Hymenolepis diminuta as a model for studying stem cells in parasitic flatworms

 描述（由申请人提供）：寄生扁虫（绦虫和吸虫）感染人类和牲畜，引起各种疾病，病理从良性到致命。这些寄生虫具有复杂的生命周期和显著的生长、繁殖、长寿和修复/再生能力。尽管它们的生命周期复杂多变，但寄生扁虫有着共同的特征：也许最值得注意的是，它们拥有多能成体干细胞。对自由生活的扁形虫的研究表明，这些干细胞（称为新生细胞）对正常组织的稳态、有性生殖和再生能力至关重要。细胞学研究表明，寄生扁虫保持neoblast，最近的分子研究表明，有共同的neoblast调控基因保守的扁虫。我们的长期目标是确定新的治疗靶点，这些靶点有可能治疗多种寄生性扁形虫病。我们目前的目标是确定和验证neoblast调节基因是保守的寄生扁形虫和寄生虫的生长和/或繁殖所必需的。我们已经确定了一个合适的实验室模型（大鼠肠道绦虫，Hymenolepis diminuta），适合基因发现和功能验证的高通量的方式。我们假设，有寄生虫保守的neoblast调节基因的寄生虫的生长和/或繁殖的关键。为了解决这一假设，我们提出了以下两个具体目标：（1）鉴定寄生扁虫中保守的基因，并在neoblasts中表达;（2）功能性筛选生长和/或繁殖所需的寄生neoblasts的调节因子。将通过RNA测序比较正常和新胚细胞缺失的H.减少。将通过原位杂交验证候选基因在新成细胞中的共表达。利用可获得的感染人类和牲畜的扁形虫的基因组和转录组组装，我们将编制寄生虫特异性的新胚富集基因的候选列表。对于功能验证，我们将利用我们培养H.体外缺陷。我们已经成功地建立了体外再生试验和筛选程序，以监测绦虫的生长和发育到生殖成熟。我们将建立方法，敲低目标1的候选基因的表达，结合我们的体外再生试验，以确定寄生虫生长和繁殖所需的基因。这些研究具有创新性，因为我们设计了一种高通量和公正的方法，利用H. diminuta作为一个模型。拟议的工作是重要的，因为我们将确定高置信度的目标，对多种寄生虫物种的广泛疗效的潜力。总之，我们的研究将填补重要的空白， 寄生扁虫的功能基因组研究，并有可能确定新的治疗目标。

 DESCRIPTION (provided by applicant): Parasitic flatworms (tapeworms and flukes) infect humans and livestock, causing a variety of diseases with pathologies that range from benign to deadly. These parasites have complex life cycles and remarkable capacity for growth, reproduction, longevity, and repair/regeneration. Despite their complexity and variations in their life cycles, parasitic flatworms share common features: perhaps most notably, they possess pluripotent adult somatic stem cells. Studies from free-living flatworms have shown that these stem cells (called neoblasts) are critical for normal tissue homeostasis, sexual reproduction, and regenerative capacity. Cytological studies revealed that parasitic flatworms maintain neoblasts, and recent molecular studies indicate that there are shared neoblast regulatory genes conserved among flatworms. Our long-term goal is to identify novel therapeutic targets against neoblasts that have the potential to treat multiple parasitic flatworm diseases. Our current objective is to identify and validate neoblast regulatory genes that are conserved among parasitic flatworms and that are necessary for parasite growth and/or reproduction. We have identified a suitable laboratory model (the rat intestinal tapeworm, Hymenolepis diminuta) that is amenable to gene discovery and functional validation in a high-throughput manner. We hypothesize that there are parasite-conserved neoblast regulatory genes critical for parasite growth and/or reproduction. To address this hypothesis, we propose the following two specific aims: (1) to identify genes that are conserved among parasitic flatworms and expressed in neoblasts; and (2) to functionally screen for regulators of parasite neoblasts that are required fo growth and/or reproduction. Neoblast-enriched genes will be identified using RNA-sequencing comparison of normal and neoblast-depleted H. diminuta. Candidate genes will be validated for co-expression in neoblasts by in situ hybridization. Using publically available genome and transcriptome assemblies of flatworms that infect humans and livestock, we will compile a candidate list of parasite-specific, neoblast-enriched genes. For functional validation, we will take advantage of our ability to culture H. diminuta in vitro. We have successfully established an in vitro regeneration assay and screening procedure to monitor tapeworm growth and development to reproductive maturity. We will establish methods to knock down expression of candidate genes from Aim 1 coupled to our in vitro regeneration assay to identify genes that are required for parasite growth and reproduction. These studies are innovative because we have designed a high-throughput and unbiased approach to uncover and functionally validate parasite-specific genes as regulators of parasite health, using H. diminuta as a model. The proposed work is significant, as we will identify high-confidence targets with the potential for broad efficacy against multiple parasite species. Together, our studies will fill important gaps in functional genomic studies of parasitic flatworms and have the potential to identify new therapeutic targets.