Oocyte polarity and mRNA localization in Zebrafish

斑马鱼卵母细胞极性和 mRNA 定位

• 批准号: 8038543
• 负责人: Florence Louise Marlow
• 金额: $ 32.37万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-27 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8038543
• 关键词: Oocyte; polarity; mRNA; localization; Zebrafish

DESCRIPTION (provided by applicant): Asymmetry in oocytes is a well-documented and conserved feature among animals including vertebrates and humans. In vertebrates, the earliest indicator of cell polarity is an asymmetric aggregate, known as the Balbiani body, that includes organelles, proteins, and, in some animals, mRNAs encoding germline determinants. In non-mammalian vertebrates, this early asymmetry is known to indicate the animal-vegetal axis, but the relationship between the Balbiani body and the animal-vegetal axis in mammals is not understood. Although the animal- vegetal axis is the first axis to form in vertebrates, and is crucial for normal development of the embryonic axes that form later in development, its specification is poorly understood. In a maternal-effect genetic screen we isolated 2 alleles of bucky ball (buc), mutants that lacks oocyte asymmetry and fails to establish the axes in embryos. The Buc protein does not contain any characterized or known functional domains based on sequence comparison, but other vertebrates including humans have bucky ball genes. The buc gene, and our mutant alleles provide the first genetic access and a unique entry point to the developmental pathway regulating oocyte polarity. Here three aims are proposed to study how cell polarity is established and maintained in the vertebrate ovary. 1) We will test the hypothesis that buc specifies the oocyte axis upstream or at the level of Balbiani body assembly. 2) We have identified Buc interacting proteins. We will study these interacting proteins, and conduct rescue based structure function analysis to identify Buc functional domains to understand the mechanism by which Buc regulates animal-vegetal polarity. 3) We will determine which factors mediate asymmetric buc mRNA localization and contribute to oocyte polarity. Understanding how the Balbiani body, a conserved oocyte asymmetric structure, forms in vertebrates will break new ground in the field of axis formation. Studies of the genetic and molecular control of axis formation in zebrafish will clarify the mechanisms establishing these earliest oocyte asymmetries, which are conserved. In humans, mutations disrupting genes required to specify oocyte polarity or the first embryonic axis are expected to result in failed implantation or miscarriage due to severe developmental abnormalities. These most severe birth defects often are not detected in humans. In model systems such as zebrafish where fertilization and development of the embryo occur externally every egg that is produced can be examined for developmental abnormalities. Thus, this vertebrate genetic system allows access to maternally regulated developmental processes. An improved understanding of the essential maternal genes regulating early embryonic development in zebrafish will provide insight into the basis of birth defects and miscarriage, and facilitate comparison with human proteins. Studies of the Buc pathway are expected to be particularly relevant to abnormalities arising in very early pregnancy since buc mutant females produce eggs that are fertilized, but fail to specify the embryonic germ layers or axes. Completing these studies will provide insight into how Bucky ball regulates Balbiani body formation and oocyte asymmetry. These studies represent a first step toward deciphering the genes and mechanisms, mediating an evolutionarily conserved feature of primary oocyte development that is predicted to play fundamental roles in fertility, and in some vertebrates, establishment of the embryonic axes. PUBLIC HEALTH RELEVANCE: Prior to zygotic genome activation, vertebrate development depends on maternally supplied factors. However, the identity of the essential components and the molecular mechanisms underlying many maternally driven processes are not known. Mutations disrupting strict maternal-effect genes are viable. The mutant females are overtly normal, due to maternal function supplied by their mother. However, all of their progeny display the mutant phenotype regardless of their genotype. Although maternal products are essential for vertebrate development, only a small fraction of the vast numbers of vertebrate genes with maternal expression have been experimentally evaluated through genetic or by interference technologies. In each of these cases, insufficient maternal contribution results in early embryonic arrest, or profound developmental abnormalities. Similar genetic defects in humans would be expected to result in failed implantation or miscarriage before pregnancy is detected. Ten to twenty percent of known pregnancies result in miscarriage; however, when combined with undetected pregnancies, the actual percentage of pregnancies ending in miscarriage is estimated to be as high as 40-50% of all pregnancies, according to The March of Dimes, The American College of Obstetricians and Gynecologists, The Mayo clinic, and the National Institutes on Child Health and Development. Our research goal is to elucidate the genetic pathways and cell biological events that establish the first embryonic axis. We will use a combination of genetic, molecular, and cell biological approaches in the zebrafish model system. In humans, loss of function mutations in genes whose products are required to specify the first embryonic axis are expected to result in miscarriage due to severe developmental abnormalities. Studies of the genetic and molecular control of axis formation in zebrafish will clarify the genetic basis of animal-vegetal axis formation, potentially illuminating the genetic basis of human birth defects, and early miscarriages of unknown etiology.

