RUI: Stem Cell Genesis in Leech

一个单细胞，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。因为所有的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，特别是它们在发育过程中使用的基因网络。然而，尽管动物拥有一套惊人的保守的发育基因，但在形态上它们彼此之间表现得非常不同（例如，将海星与人类进行比较）。为了解决这一差异，我们将重点放在一种尚未得到充分研究但实验上很优雅的生物上，水蛭，它提供了一个独特的系统来解剖胚胎发生关键早期阶段的基因表达模式。具体来说，水蛭胚胎是非典型的大，并且以不对称的方式分裂，因此胚胎干细胞及其前体细胞可以很容易地识别并从胚胎中物理分离出来，这是目前在更传统的模式生物（例如，小鼠，苍蝇，线虫）中不可能实现的技术。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、规范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目标基因来直接测试，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的，从而产生我们目前观察到的动物生命的显著多样性。在传统的模式生物中，全能性受精卵可再生地产生具有不同程度的更受限制的细胞命运潜力的适当位置的细胞（包括干细胞）的时空精度在很大程度上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的现代方法。具体来说，舌音水蛭提供了实验上可处理的胚胎，其定型裂解产生五对双边大的、可识别的、可接近的、谱系受限的四种不同效价的干细胞，从中产生节段性中胚层和外胚层。因此，人们可以在每个发育阶段，从受精卵到功能干细胞，从少量可识别的细胞中定期收集方便数量的RNA。该项目将生成一组独特定义的干细胞、非干细胞和水蛭受精卵中产生的选定前体的转录谱。Illumina测序和经过验证的生物信息学管道将用于深度组装从受精卵到四种不同类型的谱系限制干细胞（M、N、O/P和Q端母细胞）形成阶段的转录组。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来描述导致干细胞形成的转录动力学。由于水蛭代表了一种未被充分研究但在进化上具有战略意义的巨噬动物超门成员，这项工作将为分子遗传途径的范围提供一个关键的视角，这些途径导致我们在操作上认识到干细胞。这些实验是专门为许多本科生提供有意义的训练活动而设计的。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.