Investigating Microglia Colonization and Differentiation in the Embryonic Brain

摘要 小胶质细胞是中枢神经系统（CNS）的常驻免疫细胞。它们位于 通过在神经元网络结构和稳态监测中发挥关键作用来促进大脑的发育和功能。 与其他CNS驻留细胞不同，小胶质细胞起源于脑外，特别是胚胎卵黄囊（YS）。 在小胶质细胞发挥其关键功能之前，它们必须首先迁移并渗透到发育中的大脑。的 控制这一过程的细胞和分子动力学尚未完全了解，我们所知道的是基于YS- 衍生的小胶质细胞。然而，cre/lox命运定位研究仅将约30%的所有小鼠小胶质细胞定位到YS。这 表明可能存在其他来源和小胶质细胞群体。这一点在斑马鱼身上得到了证实， 小胶质细胞具有额外的非YS起源。发现了更多的小胶质细胞种群， 了解小胶质细胞前体是如何在大脑中播种的，特别是考虑到我们所知道的是来自YS衍生的 只有小胶质细胞。为了开始填补这一关键空白，我们研究了在胚胎大脑中播种的小胶质细胞。因为 小胶质细胞在大脑中的播种是一个高度动态的过程，我们利用斑马鱼的时间推移成像来观察细胞 live.我们在大脑中发现了一种未描述的细胞，它表达典型的小胶质细胞标志物，清除碎片， 在伤害中扩大。这些小胶质细胞样细胞被甘露糖受体C，1a型（mrc 1a）标记，并在 大脑比已知的小胶质细胞前体更早。mrc 1a+小胶质细胞依赖于mrc 1a+淋巴管， 就在中枢神经系统边界之外这些mrc 1a+细胞位于脑实质内，不与 这表明它们不是巨噬细胞、血管周围细胞或脑淋巴管内皮细胞 细胞尽管我们发现了这种早期渗透的种群，但这种mrc 1a+种群如何殖民的动力学 大脑和扩大作为小胶质细胞是未知的。mrc 1a+脑边界细胞学在这些过程中的意义 也未确定。这项研究的目的是研究细胞和分子特征， 一个新的，mrc 1a+小胶质细胞亚群，因为它的殖民，扩大，并在发育中的大脑分化。我 将通过使用体内成像，光遗传学工具和光活化药物的组合来实现这一点， 提出以下目标：1.确定脑缘淋巴管是否为脑胶质细胞祖细胞的形成提供了帮助。 大脑发育2.描述mrc 1a+细胞分化为小胶质细胞的分子特征。这项工作 有可能影响广泛的领域，包括基础神经发育生物学，神经发育 疾病和疾病，神经胶质生物学和CNS稳态，通过研究早期 小胶质细胞前体，因为他们渗透发育中的大脑，扩大，并打开典型的小胶质细胞标记。

ABSTRACT Microglia are the resident immune cells of the Central Nervous System (CNS). They are positioned at the center of brain development and function by playing crucial roles in neuronal network architecture and homeostatic surveillance. Unlike other CNS-resident cells, microglia originate outside the brain, specifically in the embryonic yolk sac (YS). Before microglia can serve their crucial functions, they must first migrate to and infiltrate the developing brain. The cellular and molecular dynamics governing this process are not fully understood, and what we do know is based on YS- derived microglia. However, cre/lox fate mapping studies only map ~30% of all mouse microglia to the YS. This suggests additional sources and populations of microglia could exist. This has been confirmed in zebrafish, where microglia have additional, non-YS origins. The discovery of additional microglia populations leaves us with even less understanding of how microglial precursors seed the brain, especially given that what we do know is from YS-derived microglia only. To begin to fill this critical gap, we investigated the microglia that seed the embryonic brain. Because microglia seeding the brain is a highly dynamic process, we utilized timelapse imaging in zebrafish to watch the cells live. We identified an undescribed cell in the brain that expresses canonical microglia markers, clears debris, and expands in injury. These microglia-like cells are labeled with Mannose Receptor C, type 1a (mrc1a) and colonize the brain earlier than known microglia precursors. mrc1a+ microglia are dependent on the mrc1a+ lymphatic vessels sitting just outside the CNS boundary. These mrc1a+ cells are located within the brain parenchyma and do not associate with vessels or the brain-border, suggesting they’re not macrophages, perivascular cells, or brain lymphatic endothelial cells. Despite our discovery of this early-infiltrating population, the dynamics of how this mrc1a+ population colonizes the brain and expands as microglia are unknown. The implication of mrc1a+ brain-border lymphatics in these processes is also undetermined. The goal of the proposed study is to investigate the cellular and molecular characteristics of a novel, mrc1a+ microglia subpopulation as it colonizes, expands, and differentiates in the developing brain. I will accomplish this by using a combination of in vivo imagine, optogenetic tools, and photoactivatable drugs to carry out the following aims: 1. Determine if brain-border lymphatic vessels contribute microglial progenitors to the developing brain. 2. Characterize the molecular profile of mrc1a+ cells as they differentiate into microglia. This work has the potential to impact a broad spectrum of fields including basic neurodevelopmental biology, neurodevelopmental disorder and disease, glial biology, and CNS homeostasis by investigating the fundamental dynamics behind early microglial precursors as they infiltrate the developing brain, expand, and turn on canonical microglia markers.