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

 描述(申请人提供)：寄生扁虫(绦虫和吸虫)感染人和牲畜，导致各种疾病，病理范围从良性到致命。这些寄生虫具有复杂的生活史和非凡的生长、繁殖、寿命和修复/再生能力。尽管寄生扁虫的生命周期复杂多变，但它们有共同的特征：或许最值得注意的是，它们拥有多能的成体干细胞。来自自由生活扁虫的研究表明，这些干细胞(称为新生细胞)对正常组织的动态平衡、有性繁殖和再生能力至关重要。细胞学研究表明，寄生的扁虫维持着新胚细胞，最近的分子研究表明，在扁虫之间存在保守的共享的新胚细胞调控基因。我们的长期目标是确定新的治疗靶点，以治疗具有治疗多种寄生虫疾病的潜力的新生母细胞。我们目前的目标是鉴定和验证寄生扁虫中保守的、对寄生虫生长和/或繁殖所必需的新生细胞调控基因。我们已经确定了一个合适的实验室模型(大鼠肠道绦虫)，它可以高通量地进行基因发现和功能验证。我们假设存在寄生虫保守的新生细胞调控基因，这些基因对寄生虫的生长和/或繁殖至关重要。为了解决这一假设，我们提出了以下两个具体目标：(1)鉴定在寄生虫中保守并在新生细胞中表达的基因；(2)从功能上筛选生长和/或繁殖所需的寄生虫新生细胞的调节因子。新生细胞丰富的基因将通过对正常和新胚细胞耗尽的微小毛虫进行RNA测序比较来识别。候选基因将通过原位杂交来验证在新生细胞中的共表达。利用可公开获得的感染人类和家畜的扁虫基因组和转录组组合，我们将汇编一份寄生虫特异的、新胚细胞丰富的候选基因清单。为了进行功能验证，我们将利用我们的能力在体外培养微小毛藻。我们已经成功地建立了体外再生试验和筛选程序，以监测绦虫的生长发育到生殖成熟。我们将建立与体外再生实验相结合的方法来抑制来自Aim 1的候选基因的表达，以确定寄生虫生长和繁殖所需的基因。这些研究是创新的，因为我们设计了一种高通量和无偏见的方法来发现并从功能上验证寄生虫特异性基因作为寄生虫健康调节器的作用，以H.minuta为模型。拟议的工作意义重大，因为我们将确定具有广泛疗效的高置信度目标，以对抗多种寄生虫物种。我们的研究将共同填补在以下领域的重要空白 寄生扁虫的功能基因组研究，并有可能确定新的治疗靶点。

 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.