Oocyte polarity and mRNA localization in Zebrafish

描述(由申请人提供)：卵母细胞的不对称性是包括脊椎动物和人类在内的动物中一个有充分证据和保守的特征。在脊椎动物中，细胞极性的最早指示物是被称为巴比亚尼小体的不对称聚集体，它包括细胞器、蛋白质，在一些动物中，还包括编码生殖系决定因素的mRNAs。在非哺乳动物脊椎动物中，这种早期的不对称表明动物-植物轴，但哺乳动物的Balbiani小体和动物-植物轴之间的关系尚不清楚。虽然动植物轴是脊椎动物中第一个形成的轴，并且对发育后期形成的胚轴的正常发育至关重要，但人们对它的具体规定知之甚少。在母体效应遗传筛选中，我们分离到了Bucky ball(Buc)的2个等位基因，这是一种缺乏卵母细胞不对称且无法在胚胎中建立轴的突变体。根据序列比较，Buc蛋白不包含任何特征或已知的功能结构域，但包括人类在内的其他脊椎动物都有Bucky ball基因。Buc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通道和一个独特的入口点。这里提出了三个目标来研究细胞极性是如何在脊椎动物卵巢中建立和维持的。1)我们将检验Buc指定上游或Balbiani身体组装水平的卵子轴的假设。2)我们鉴定了Buc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RESPECT的结构功能分析，以确定Buc的功能结构域，以了解Buc调节动植物极性的机制。3)我们将确定哪些因素介导了Buc mRNA的不对称定位，并对卵母细胞的极性做出了贡献。了解脊椎动物的巴比亚尼小体(一种保守的卵母细胞不对称结构)是如何形成的，将在轴形成领域开辟新的天地。对斑马鱼轴形成的遗传和分子控制的研究将阐明建立这些最早的卵母细胞不对称的机制，这些不对称是保守的。在人类中，突变破坏了指定卵母细胞极性或第一个胚胎轴所需的基因，预计会由于严重的发育异常而导致着床失败或流产。这些最严重的出生缺陷通常在人类身上检测不到。在斑马鱼等体外受精和胚胎发育的模型系统中，可以检查产生的每个卵子是否发育异常。因此，这种脊椎动物的遗传系统允许进入母体调节的发育过程。对控制斑马鱼早期胚胎发育的基本母体基因的进一步了解将有助于深入了解出生缺陷和流产的基础，并有助于与人类蛋白质进行比较。Buc途径的研究预计将与妊娠早期出现的异常特别相关，因为Buc突变雌性产生受精的卵子，但未能具体说明胚胎胚层或胚轴。完成这些研究将有助于深入了解Bucky ball如何调节Balbiani小体的形成和卵母细胞的不对称。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，据预测，这一特征将在生育和某些脊椎动物胚轴的建立中发挥基础性作用。 公共卫生相关性：在合子基因组激活之前，脊椎动物的发育取决于母体提供的因素。然而，许多母性驱动的过程背后的基本成分和分子机制的一致性尚不清楚。破坏严格的母系效应基因的突变是可行的。由于母亲提供的母性功能，突变的雌性动物明显是正常的。然而，无论它们的基因型如何，它们的后代都表现出突变表型。尽管母性产物对脊椎动物的发育至关重要，但在大量具有母性表达的脊椎动物基因中，只有一小部分通过基因或干扰技术进行了实验评估。在每一种情况下，母亲的贡献不足都会导致早期胚胎停滞或严重的发育异常。人类中类似的基因缺陷预计会导致植入失败或在怀孕被检测到之前流产。已知的怀孕中有10%到20%会导致流产；然而，根据Dimes杂志、美国妇产科医生学会、梅奥诊所和美国国家儿童健康和发展研究所的数据，如果与未被发现的怀孕结合在一起，以流产告终的实际怀孕百分比估计高达所有怀孕的40%到50%。我们的研究目标是阐明建立第一个胚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，其产物需要指定第一个胚胎轴的基因功能突变的丧失预计会导致流产。对斑马鱼轴形成的遗传和分子控制的研究将阐明动物-植物轴形成的遗传基础，可能阐明人类出生缺陷和原因不明的早期流产的遗传基础。

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.