RUI: Stem Cell Genesis in Leech

单细胞（如受精卵）发育成复杂的多细胞动物的过程可能是现代生物学中最令人着迷的问题之一。因为所有动物都有共同的祖先，所以它们发育的许多方面都是保守的，特别是关于它们在发育过程中使用的基因网络。尽管如此，动物在形态上彼此非常不同（例如，比较海星和人类），尽管它们有一组非常保守的发育基因。为了解决这一差距，我们专注于一个研究不足，但实验优雅的生物体，水蛭，它提供了一个独特的系统解剖基因表达模式在胚胎发生的关键早期阶段。具体地，水蛭胚胎是巨大的，并且以不对称的模式分裂，使得胚胎干细胞及其前体细胞可以容易地被识别并与胚胎物理分离，这是目前在更常规的模式生物中不可能的技术（例如，小鼠、苍蝇、线虫）。利用现代分子和基因组学方法，我们将重建胚胎内发生的导致水蛭胚胎干细胞形成、特化和维持的详细分子事件。我们的假设将通过在发育过程中操纵靶基因来直接测试，利用水蛭胚胎作为实验平台。这些信息将有助于更广泛地了解祖先遗传途径是如何进化成我们目前观察到的动物生命的显著多样性的。全能合子可再生地产生具有不同程度的更受限制的细胞命运潜能的适当定位的细胞（包括干细胞）的时空精度在常规模式生物中仍然是一个站不住脚的问题。该项目通过将生物信息学和分子技术应用于一个非常适合这种方法的经典系统，为这个问题提供了一种替代和现代的方法。具体而言，glossiphoniid水蛭提供实验上听话的胚胎，其定型分裂产生五个双边对大的，可识别的和可访问的，谱系限制的干细胞的四个不同的效力，从节段性中胚层和外胚层出现。因此，在每个发育阶段，从受精卵到功能性干细胞，人们可以常规地从少量可识别的细胞中收集方便数量的RNA。该项目将产生一组独特定义的干细胞，非干细胞和选定的前体细胞，它们在水蛭受精卵中产生的转录谱。Illumina测序和经过验证的生物信息学管道将用于组装从受精卵到形成四种不同类型的谱系限制性干细胞（M，N，O/P和Q端细胞）的阶段的深度转录组。再加上全基因组序列的可用性，这提供了一个机会，以前所未有的细节和完整性，在逐个细胞的基础上分析导致干细胞形成的转录动力学。由于水蛭是超门Lophotrochozoa的一个未被充分研究但在进化上具有战略意义的成员，这项工作将提供一个关键的视角，探讨导致我们在操作上识别为干细胞的分子遗传途径的范围。 该奖项反映了NSF的法定使命，并被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

The process by which a single cell, such as a fertilized egg, develops into a complex, multicellular animal is perhaps one of the most fascinating questions in modern biology. Because all animals share a common ancestry many aspects of their development are conserved, particularly with regard to the network of genes they employ during the developmental process. Nonetheless, animals appear morphologically very different from each other (e.g., compare a starfish with a human) despite having a strikingly conserved set of developmental genes. To address this disparity, we focus on an understudied yet experimentally elegant organism, the leech, which offers a unique system for dissecting gene expression patterns in the critical early stages of embryogenesis. Specifically, leech embryos are atypically large and divide in asymmetrical patterns such that embryonic stem cells and their precursor cells can be readily identified and physically separated from the embryo, a technique that is not currently possible in more conventional model organisms (e.g., mouse, fly, nematode). Using modern molecular and genomics approaches, we will reconstruct the detailed molecular events that occur inside the embryo that lead to the formation, specification and maintenance of embryonic stem cells in leech. Our hypotheses will be tested directly by manipulating target genes during development, utilizing leech embryos as an experimental platform. This information will contribute to a broader understanding of how ancestral genetic pathways have evolved to generate the remarkable diversity of animal life we currently observe. The spatiotemporal precision by which totipotent zygotes reproducibly generate properly situated cells (including stem cells) with varying degrees of more restricted cell fate potentials remains mostly an untenable question in conventional model organisms. The project offers an alternative and modern approach to this problem by applying bioinformatic and molecular techniques to a classical system that is uniquely well-suited for such methodologies. Specifically, glossiphoniid leeches provide experimentally tractable embryos whose stereotyped cleavages yield five bilateral pairs of large, identifiable and accessible, lineage-restricted stem cells of four distinct potencies, from which segmental mesoderm and ectoderm arise. Thus, one can routinely collect convenient quantities of RNA from small numbers of identifiable cells at each developmental step, from the zygote up to functioning stem cells. This project will generate transcriptional profiles for a set of uniquely defined stem cells, non-stem cells and selected precursors from which they arise in the leech zygote. Illumina sequencing and proven bioinformatic pipelines will be used to assemble in-depth transcriptomes for stages leading from zygote through the formation of four distinct classes of lineage-restricted stem cells (the M, N, O/P and Q teloblasts). Coupled with the availability of whole genome sequence, this presents an opportunity to profile transcriptional dynamics leading to stem cell formation on a cell-by-cell basis with unprecedented detail and completeness. Since the leech represents an understudied yet evolutionarily strategic member of the super-phylum Lophotrochozoa, this work will provide a critical perspective on the range of molecular genetic pathways leading to what we recognize operationally as stem cells. The experiments are specifically designed to provide meaningful training activities to many undergraduate students.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.