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 regionalization in the pallial anatomical compartments, and that their changes has been the driving force for evolutionary diversity.

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 brains were washed twice with PBST (0.5% Triton-X100 in PBS) and immersed into ScaleA2 solution for about three hours on ice ^26. When the brains were confirmed to be transparent, they were transferred into a new Sca/eA2 solution with DAPI (0.5 ul/2ml) overnight at 4°C. The next day, the brains were transferred into the new ScaleA2 solution and washed with PBST. To prepare samples for signal detection, the brains were embedded in 0.75% agarose gel with Sca/A2-Triton solution and fixed on glass coverslips. Fluorescent signal detection was performed using a hand-made light sheet microscopy Digital Light-sheet Microscope (DSLM) ⁵⁵ and a commercial one (Zeiss, Lightsheet Z.1).

In order to define the anatomical regions, we used DAPI signals as landmarks. For whole-brain imaging, we dissected adult medaka brains, fixed with 4% paraformaldehyde/PBS, stained with DAPI in ScaleA4 (0.5/2000) for two days and soaked with Sca/eA4 for several days.

All data can be opened in Fiji. In order to do 3d reconstruction, we used FluoRender.