Single Centriole-Mediated Gynogenesis in the Zebrafish

描述（由研究者提供）：单性生殖，从单个生物体产生可存活二倍体后代的能力，允许单个个体中以杂合形式存在的等位基因直接纯合（在单个世代中）。这潜在地为斑马鱼模型提供了类似于基于雌雄同体的C。elegans遗传学这种能力将是特别有用的成人性状的生物医学相关性，这是很难的方法，使用纯粹基于自然杂交的正向遗传学分析。目前的雌核发育方法似乎受到这样一个事实的限制，即它们涉及抑制细胞分裂的物理治疗，这只是部分有效，也导致大部分发育异常。该提案的目的是开发一种有效的基于遗传的雌核发育方法（从女性单性生殖）。我们发现，父亲的中心粒生物发生基因cellular atoll/sas-6（cea）功能障碍导致第一次胚胎细胞分裂的一个周期延迟，这可能是因为精子提供了一个单一的中心粒，而不是正常的两个中心粒。第一次细胞分裂延迟导致基因组的精确复制。当与单倍体产生结合时，依赖cea的基因组复制促进雌核发育。基于单中心粒父本成分的倍性复制具有高效促进雌核发育的潜力，并且不存在胚胎综合征。这项研究的目的是优化这种单中心粒介导的雌核发育。我们将尝试通过多种方法。在目的1中，我们将测试各种遗传背景对父本cea表型的遗传率的影响。在目标2中，我们将使用F1遗传筛选来鉴定温度敏感性突变，这些突变要么增强父代效应cea表型，要么自身引起类似的第一次细胞分裂延迟。在目标3中，我们将联合收割机结合我们从以前的目标中获得的发现，以高效率实现单中心粒介导的雌核发育。这种组合方法旨在提供一种功能强大、易于应用和广泛可用的新方法，以促进斑马鱼中的正向遗传筛选和其他遗传操作。这项工作也将提供遗传切入点的途径参与精子发生和早期胚胎发生。 公共卫生相关性：这项研究将开发一种简单、有效和广泛使用的遗传工具包，以诱导斑马鱼的倍性操作，特别是雌核发育。使用遗传介导的雌核发育的能力将通过促进在单个个体中以杂合形式存在的等位基因的直接纯合来促进该生物体中的遗传分析。从本质上讲，有效的雌核发育应该为斑马鱼提供一种类似于雌雄同体为秀丽隐杆线虫模型系统提供的遗传能力。这项技术的应用将特别影响生物医学相关的成人特征的遗传分析。在斑马鱼的这一过程中获得的知识也可以应用于在其他脊椎动物模型系统中生成类似的方法。此外，我们的研究将确定参与精子发生和早期胚胎发育的基因。

DESCRIPTION (provided by investigator): Parthenogenesis, the ability to generate viable diploid offspring from a single organism, allows the direct homozygosis (in a single generation) of alleles present in the heterozygous form in a single individual. This potentially provides to the zebrafish model a power similar to that of the hermaphroditism-based C. elegans genetics. Such ability would be particularly useful for the analysis of adult traits of biomedical relevance, which are difficult to approach using forward genetics based solely on natural crosses. Current gynogenesis methods appear to be limited by the fact that they involve physical treatments to inhibit cell division, which are only partially effective and also result in a large fraction of developmental abnormalities. The objective of this proposal is to develop an efficient genetically-based method of gynogenesis (parthenogenesis from a female). We have found that paternal dysfunction in the centriolar biogenesis gene cellular atoll/sas-6 (cea) causes a one-cycle delay in the first embryonic cell division, presumably because the sperm provides a single centriole instead of the normal two-centriole component. This first cell division delay results in the exact duplication of the genome. When coupled to haploid production, cea-dependent genome duplication promotes gynogenetic development. Ploidy duplication based on a single-centriole paternal component has the potential to promote gynogenesis with a high efficiency and in the absence of embryonic syndromes. The objective of the proposed research is to optimize such Single Centriole-mediated Gynogenesis. We will attempt this through multiple approaches. In Aim 1 we will test the effect of various genetic backgrounds on the penetrance of the paternal cea phenotype. In Aim 2, we will use an F1 genetic screen to identify temperature-sensitive mutations that either enhance the paternal-effect cea phenotype or cause on their own a similar first cell division delay. In Aim 3, we will combine our findings from previous Aims to implement Single Centriole-mediated Gynogenesis with a high efficiency. This combined approach is intended to provide a powerful, easily-applicable and widely- available new method to facilitate forward genetic screens and other genetic manipulations in the zebrafish. This work will also provide genetic entry points into pathways involved in spermatogenesis and early embryogenesis. PUBLIC HEALTH RELEVANCE: The proposed studies will develop a simple, effective and widely-available genetic tool kit to induce ploidy manipulation, specifically gynogenesis, in the zebrafish. The ability to use genetically-mediated gynogenesis will facilitate genetic analysis in this organism by promoting the direct homozygosis of alleles present in the heterozygous form in a single individual. In essence, effective gynogenesis should provide to the zebrafish a similar genetic power to that which hermaphroditism has provided to the Caenorhabditis elegans model system. The application of this technology will particularly impact the genetic analysis of adult traits of biomedical relevance. Knowledge gained on this process in the zebrafish could also be applied to generate similar methods in other vertebrate model systems. In addition, our studies will identify genes involved in spermatogenesis and early embryonic development.