Epigenetically distinct synaptic architecture in clonal compartments in the teleostean dorsal pallium

The dorsal telencephalon (i.e. the pallium) exhibits high anatomical diversity across vertebrate classes. The non-mammalian dorsal pallium accommodates various compartmentalized structures among species. The developmental, functional and evolutional diversity of the dorsal pallium remain unillustrated. Here, we analyzed the structure and epigenetic landscapes of cell lineages in the telencephalon of medaka fish (Oryzias latipes) which possesses a clearly delineated dorsal pallium (Dd2). We found that pallial anatomical regions, including Dd2, are formed by mutually exclusive clonal units, and that each pallium compartment exhibits a distinct epigenetic landscape. In particular, Dd2 possesses a unique open chromatin pattern that preferentially targets synapse-related genes. Indeed, Dd2 shows a high density of synapses. Finally, we identified several transcription factors as candidate regulators. Taken together, we suggest cell lineages are the basic components for the functional regional..., Fish husbandry
Medaka fish are kept at 14h/10h on light at 28 °C. The transgenic line　(Tg) (HuC:loxp-DsRed-loxp-GFP) and Tg (HSP:Cre/Crystallin-CFP) were generated as previously reported 19. Both males and females were used in the experiments. We used more than 2 months old fish as adult fish.
Clonal units visualization
To induce Cre/loxP recombination, embryos of the double Tg line, being crossed from female Tg (HuC:loxp-DsRed-loxp-GFP) and male Tg (HSP:Cre) line, were mildly heated at 38 °C for 15 min. Heat shock was applied using a thermal cycler to polymerase chain reaction tubes containing two eggs each. After the mild heat shock treatment, the embryos were maintained at 26°C until they hatched. Then the fish were kept in the fish tank and raised till the adult stage.
Whole-brain clearing and imaging
Adult fish brains were dissected by the usual procedure 54. After  the dissection, the brains were fixed in 4% paraformaldehyde/phosphate-buffered saline (PBS) overnight. Then the brai..., All data can be opened in Fiji. In order to do 3d reconstruction, we used FluoRender.

背侧端脑（dorsal telencephalon）即大脑皮层（pallium），在脊椎动物各纲中展现出高度的解剖学多样性。非哺乳类的背侧大脑皮层（dorsal pallium）在不同物种中包含多种分区结构，但其发育、功能与进化多样性至今仍未被阐明。本研究中，我们对具有清晰界定的背侧大脑皮层分区Dd2的青鳉（Oryzias latipes）端脑细胞谱系的结构与表观基因组景观进行了分析。我们发现，包括Dd2在内的大脑皮层解剖区域均由互斥的克隆单位构成，且每个大脑皮层分区都展现出独特的表观基因组景观。尤为重要的是，Dd2拥有独特的开放染色质模式，可优先靶向与突触相关的基因。事实上，Dd2区域的突触密度极高。最后，我们鉴定出若干转录因子作为潜在调控因子。综上，我们认为细胞谱系是功能区域化的基本组成成分……【原文此处截断】，鱼类饲养。

鱼类饲养：青鳉饲养于14小时光照/10小时黑暗的循环中，饲养温度为28℃。转基因品系（Tg）(HuC:loxp-DsRed-loxp-GFP)与Tg(HSP:Cre/Crystallin-CFP)的构建方法参照此前发表的文献19。实验中使用了雌性与雄性青鳉，选取月龄超过2个月的个体作为成鱼。

克隆单位可视化：为诱导Cre/loxP重组，将由雌性Tg(HuC:loxp-DsRed-loxp-GFP)与雄性Tg(HSP:Cre)品系杂交获得的双转基因胚胎置于38℃下温和加热15分钟。使用聚合酶链式反应仪作为热循环装置，对每管含2枚胚胎的样本进行热激处理。温和热激处理后，将胚胎置于26℃环境中培养至孵化，随后转入饲养缸中饲养至成体阶段。

全脑透明化与成像：成鱼脑组织的解剖操作参照标准流程54。解剖完成后，将脑组织置于4%多聚甲醛/磷酸盐缓冲液（PBS）中固定过夜。随后脑组织[原文此处截断]，所有数据均可通过Fiji软件打开。进行三维重建时，我们使用了FluoRender软件。