Morphogenetic Role of Serotonin in Very Early Embryogenesis

了解发育模式的形成是细胞和进化生物学的基础；它将是生物医学的重要组成部分，通过实现对活组织结构/功能的精细控制来解决出生缺陷、癌症和器官/组织再生问题。后基因组时代的研究需要遗传学和分子生物学与体内功能生理学的综合。通过跨学科的分子和计算机建模技术，莱文实验室寻求详细了解通过调节基因活性和膜通量的生物物理来控制大规模图案形成的细胞信号。脊椎动物的身体平面具有两侧对称性；然而，胚胎也表现出明显的内脏和脑的左右不对称(LRA)，这在物种之间是高度保守的。为什么会存在不对称呢？非对称性对心脏、肠道和大脑的正常生理有何影响？为什么所有正常个体不仅不对称，而且朝着同一方向不对称？在进化论中，手不对称是什么时候出现的？它与植物和软体动物贝壳中的手性有关吗？脊椎动物胚胎在什么发育阶段开始不对称？大脑不对称对人类认知的影响是什么？而且，在没有任何区分左右的世界宏观特征的情况下，左右轴如何相对于另外两个轴一致地定向呢？量子宇称破坏与介观生物形状有关吗？这些问题有待在分子、遗传和生化水平上对胚胎偏向不对称性的详细了解，因为早期的LR步骤知之甚少。在探索LRA机制的过程中，发现了一个新的重要方面：5-羟色胺(5-HT)。5-羟色胺是中枢神经系统中调节心理动力功能的关键神经递质，与正常认知和诸如记忆障碍、睡眠障碍、精神分裂症、痴呆症、攻击性、抑郁症等症状有关。因此，5-羟色胺可能是意识状态的一种生化关联，可用于探索认知的物理基础。我们实验室的初步研究表明：(1)在卵裂期胚胎(即早于神经元形成之前)，5HT途径的一些成员以动态的方式存在于卵裂期胚胎中，其中一些成员表现出一致的LRA；2)通过药理学实验，5HT信号在LRA的早期步骤中具有功能意义；以及3)胚胎的L和R侧对细胞内5-羟色胺表现出不同的反应。因此，5-羟色胺通路是进入LR模式早期步骤的一个很有前途的全新的切入点。这项拟议的研究将寻求了解早期胚胎中5-羟色胺能信号的极其基本的特性，方法是利用非洲爪哇胚胎相对于啮齿动物和人类系统的许多优势进行这些研究，并在神经生物学领域进行合作。其目的是：(1)在胚胎和亚细胞水平上表征所有与LR相关的5HT通路成员在早期发育的每个阶段的定位；(2)准确地确定LR模式中的哪些胚胎阶段利用5HT信号；(3)表征一个新的功能性5HT受体的细胞质位置；以及(4)利用已知的LR模式的早期步骤来测试5HT信号作用的两个特定的分子模型。智力优势和更广泛的影响：一个直接的好处将是在已知的LR图案化步骤的背景下，描述胚胎左右不对称的新机制。重要的是，由于这项工作在受精后2小时内确定了LR不对称的标记，这表明胚胎知道从左到右的时间比迄今在现场怀疑的时间要早得多，并提供了直接攻击难以捉摸的“第一步”的试剂。也许最有趣的是这项跨学科工作的众多长期、更广泛的影响。将开发和引入用于体内小分子通量功能发育研究的技术。这些新的胚胎学技术和方法将直接教授给我们实验室的研究生和博士后，并将发表在科学会议上，以帮助向科学界传播有价值的想法，这些想法可以形成许多实验室未来新工作的基础。特别令人兴奋的是，这种神经递质的早期作用可能表明，5-羟色胺能突触是更原始的细胞：细胞信号事件的进化产物，这将对进化发育生物学和神经生物学产生有趣的影响。这提供了一个现成的背景，在其中培训未来的科学家以跨学科的方法解决生物学问题。因此，这些研究将启动对信号分子新角色的理解，该信号分子以其在成人组织中的生理相关性而闻名。这些信息将有助于揭示神经科学、胚胎学、进化以及怀孕期间流行的药物5-羟色胺调节药物的安全性等非常基本的问题，从而造福社会。

0234388LevinUnderstanding developmental pattern formation is fundamental to cell and evolutionary biology; it will be a vital part of biomedicine addressing birth defects, cancer, & organ/tissue regeneration by achieving fine-scale control over the structure/function of living tissues. Research in the post-genomic age requires a synthesis of genetics and molecular biology with functional physiology in vivo. Through interdisciplinary molecular and computer modeling techniques, the Levin lab seeks a detailed understanding of cellular signals that control large-scale pattern formation by modulation of gene activity and the biophysics of membrane flux. The vertebrate body-plan possesses bilateral symmetry; however, embryos also exhibit a striking left-right asymmetry (LRA) of the viscera and brain which is highly conserved across species. Why does asymmetry exist at all? What are the implications of asymmetry for the normal physiology of the heart, gut, and brain? Why are all normal individuals not only asymmetric, but asymmetric to the same direction? When, in evolution, did handed asymmetry appear? Is it connected to chirality in plants and mollusc shells? At what developmental stages is asymmetry initiated in vertebrate embryos? What are the implications of brain asymmetry for human cognition? And, how can the left-right axis be consistently oriented with respect to the other two axes in the absence of any macroscopic feature of the world which distinguishes left from right? Are quantum parity violations relevant to mesoscopic biological shape? These issues await a detailed understanding, at the molecular, genetic, and biochemical levels, of biased asymmetry in embryos since early LR steps are poorly understood. In pursuing the mechanisms of LRA, the involvement of a novel and important aspect was discovered: serotonin (5HT). 