Effects of Hypoxia on Craniofacial Morphology and Molecular Signaling

描述（由申请人提供）：颅面畸形可由遗传原因和/或环境因素引起，包括妊娠期缺氧。为了了解缺氧导致颅面畸形的机制，我设计了一个缺氧鸡胚胎模型，通过降低鸡蛋中的氧气水平。为了测试缺氧与颅面畸形之间的相关性，我将通过缺氧诱导因子-1 （HIF-1）途径和活性氧（ROS）来研究发育形态发生信号的作用和细胞对缺氧的反应。受最近一份关于缺氧的人类心脏双胞胎胎儿全前脑畸形的临床报告的启发，我的初步研究表明，缺氧降低了鸡胚胎的存活率，延缓了发育，破坏了细胞增殖，改变了面部形态。缺氧导致生存率随缺氧程度的增加大致呈剂量依赖性下降，发育迟缓的严重程度随幸存者年龄的增加而降低（即幸存者越少，发育迟缓越严重）。缺氧胚胎也显示出一系列的头和颅面畸形，从轻微的不对称和眼睛缺陷到更严重的额鼻结构缺陷和暴露的头侧组织，这是前脑畸形的特征。二维几何形态测量显示，与质心大小和年龄相关的面部形状异常，在缺氧对照组与常氧对照组之间，在缺氧组与常氧组之间。形态测量数据表明，缺氧会导致严重的发育迟缓，在较小程度上，胚胎畸形能够在缺氧损伤下存活。缺氧破坏细胞增殖。在发育早期，神经嵴祖细胞有不同程度的凋亡。缺氧如何导致颅面畸形尚不清楚。我假设缺氧通过hif -11介导的细胞凋亡和由于前脑和额鼻突之间的分子信号传导改变而导致的细胞增殖减少而导致颅面形态异常。为了验证我的假设，我将通过改变细胞增殖、细胞死亡和分子变化来确定缺氧在多大程度上产生了显著的形态学改变。此外，我将进行功能增益和功能丧失的研究，以确定缺氧适应分子信号与畸形的关系。在我的两个具体目标中，我将揭示缺氧导致畸形的可能的细胞和分子机制，以及HIF-1和ROS活性在缺氧应激下细胞中的作用。在Aim 1中，我将通过量化面部形态学的变化并将这些变化与细胞行为和分子信号传导联系起来，确定缺氧引起的细胞和分子变化在多大程度上导致类似前脑全裂的面部畸形；这将直接验证缺氧导致前脑畸形的假说。在Aim 2中，我将测试HIF-11、ROS和面部畸形之间的关系，通过评估HIF-11在多大程度上是引起缺氧导致的缺陷的必要和充分条件，并通过测试缺氧传感器（缺氧时电子传递链产生的ROS）可以靶向降低死亡率和逆转畸形的假设。我的研究结果将为未来的治疗策略如何预防和逆转缺氧引起的颅面畸形提供见解。

DESCRIPTION (provided by applicant): Craniofacial malformations can arise from genetic causes and/or environmental factors, including hypoxia in pregnancy. To understand the mechanisms underlying craniofacial malformations induced by hypoxia, I have designed a hypoxic chick embryo model by decreasing the level of oxygen in ovo. To test the correlation between hypoxia and craniofacial malformations, I will examine the role of developmental morphogenetic signaling and the cellular response to hypoxia via the hypoxia-inducible factor-1 (HIF-1) pathway and reactive oxygen species (ROS). Inspired by a recent clinical report of holoprosencephalic anomalies in an acardiac human twin fetus that was hypoxic, my preliminary studies demonstrate that hypoxia reduces survival and delays development in chick embryos, disrupts cell proliferation, and alters facial morphology. Hypoxia led to a roughly dose-dependent decrease in survival with increasing hypoxia, and the severity of developmental delay decreased with the age of survivors (ie, fewer survivors with more severe delay). Hypoxic embryos also showed a spectrum of cephalic and craniofacial malformations ranging from mild asymmetry and eye defects to more severe defects in frontonasal structures and exposed cephalic tissues, characteristic of holoprosencephaly. 2-dimensional geometric morphometrics showed significant abnormal facial shape variation in relation to centroid size and age, among individuals in hypoxic vs. normoxic control groups, and among hypoxic groups compared to the normoxic group. The morphometric data indicate that hypoxia leads to severe developmental delay, and to a lesser degree, malformations in embryos that are able to survive the hypoxic insult. Hypoxia disrupted cell proliferation. In early stages of development, apoptosis of neural crest progenitor cells was observed in varying degrees. How hypoxia causes craniofacial malformations is not understood. I hypothesize that hypoxia creates abnormal craniofacial morphology via Hif-11-mediated apoptosis and by decreased cell proliferation due to altered molecular signaling between the forebrain and frontonasal process. To test my hypothesis, I will determine the extent to which hypoxia generates significant morphological alterations by altering cell proliferation, cell death and molecular changes. In addition, I will undertake gain- and loss-of-function studies to define the relationship of hypoxia-adaptive molecular signaling to malformations. In my two Specific Aims, I will uncover the possible cellular and molecular mechanisms underlying malformations due to hypoxia, as well as the role of HIF-1 and ROS activity in cells under hypoxic stress. In Aim 1, I will determine the extent to which cellular and molecular changes due to hypoxia cause facial malformations that resemble holoprosencephaly, by quantifying changes in facial morphology and relating these changes to cellular behavior and molecular signaling; this will test directly the hypothesis that hypoxia produces holoprosencephaly. In Aim 2, I will test the relationship between HIF-11, ROS, and facial malformations, by assessing the extent to which HIF-11 is necessary and sufficient to cause defects resulting from hypoxia and by testing the hypothesis that the hypoxia sensor, ROS production by the electron transport chain in hypoxia, can be targeted to reduce mortality rates and reverse malformations. My findings will provide insights into how craniofacial malformations caused by hypoxia can be prevented and reversed with future therapeutic strategies. PUBLIC HEALTH RELEVANCE: How can an environmental stressor, such as oxygen deprivation, affect the development of craniofacial structures in the embryo? Answering this question is important for the prevention and treatment of craniofacial birth defects that at least partially arise from environmental stresses including the lack of oxygen during embryonic and fetal development, as well as for devising new therapies to reverse these malformations. This project aims to identify the cellular and molecular events that create craniofacial defects in a low-oxygen environment.