Control of neural stem cells by the nascent cerebrospinal fluid

描述(由申请人提供)：我们建议研究羊水(AF)和新生脑脊液(CSF)中的信号如何指导神经管关闭前后早期大脑发育阶段的神经干细胞行为(E8.5-E10.5)。在这些早期阶段，迅速变化和增长的神经干种群将在成人大脑中形成所有神经元和神经胶质细胞，但相对较少的研究涉及推动这些早期发育阶段的内在基因ETC程序或外部基于液体的信号，这主要是由于技术限制。我们和其他人已经展示了脑脊液和血管液体生态位在指导大脑发育的后期阶段的广泛影响。然而，在脉络丛和血管系统尚未形成的早期大脑发育期间，关于房颤和早期脑脊液的蛋白质组成几乎一无所知。我们的主要假设是，房颤和新生脑脊液中的分泌信号对于指导和同步浸泡在这些液体中的胚胎E8.5-E10.5神经干细胞的增殖和命运至关重要。我们将首先通过比较AF、早期脑脊液和缓冲介质对E8.5和E10.5岁的干细胞外植体的影响来检验这一假设。我们预测，就像在发育后期一样，早期外植体只有在沐浴在与年龄相适应的液体中才能正常发育，这表明AF/CSF蛋白质组的变化反映了神经干细胞顶端表面相关受体表达的变化。因此，我们预计正常的干细胞功能应该受到与AF/CSF直接接触的神经干细胞顶端表面纤毛和膜的遗传扰动的影响。然后将使用定量质谱仪和RNAseq技术，结合免疫染色，推断出几乎完整的脑脊液蛋白及其在根尖膜上的相关受体的清单。然后，我们将确定CSF-LIF和其他顶级候选蛋白在指导E10.5干细胞行为的特定方面的作用，以及这些CSF信号的胚胎来源。这一建议具有重要的临床意义：目前，我们对早期发育障碍(由于错误的神经管闭合、脑积水和畸形物渗透造成的缺陷)的起源了解相对较少，缺乏早期诊断或干预的能力。这一建议应该为研究这些早期的扰动最终如何使正常脑发育的某些或全部方面脱轨，以及为疾病诊断和神经干细胞的重新编程开发微创的AF/CSF采样和替换策略，以便使发育的大脑回到正轨提供基础。

DESCRIPTION (provided by applicant): We propose to investigate how signals in the amniotic fluid (AF) and nascent cerebrospinal fluid (CSF) instruct neural stem cell behavior during early brain developmental stages surrounding the time of neural tube closure (E8.5-E10.5). The rapidly changing and growing population of neural stems at these early stages will give rise to all neurons and glia in the adult brain, yet comparatively few studies exist on the intrinsic genetc programs or the extrinsic fluid-based signals involved in driving these early stages of development, largely due to technical limitations. We and others have demonstrated broad influences of CSF and vascular fluid niches in instructing later stages of brain development. Yet virtually nothing is known about the protein composition of AF and early CSF during early brain development, at a time when the choroid plexus and vasculature have yet to form. Our overarching hypothesis is that secreted signals in the AF and nascent CSF are critical in instructing and synchronizing the proliferation and fate of embryonic E8.5-E10.5 neural stem cells bathed in these fluids. We will first test this hypothesis by comparing the effects of AF, early CSF and buffered media on stem cell explants at ages E8.5 and E10.5. We predict that, as in later development, early explants only develop normally when bathed in the age-appropriate fluid, suggesting that changes in the AF/CSF proteome are mirrored by changes in expression of associated receptors at the apical surface of neural stem cells. As such, we expect that normal stem cell function should be impaired by genetic perturbations to the cilia and membrane of neural stems cells at their apical surface, which is in direct contact with the AF/CSF. A near- complete list of CSF proteins and their associated receptors on apical membranes will then be deduced using quantitative mass-spec and RNAseq technologies, together with immunostaining. We will then determine the contributions of CSF-LIF and other top candidate proteins in instructing specific aspects of E10.5 stem cell behavior, and the embryonic sources of these CSF signals. This proposal has important clinical significance: We currently have relatively little understanding of the origins of early developmental disorders (defects due to errant neural tube closure, hydrocephalus, and infiltration by teratogens), and scant capacities for early diagnosis or intervention. This proposal should provide a foundation for asking how these early perturbations ultimately derail some or all aspects of normal brain development, and for developing minimally invasive AF/CSF sampling and replacement strategies for disease diagnosis and for reprogramming of neural stem cells in order to bring the development brain back on track.