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干细胞在神经管闭合时间（E8.5-E10.5）周围的早期脑发育阶段的行为。在这些早期阶段，神经干的快速变化和增长将产生成人大脑中的所有神经元和神经胶质细胞，但相对较少的研究存在内在的基因程序或外在的基于液体的信号参与驱动这些早期阶段的发展，主要是由于技术限制。我们和其他人已经证明了CSF和血管液小生境在指导大脑发育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过程中，对AF和早期CSF的蛋白质组成几乎一无所知。我们的首要假设是，AF和新生CSF中的分泌信号在指导和同步沐浴在这些液体中的胚胎E8.5-E10.5神经干细胞的增殖和命运方面至关重要。我们将首先通过比较AF、早期CSF和缓冲培养基对E8.5和E10.5年龄的干细胞外植体的影响来检验这一假设。我们预测，在以后的发展，早期外植体只有正常发展时，沐浴在年龄适当的液体，这表明AF/CSF蛋白质组的变化反映了相关受体在神经干细胞的顶端表面的表达的变化。因此，我们预计正常干细胞功能应受到神经干细胞顶面纤毛和细胞膜遗传扰动的损害，该顶面与AF/CSF直接接触。然后将使用定量质谱和RNAseq技术以及免疫染色来推导CSF蛋白及其在顶膜上的相关受体的接近完整的列表。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贡献，以及这些CSF信号的胚胎来源。这一提议具有重要的临床意义：我们目前对早期发育障碍（由于神经管错误闭合、脑积水和致畸剂渗透造成的缺陷）的起源了解相对较少，并且早期诊断或干预的能力也很有限。这项建议应该为询问这些早期扰动最终如何破坏正常大脑发育的某些或所有方面提供基础，并为开发用于疾病诊断的微创AF/CSF采样和替代策略以及神经干细胞的重编程提供基础，以便使发育大脑回到正轨。

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.