：本修订申请的母方拨款(前胶原C-蛋白酶增强剂：体内作用，R01 AR53815)涉及对PCOLCE1缺失、PCOLCE2缺失和PCOLCE1/PCOLCE2双缺失敲除小鼠的深入鉴定，以在体内研究两种前胶原C-蛋白酶增强蛋白PCOLCE1和PCOLCE2在体内所扮演的角色，这两种蛋白质可以增强BMP1样胞外蛋白酶生物合成地切割主要纤维前胶原蛋白中的C前肽的能力。母基金是主要研究人员以前拨款工作的继续，该基金专注于BMP1样蛋白酶、它们的底物、生物学功能和这些功能的调节器。然而，除了受到PCOLCE1和2的影响外，目前的父母拨款并没有调查BMP1样蛋白酶的额外生物学作用。在这里，我们建议直接研究BMP1样蛋白酶的额外功能，这将通过使用本实验室创造的新试剂来利用，以及我们在以前的研究中获得的技能。具体地说，虽然Tll1基因的敲除是胚胎致死的，但我们最近成功地创造了具有Tll1等位基因的小鼠，这将允许组织特异性的Tll1敲除。Tll1基因编码BMP1样蛋白水解酶，哺乳动物Tolloid样1(MTLL1)。我们已经证明，Tll1在发育中的和成人中枢神经系统(CNS)的特定结构中高度表达。因此，我们已经将我们的TLL1小鼠与Cre重组酶由Nestin基因的CNS特异性增强子序列驱动的小鼠杂交，并在这里展示了CNS特异性的TLL1基因敲除。我们建议使用这些小鼠在体内确定T111CNS的功能。我们要求为一名实验室成员提供工资支持，以进行此类研究。此外，我们已经开始使用功能强大的斑马鱼系统来进一步确定BMP1样金属蛋白酶的功能。然而，我们之前在这个系统中的工作使用了另一个实验室的设备和鱼，大大推迟了我们的研究。我们建议购买设备，使我们能够在实验室进行斑马鱼工作。我们建议首先利用这样的设备在体内确定未知蛋白斑马鱼脊索素样蛋白(ZCHL)的作用，我们已经发现它是早期胚胎中的一个母性因子，并假设它是BMP1样蛋白酶的底物，参与调节BMP信号。获得该设备不仅将极大地增强成功完成zCHL研究的能力，而且将极大地增强我们利用强大的斑马鱼系统进一步探索BMP1样蛋白酶和ECM成分的能力，这些也是本实验室一直以来的研究重点。 与公共健康相关：我们发现，移除老鼠体内的特定基因会导致心脏处于错误的位置，从而不能很好地维持胚胎的存活。我们还发现，该基因在发育中的中枢神经系统和成年中枢神经系统中高水平表达。我们建议使用一种方法，只在中枢神经系统中使这种基因失活，这样做的动物不会在胚胎阶段死于心脏缺陷，而是发育成成年人，我们可以在其中研究大脑结构中这种基因的丧失及其在行为和生理测试中的功能的影响。此外，我们建议使用一种被称为斑马鱼的小型热带鱼来研究我们发现的一种基因在早期发育中的作用。我们认为，这种蛋白质在决定胚胎的正面和背面是很重要的。斑马鱼被选为这项研究的对象，因为它们产生的卵在外部受精，并在母亲之外发育。因此，胚胎在发育的所有阶段都可以进行观察和基因操作，而不需要对母亲进行任何侵入性程序。斑马鱼胚胎是光学透明的，发育迅速，使人们能够分析在完整胚胎中发生的发育事件。我们相信，通过对老鼠和斑马鱼系统的研究获得的见解将为相关人类基因的功能提供重要的见解，从而为这些基因的缺陷可能如何与人类疾病有关提供重要的见解。

DESCRIPTION (provided by applicant): The parent grant (Procollagen C-proteinase Enhancers: In vivo Roles, R01 AR53815) for this Revision Application involves in depth characterization of PCOLCE1-null, PCOLCE2-null, and PCOLCE1/PCOLCE2 doubly null knockout mice to study in vivo roles of the two procollagen C-proteinase enhancer proteins PCOLCE1 and PCOLCE2 which enhance the ability of BMP1-like extracellular proteinases to biosynthetically cleave the C-propeptides from the major fibrillar procollagens. The parent grant is a continuation of work from previous grants of the principal investigator that focused on the BMP1-like proteinases, their substrates, biological functions, and modulators of those functions. However, the present parent grant does not investigate additional biological roles of BMP1-like proteinases, other than those affected by PCOLCE1 and 2. Here we propose direct studies of additional functions of the BMP1-like proteinase, that will be leveraged by use of novel reagents created in this lab, and skills obtained in our previous studies. Specifically, although knockout of the Tll1 gene, which encodes the BMP1-like proteinase, mammalian tolloid-like 1 (mTLL1), is embryonic lethal, we have recently been successful in creating mice with floxed Tll1 alleles, which will allow tissue-specific Tll1 knockout. We've shown that Tll1 is highly expressed in specific structures of the developing and adult central nervous system (CNS). Thus, we've crossed our floxed Tll1 mice to mice in which Cre recombinase is driven by the CNS-specific enhancer sequences of the nestin gene, and demonstrate herein CNS-specific knockout of Tll1. We propose using these mice to determine in vivo Tll1 CNS functions. We request salary support for a lab member to conduct such studies. In addition, we have begun using the powerful zebrafish system to further define functions of the BMP1-like metalloproteinases. However, our previous work in this system employed the equipment and fish of another lab, greatly delaying our studies. We propose acquiring equipment that will enable us to perform zebrafish work in our lab. We propose first using such equipment to determine in vivo roles of the uncharacterized protein zebrafish Chordin-like (zCHL), which we have found to be a maternal factor in earliest embryos, and hypothesize to be a substrate of BMP1-like proteinases, involved in regulating BMP signaling. Obtaining the equipment will not only greatly enhance ability to successfully complete the zCHL study, but will greatly enhance our ability to use the powerful zebrafish system to further explore the BMP1-like proteinases, and ECM components that have also been the focus of research in this laboratory. PUBLIC HEALTH RELEVANCE: We've found that removing a particular gene in mice results in hearts that are in the wrong place, and which don't work sufficiently well to keep the embryo alive. We also have found that this gene is expressed at high levels in the developing and adult central nervous system. We propose using a method that will inactivate this gene only in the central nervous system, so that animals in which this is done will not die of heart defects in the embryonic stage, but will develop into adults in which we can study the effects of loss of this gene in the structure of the brain and its functioning in behavioral and physiological tests. In addition, we propose using a small tropical fish, known as the zebrafish, to examine the role in early development of a gene we've discovered. We believe that this protein is important in determining what will be the front and what will be the back of the embryo. Zebrafish were chosen for this study, as they generate eggs that are fertilized externally and develop outside the mother. The embryos are therefore accessible for observation and genetic manipulation at all stages of development without requiring any invasive procedures on the mother. Zebrafish embryos are optically transparent and develop rapidly, allowing one to analyze developmental events as they occur in the intact embryo. We believe that insights obtained from study in the mouse and zebrafish systems will provide important insights into the functions of the related human genes, and thus into how defects in such genes might be involved in human disease.