Deconstructing the hypothalmic ontogeny and plasticity via clonal analysis

 描述(申请人提供)：哺乳动物的大脑功能在胚胎发育过程中关键依赖于复杂的细胞结构组织。细胞的产生、迁移、突触形成和电路整合通常遵循高度刻板的模式，在大脑中形成复杂的层状或核结构。最近发展起来的克隆谱系分析揭示了干细胞的行为导致了层状结构，如大脑皮层、小脑和视网膜，具有前所未有的单细胞分辨率。然而，神经干细胞(NSCs)形成核结构的机制尚不清楚，尚未进行系统的研究。哺乳动物下丘脑是一个异质的核结构，对内分泌、自主神经和行为功能的整合和动态平衡的维持至关重要。重建下丘脑神经元是如何在发育早期由单个神经干细胞产生并组织成离散的核的，对于了解不同下丘脑核的结构-功能关系以及环境条件对这种关系的调节程度是至关重要的。我们开发了一种基于遗传的单细胞谱系追踪技术，该技术使用MADM(马赛克双标记分析)动物来标记发育中的胚胎中的神经干细胞，并开始解决这些突出的下丘脑组织问题。这项研究的目标是在体内重建和量化单独标记的神经干细胞的行为，破译组成下丘脑核团的一般原理，破译单个下丘脑核团的个体发育，并在母体挑战的背景下探索核组织的个体发育可塑性。到目前为止，下丘脑的解剖和分子细分的复杂性，以及缺乏适当的遗传工具，阻碍了对这种核结构的组织和个体发育的深入了解。我们的研究的成功完成将产生单个神经干细胞及其后代在区域、区带和核水平的全面图谱，在三维环境中兄弟神经元的克隆组织以确定新生神经元的迁移模式，以及单个干细胞对功能不同的核的贡献能力。我们还将验证一个实验平台，用于未来病理条件下下丘脑失调的机制研究，这可以导致有针对性的诊断和治疗策略，以保护关键的生理功能。

 DESCRIPTION (provided by applicant): Mammalian brain function critically relies on sophisticated cytoarchitectonic organization during embryonic development. Cell generation, migration, synapse formation and circuit integration often follow highly stereotyped patterns to form complex laminated or nuclear structures in the brain. Recently developed clonal lineage analysis has revealed stem cell behavior giving rise to laminar structures, such as the cerebral cortex, cerebellum and retina, with unprecedented single-cell resolution. However, the formation of nuclear structures by neural stem cells (NSCs) remains unclear and has yet to be systematically investigated. The mammalian hypothalamus is a heterogeneous nuclear structure that is critical for the integration and homeostatic maintenance of endocrine, autonomic and behavioral functions. Reconstructing how hypothalamic neurons are generated from individual NSCs and organized into discrete nuclei during early development is essential to understand the structure-function relationship of different hypothalamic nuclei and the extent to which this can be modulated by environmental conditions. We have developed a genetically-based single-cell lineage tracing-technique that employs MADM (mosaic analysis of double marker) animals to label NSCs in the developing embryo and begin to address these outstanding questions of hypothalamic organization. The goal of the proposed research is to reconstruct and quantify the behavior of individually-labeled NSCs in vivo, decipher the general principles organizing hypothalamic nuclei, decode the ontogeny of individual hypothalamic nuclei and explore the ontogenetic plasticity of nuclear organization in the context of a maternal challenge. The complexity of the anatomical and molecular subdivisions of the hypothalamus, and lack of appropriate genetic tools, has thus far prevented a deep understanding of the organization and ontogeny of this nuclear structure. Successful completion of our study will result in a comprehensive map of single NSCs and their progeny at regional, zonal and nuclear levels, the clonal organization of sibling neurons in a three-dimensional context to determine migratory patterns of newly born neurons, and the capacity of single stem cells to contribute to functionally distinct nuclei. We will also have validated an experimental platform for future mechanistic investigations of hypothalamic dysregulation under pathological conditions, which can lead to targeted diagnostic and therapeutic strategies to preserve critical physiological functions.