Stem cell and regeneration regulatory genes in planarians

描述（由申请人提供）：涡虫以其从动物的微小碎片迅速再生新头部甚至整个生物体的能力而闻名。涡虫再生涉及一群增殖细胞（新生细胞），其中包括能产生成年动物所有细胞的多能成体干细胞（新生细胞）。尽管几个世纪以来人们一直对再生着迷，但机械的解释仍有待阐明。本研究的长远目标是利用涡虫作为一个模型系统来识别和理解调节干细胞促进再生的分子机制。具体目的是：1)确定新母细胞调节基因的功能，2)确定再生的细胞基础，3)确定激活新母细胞再生反应的伤口诱导机制。干细胞和再生生物学是近年来再生医学研究的热点。此外，干细胞的失调可能是许多类型癌症的核心。现在存在一种新开发的用于涡虫分子遗传学研究的工具库。例如，用RNA干扰（RNAi）进行系统的基因扰动现在是可能的，涡虫基因组已经测序。超过一半的涡虫基因在人类基因组中有对应的基因；因此，涡虫研究应该确定保守的干细胞调控基因。目的1将利用RNAi、流式细胞术和定量mRNA测序（RNA-seq）确定转录因子SoxP-1在调节新细胞基因表达中的作用。新生细胞可以产生后代细胞的菌落，包括新生细胞和跨越胚层的分化细胞。目的1将利用cNeoblast菌落的特征来描述在新母细胞群体中表达丰富的基因的作用。Aim #2将利用RNA测序和原位杂交来识别cNeoblast的分子特征，这些特征将其与谱系承诺的后代细胞区分开来。Aim #2还将利用新母细胞分离和移植来开发新母细胞遗传操作工具。目标3将确定器官前体对移除目标组织的损伤的反应。具体来说，将眼睛部分或完全受伤，以研究眼部前体是否由眼部小损伤诱导，并根据损伤类型定制适当数量。最后，我们将利用RNAi和细胞实验来确定损伤后诱导在新细胞中表达的基因的作用，包括两个编码保守runx家族转录因子的基因。上述目标的成功完成将极大地促进我们对再生机制基础的理解，并将涡虫作为研究人类干细胞和再生生物学中人类保守基因的模式系统，这些领域对人类健康至关重要。

DESCRIPTION (provided by applicant): Planarian flatworms are famous for their ability to rapidly regenerate new heads or even entire organisms from a tiny fragment of the animal. Planarian regeneration involves a population of proliferative cells (neoblasts) that include pluripotent adult stem cells (cNeoblasts) that can produce every cell of the adult animal. Despite centuries of fascination with regeneration, mechanistic explanations await elucidation. The broad, long-term objectives of this proposal are to use planarians as a model system to identify and understand the molecular mechanisms that regulate stem cells to promote regeneration. The specific aims are: 1) to determine the functions of neoblast regulatory genes, 2) to identify the cellular basis for regeneration, and 3) to identify wound-induced mechanisms that activate neoblast regenerative responses. Stem cells and regenerative biology are the subjects of recent and intense interest for regenerative medicine. In addition, the misregulation of stem cells may be central to many types of cancer. A newly developed arsenal of tools for molecular genetic study of planarians now exists. For example, systematic gene perturbation with RNA interference (RNAi) is now possible and the planarian genome has been sequenced. Greater than half of planarian genes have counterparts in the human genome; therefore, planarian studies should identify conserved stem cell regulatory genes. Aim #1 will determine the role of the transcription factor SoxP-1 in regulating gene expression in the neoblasts, utilizing RNAi, flow cytometry, and quantitative mRNA sequencing (RNA-seq). cNeoblasts can produce colonies of descendant cells including neoblasts and differentiating cells spanning germ layers. Aim #1 will utilize features of cNeoblast colonies to characterize roles for genes with enriched expression in the neoblast population. Aim #2 will utilize RNA seq and in situ hybridizations to identify the molecular features of the cNeoblast that distinguish it from lineage-committed descendant cells. Aim #2 will also utilize cNeoblast isolation and transplantation to develop neoblast genetic manipulation tools. Aim #3 will determine how organ precursors respond to injuries that remove target tissues. Specifically, eyes will be injured partially or completely to investigate whether eye precursors are induced by small eye injuries and in appropriate numbers tailored to the injury type. Finally, the roles of genes induced to be expressed in neoblasts following injury, including two genes encoding conserved Runx-family transcription factors, will be determined using RNAi and cellular assays for neoblast response to wounds. Successful completion of proposed aims will greatly advance our understanding of the mechanistic basis for regeneration and advance planarians as a model system for the study of genes conserved in humans in stem cell and regenerative biology, areas of great importance in human health.