5HT is a key neurotransmitter in the central nervous system that regulates psychodynamic function and is directly implicated in both normal cognition and syndromes such as memory impairment, sleep disorders, schizophrenia, dementia, aggression, depression, etc. Thus serotonin is, potentially, a biochemical correlate of conscious states which can be used to probe the physical basis of cognition. Preliminary studies in our lab utilized the frog embryo (Xenopus) to show that (1) some members of the 5HT pathway are present in the cleavage-stage embryo (i.e., long prior to the formation of neurons) in dynamic patterns some of which display a consistent LRA, 2) 5HT signaling is functionally implicated in early steps of LRA by pharmacological experiments, and 3) the L and R sides of the embryo show differential responses to intracellular serotonin. Thus, the 5HT pathway is a promising and completely novel entrypoint into early steps of LR patterning. The proposed research will seek to understand extremely basic properties of serotonergic signaling in early embryos by capitalizing on the many advantages of Xenopus embryos over rodent and human systems for these studies, and collaborations in the neurobiology community. The aims are: (1) characterize at the embryonic and subcellular level, the localization of all LR-relevant 5HT pathway members at each stage of early development, (2) determine precisely which embryonic stages in LR patterning utilize 5HT signaling, (3) characterize a novel cytoplasmic site of functional 5HT receptors, and (4) test two specific molecular models of the role of 5HT signaling with known early steps in LR patterning. Intellectual merit and broader impacts: A direct benefit will be the characterization of a novel mechanism in embryonic left-right asymmetry, in the context of known steps in LR patterning. Importantly, since this work identifies a marker of LR asymmetry within 2 hours of fertilization, it shows that embryos know left from right far earlier than heretofore suspected in the field, as well as providing reagents which set up a direct attack on the elusive "step 1". Perhaps the most interesting are the numerous long-term, wider impacts of this interdisciplinary work. Techniques will be developed and introduced for functional developmental studies of small molecule flux in vivo. These novel embryological techniques and approaches will be taught directly to graduate students and post-docs in our lab, as well as be published and presented at scientific meetings to help disseminate valuable ideas to the scientific community which can form the basis of new future work in many labs. Particularly exciting is the fact that an early role for this neurotransmitter may suggest that serotonergic synapses are an evolutionary co-opting of far more primitive cell:cell signaling events, which will have interesting implications for evolutionary developmental biology as well as neurobiology. This provides a ready context within which to train future scientists in interdisciplinary approaches to biological problems. These studies will thus initiate an understanding of a new role for a signaling molecule well-known for its physiological relevance in adult tissue. This information will benefit society by shedding light on very basic problems of neuroscience, embryology, evolution, and the safety of pharmaceutically popular 5HT- modulating drugs during pregnancy.