Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan ; National Institute for Basic Biology, Myodaiji, Okazaki, Aichi, Japan.
PLoS One. 2013 Jun 25;8(6):e66597. doi: 10.1371/journal.pone.0066597. Print 2013.
Genetic mosaic techniques have been used to visualize and/or genetically modify a neuronal subpopulation within complex neural circuits in various animals. Neural populations available for mosaic analysis, however, are limited in the vertebrate brain.
METHODOLOGY/PRINCIPAL FINDINGS: To establish methodology to genetically manipulate neural circuits in medaka, we first created two transgenic (Tg) medaka lines, Tg (HSP:Cre) and Tg (HuC:loxP-DsRed-loxP-GFP). We confirmed medaka HuC promoter-derived expression of the reporter gene in juvenile medaka whole brain, and in neuronal precursor cells in the adult brain. We then demonstrated that stochastic recombination can be induced by micro-injection of Cre mRNA into Tg (HuC:loxP-DsRed-loxP-GFP) embryos at the 1-cell stage, which allowed us to visualize some subpopulations of GFP-positive cells in compartmentalized regions of the telencephalon in the adult medaka brain. This finding suggested that the distribution of clonally-related cells derived from single or a few progenitor cells was restricted to a compartmentalized region. Heat treatment of Tg(HSP:Cre x HuC:loxP-DsRed-loxP-GFP) embryos (0-1 day post fertilization [dpf]) in a thermalcycler (39°C) led to Cre/loxP recombination in the whole brain. The recombination efficiency was notably low when using 2-3 dpf embyos compared with 0-1 dpf embryos, indicating the possibility of stage-dependent sensitivity of heat-inducible recombination. Finally, using an infrared laser-evoked gene operator (IR-LEGO) system, heat shock induced in a micro area in the developing brains led to visualization of clonally-related cells in both juvenile and adult medaka fish.
CONCLUSIONS/SIGNIFICANCE: We established a noninvasive method to control Cre/loxP site-specific recombination in the developing nervous system in medaka fish. This method will broaden the neural population available for mosaic analyses and allow for lineage tracing of the vertebrate nervous system in both juvenile and adult stages.
遗传嵌合体技术已被用于在各种动物的复杂神经回路中可视化和/或遗传修饰神经元亚群。然而，在脊椎动物大脑中，可用于嵌合分析的神经群体是有限的。
方法/主要发现：为了在斑马鱼中建立遗传操纵神经回路的方法，我们首先创建了两个转基因（Tg）斑马鱼品系，Tg（HSP：Cre）和 Tg（HuC：loxP-DsRed-loxP-GFP）。我们证实了幼鱼斑马鱼全脑和成年脑神经元前体细胞中报告基因的斑马鱼 HuC 启动子衍生表达。然后，我们证明了通过在 1 细胞期将 Cre mRNA 微注射到 Tg（HuC：loxP-DsRed-loxP-GFP）胚胎中，可以诱导随机重组，这使我们能够在成年斑马鱼脑中的端脑分区化区域中观察到一些 GFP 阳性细胞的亚群。这一发现表明，源自单个或少数祖细胞的克隆相关细胞的分布局限于分区化区域。在热循环器（39°C）中对 Tg（HSP：Cre x HuC：loxP-DsRed-loxP-GFP）胚胎（受精后 0-1 天[dpF]）进行热处理导致整个大脑中的 Cre/loxP 重组。与 0-1 dpf 胚胎相比，使用 2-3 dpf 胚胎时重组效率明显较低，表明热诱导重组存在与发育阶段相关的敏感性。最后，使用红外激光诱导基因操作（IR-LEGO）系统，在发育中的大脑的微区域中诱导热休克导致在幼鱼和成年斑马鱼中均观察到克隆相关细胞的可视化。
结论/意义：我们建立了一种非侵入性方法来控制斑马鱼发育中神经系统中的 Cre/loxP 位点特异性重组。这种方法将拓宽可用于嵌合分析的神经群体，并允许在幼鱼和成年阶段对脊椎动物神经系统进行谱系追踪。
