Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers (MADM)
Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers (MADM)
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DOI：
10.3791/61147
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发表时间：
2020-05-01
影响因子：
1.2
通讯作者：
Hippenmeyer, Simon
中科院分区：
文献类型：
--
作者：
Beattie, Robert;Streicher, Carmen;Hippenmeyer, Simon
从有限的祖细胞库开始，哺乳动物的大脑皮层形成高度组织化的功能神经回路。然而，调节神经干细胞（NSCs）的谱系转换和神经元和胶质细胞在发育中的神经上皮中的最终产生的潜在细胞和分子机制仍不清楚。追踪神经干细胞分裂模式和绘制克隆相关细胞谱系的方法已经取得了显着进展。然而，许多当代谱系追踪技术缺乏后代细胞命运的细胞分辨率，这对于破译祖细胞分裂模式是必不可少的。提出了一种使用双标记镶嵌分析（MADM）进行体内克隆分析的方案。MADM同时操纵单个祖细胞，并以前所未有的单细胞分辨率可视化精确的分裂模式和谱系进展。在有丝分裂的G2-X期期间基于MADM的染色体间重组事件，连同时间诱导型CreERT 2，提供克隆的出生日期及其分裂模式的确切信息。因此，MADM谱系追踪在单细胞水平上提供了干细胞祖细胞增殖模式的前所未有的定性和定量光学读数。MADM还允许检查NSC谱系进展中候选基因的机制和功能要求。该方法的独特之处在于可以在体内相同的组织环境中进行对照和突变体亚克隆的比较分析。在这里，详细描述的协议，并采用MADM克隆分析和谱系追踪在发育中的大脑皮层的实验范例进行了演示。重要的是，该方案可以适用于在任何鼠干细胞小生境中进行MADM克隆分析，只要CreERT 2驱动程序存在。
Beginning from a limited pool of progenitors, the mammalian cerebral cortex forms highly organized functional neural circuits. However, the underlying cellular and molecular mechanisms regulating lineage transitions of neural stem cells (NSCs) and eventual production of neurons and glia in the developing neuroepithelium remains unclear. Methods to trace NSC division patterns and map the lineage of clonally related cells have advanced dramatically. However, many contemporary lineage tracing techniques suffer from the lack of cellular resolution of progeny cell fate, which is essential for deciphering progenitor cell division patterns. Presented is a protocol using mosaic analysis with double markers (MADM) to perform in vivo clonal analysis. MADM concomitantly manipulates individual progenitor cells and visualizes precise division patterns and lineage progression at unprecedented single cell resolution. MADM-based interchromosomal recombination events during the G2-X phase of mitosis, together with temporally inducible CreERT2, provide exact information on the birth dates of clones and their division patterns. Thus, MADM lineage tracing provides unprecedented qualitative and quantitative optical readouts of the proliferation mode of stem cell progenitors at the single cell level. MADM also allows for examination of the mechanisms and functional requirements of candidate genes in NSC lineage progression. This method is unique in that comparative analysis of control and mutant subclones can be performed in the same tissue environment in vivo. Here, the protocol is described in detail, and experimental paradigms to employ MADM for clonal analysis and lineage tracing in the developing cerebral cortex are demonstrated. Importantly, this protocol can be adapted to perform MADM clonal analysis in any murine stem cell niche, as long as the CreERT2 driver is present.