Kraft Karoline F, Urbach Rolf
Institute of Genetics, University of Mainz, Mainz, Germany.
Methods Mol Biol. 2020;2047:115-135. doi: 10.1007/978-1-4939-9732-9_7.
Proper functioning of the brain relies on an enormous diversity of neural cells generated by neural stem cell-like neuroblasts (NBs). Each of the about 100 NBs in each side of brain generates a nearly invariant and unique cell lineage, consisting of specific neural cell types that develop in defined time periods. In this chapter we describe a method that labels entire NB lineages in the embryonic brain. Clonal DiI labeling allows us to follow the development of an NB lineage starting from the neuroectodermal precursor cell up to the fully developed cell clone in the first larval instar brain. We also show how to ablate individual cells within an NB clone, which reveals information about the temporal succession in which daughter cells are generated. Finally, we describe how to combine clonal DiI labeling with fluorescent antibody staining that permits relating protein expression to individual cells within a labeled NB lineage. These protocols make it feasible to uncover precise lineage relationships between a brain NB and its daughter cells, and to assign gene expression to individual clonal cells. Such lineage-based information is a critical key for understanding the cellular and molecular mechanisms that underlie specification of cell fates in spatial and temporal dimension in the embryonic brain.
大脑的正常运作依赖于由神经干细胞样神经母细胞（NBs）产生的种类繁多的神经细胞。大脑每一侧约100个神经母细胞中的每一个都会产生几乎不变且独特的细胞谱系，该谱系由在特定时间段内发育的特定神经细胞类型组成。在本章中，我们描述了一种标记胚胎大脑中整个神经母细胞谱系的方法。克隆性DiI标记使我们能够追踪神经母细胞谱系从神经外胚层前体细胞到第一龄幼虫大脑中完全发育的细胞克隆的发育过程。我们还展示了如何在神经母细胞克隆内消融单个细胞，这揭示了子细胞产生的时间顺序信息。最后，我们描述了如何将克隆性DiI标记与荧光抗体染色相结合，从而将蛋白质表达与标记的神经母细胞谱系中的单个细胞联系起来。这些方案使得揭示大脑神经母细胞与其子细胞之间精确的谱系关系以及将基因表达分配给单个克隆细胞成为可能。这种基于谱系的信息是理解胚胎大脑中细胞命运在空间和时间维度上特化的细胞和分子机制的关键。
