Activity-dependent Regulation of the Choroid Plexus-Cerebrospinal Fluid Stem Cell Niche

项目摘要/摘要 构建大脑皮层需要跨神经的分子信号的时空协调 祖细胞。这些细胞中的每一个都沐浴在脑脊液(CSF)中，脑脊液是一种富含健康和生长的介质- 促进在大脑发育过程中其组成在几天内发生深刻变化的因素。脉络膜 神经丛(CHP)可能对脑脊液成分的这种动态发育调节有很大贡献，但 其潜在的机制尚不清楚。在这里，我们将使用一套现代工具来测试 假设胚胎CHP上皮细胞钙浓度升高是对 外在提示对于调节脑脊液信号因子的合成和分泌是至关重要的。钙 活性调节从神经元到唾液腺上皮等不同细胞的转录和分泌。我们 CHP上皮细胞中表达的介导钙离子进入细胞的受体，如TRPM3 受体，或驱动体内钙释放的受体，例如5-羟色胺(5-羟色胺)的5HT2C受体(Htr2C)。 HT，5-羟色胺)。我们的初步数据表明，5-羟色胺对胚胎CHP钙的调节 CHP的活性、转录和囊泡释放，以及通过5HT2C受体的完整信号是 对于正常的大脑皮层发育是必不可少的。此外，产妇分娩一种选择性激动剂用于治疗 5HT2C受体在胚胎CHP中触发快速、强劲的即刻早期基因表达。因此，我们建议 CHP钙活性是由激活5HT2C受体(和其他受体，如 AS TRPM3)调节CHP转录和信号因子分泌到脑脊液和对照 身体-大脑和母婴相互作用，从而紧密协调大脑皮层的发育。 为了验证这一点，我们将首先研究5HT2C和TRPM3受体激活的影响，通过直接应用 CHP外植体钙显影过程中上皮细胞顶端表面的配体(目标1a)和刺激 在活体内将配体(IP)输送到母体的母体后，基底面的变化 附着性胚胎的光子钙成像(目标1b)。然后，我们评估这样的转录影响 刺激(Aim 1c)，并测试胚胎CHP钙活性是否通过激活5HT2C受体和 其他受体在体外(Aim 2a)和体内(Aim 2b)触发囊泡释放和蛋白质分泌。我们还将 分析CHP受体信号中断对皮质发育的影响(目标3a)。脑脊液中断 容量、成分和脑室形成是许多神经系统疾病的共同特征。中的摄动 母婴健康可以扰乱大脑发育，部分是通过5-羟色胺能和其他信号的失调。 小路。我们的发现为阐明大脑调节失调的机制提供了一个新的平台 通过环境扰动进行发展，从而为未来提供概念和技术路线图 CHP上皮细胞活动如何影响基因表达和蛋白质分泌的研究 对许多神经发育疾病的后果。

PROJECT SUMMARY / ABSTRACT Building the cerebral cortex requires the spatial and temporal orchestration of molecular signals across neural progenitor cells. Each of these cells is bathed in cerebrospinal fluid (CSF), a medium rich in health- and growth- promoting factors whose composition changes profoundly across days during brain development. The choroid plexus (ChP) is likely to strongly contribute to this dynamic developmental regulation of CSF composition, but the underlying mechanisms are not understood. Here, we will use a suite of modern tools to test the hypothesis that elevations in calcium concentration in embryonic ChP epithelial cells in response to extrinsic cues is critical for regulating synthesis and secretion of signaling factors into the CSF. Calcium activity regulates transcription and secretion in diverse cells ranging from neurons to salivary gland epithelia. We identified receptors expressed in ChP epithelial cells that mediate calcium entry into the cell, such as the TRPM3 receptor, or that drive calcium release from internal stores, such as the 5HT2C receptor (Htr2c) for serotonin (5- HT, 5-hydroxytryptamine). Our preliminary data suggest that 5-HT regulates embryonic ChP calcium activity, transcription, and vesicle release, and that intact signaling via the 5HT2C receptor in ChP is essential for normal cerebral cortical development. Further, maternal delivery of a selective agonist for the 5HT2C receptor triggers rapid, robust immediate early gene expression in embryonic ChP. Thus, we propose that ChP calcium activity triggered by signals that activate the 5HT2C receptor (and other receptors such as TRPM3) regulates ChP transcription and secretion of signaling factors into the CSF and controls body-brain and maternal-fetal interactions, thereby tightly orchestrating cerebral cortical development. To test this, we will first investigate the effects of 5HT2C and TRPM3 receptor activation by direct application of ligand to the apical surface of epithelial cells during calcium imaging in ChP explants (Aim 1a) and by stimulation of the basal surface following intraperitoneal (IP) delivery of ligands to the maternal dam during in vivo two- photon calcium imaging of attached embryos (Aim 1b). We then assess the transcriptomic impact of such stimulation (Aim 1c), and test whether embryonic ChP calcium activity via activation of 5HT2C receptors and other receptors triggers vesicle release and protein secretion in vitro (Aim 2a) and in vivo (Aim 2b). We will also analyze the effects of disruption of ChP receptor signaling on cortical development (Aim 3a). Disrupted CSF volume, composition, and ventricle formation are common to many neurological conditions. Perturbations in maternal-fetal health can disrupt brain development, in part via dysregulation of serotonergic and other signaling pathways. Our findings provide a novel platform for elucidating the mechanisms underlying dysregulation of brain development by environmental perturbations, thereby providing a conceptual and technical roadmap for future studies on how activity in ChP epithelial cells influences gene expression and protein secretion, with consequences for many neurodevelopmental diseases.