描述（由申请人提供）：卵母细胞的不对称性是动物（包括脊椎动物和人类）中有充分记载和保守的特征。在脊椎动物中，细胞极性的最早指标是一个不对称的聚集体，称为Balbiani体，其中包括细胞器，蛋白质，以及在某些动物中，编码生殖系决定簇的mRNA。在非哺乳类脊椎动物中，这种早期的不对称性被认为是动物-植物轴，但巴尔比亚尼体和哺乳类动物-植物轴之间的关系尚不清楚。虽然动物-植物轴是脊椎动物中形成的第一个轴，并且对于发育后期形成的胚胎轴的正常发育至关重要，但其规格知之甚少。在母体效应遗传筛选中，我们分离出2个巴基球（布克）的等位基因，这是一种缺乏卵母细胞不对称性且不能在胚胎中建立中轴的突变体。基于序列比较，布克蛋白不包含任何特征性或已知的功能结构域，但包括人类在内的其他脊椎动物具有巴基球基因。布克基因和我们的突变等位基因为调节卵母细胞极性的发育途径提供了第一个遗传途径和独特的切入点。本文提出了三个研究脊椎动物卵巢细胞极性的建立和维持的目的。1)我们将测试的假设，即布克指定的卵母细胞轴上游或Balbiani体组装的水平。2)我们已经确定了布克相互作用蛋白。我们将研究这些相互作用的蛋白质，并进行基于拯救的结构功能分析，以确定布克功能结构域，以了解布克调节动物-植物极性的机制。3)我们将确定哪些因素介导不对称布克mRNA定位和卵母细胞极性。了解Balbiani体，一种保守的卵母细胞不对称结构，在脊椎动物中的形成将在轴形成领域开辟新天地。对斑马鱼中轴形成的遗传和分子控制的研究将阐明建立这些最早期卵母细胞不对称性的机制，这些机制是保守的。在人类中，破坏指定卵母细胞极性或第一个胚胎轴所需的基因的突变预计会导致植入失败或由于严重发育异常而流产。这些最严重的出生缺陷通常在人类中检测不到。在模式系统中，如斑马鱼，受精和胚胎发育发生在外部，每一个产生的卵子都可以检查发育异常。因此，这种脊椎动物遗传系统允许进入母系调节的发育过程。对调节斑马鱼早期胚胎发育的必需母体基因的更好理解将提供对出生缺陷和流产基础的深入了解，并有助于与人类蛋白质进行比较。对布克途径的研究预计与非常早期妊娠中出现的异常特别相关，因为布克突变型雌性产生受精卵，但不能指定胚胎胚层或轴。完成这些研究将有助于深入了解Bucky ball如何调节Balbiani小体形成和卵母细胞不对称性。这些研究代表了破译基因和机制的第一步，介导了初级卵母细胞发育的进化保守特征，该特征预计在生育力中发挥重要作用，并且在一些脊椎动物中，建立胚胎轴。 公共卫生相关性：在合子基因组激活之前，脊椎动物的发育依赖于母体提供的因子。然而，身份的基本组成部分和分子机制的基础上，许多母系驱动的过程是未知的。破坏严格母源效应基因的突变是可行的。突变的雌性是明显正常的，由于母性功能由母亲提供。然而，无论其基因型如何，其所有后代均显示突变表型。虽然母体产物对于脊椎动物的发育是必不可少的，但是在大量具有母体表达的脊椎动物基因中，只有一小部分通过遗传或干扰技术进行了实验评估。在每一种情况下，母亲的贡献不足导致早期胚胎停滞或严重的发育异常。人类类似的遗传缺陷预计会导致植入失败或在检测到怀孕之前流产。10%到20%的已知怀孕会导致流产;然而，根据The March of Dimes，美国妇产科医师学院，马约诊所和国家儿童健康与发展研究所的数据，当与未被发现的怀孕相结合时，流产的实际百分比估计高达所有怀孕的40-50%。我们的研究目标是阐明建立第一个胚胎轴的遗传途径和细胞生物学事件。我们将在斑马鱼模型系统中使用遗传、分子和细胞生物学方法的组合。在人类中，由于严重的发育异常，其产物被要求指定第一个胚胎轴的基因的功能突变的丧失预计会导致流产。对斑马鱼中轴形成的遗传和分子控制的研究将阐明动物-植物中轴形成的遗传基础，可能阐明人类出生缺陷和不明病因的早期流产的遗传基础。