Introduction
The practicum has been developed in RIKEN Centre of Developmental Biology in Kobe, in the laboratory of Axial Pattern Dynamics under the supervision of Inomata-sensei and Matsukawa-san.
In the laboratory they try to artificially regulate the gradient shape, they can control morphogen-dependent pattern formation. In general, the shape of a gradient is defined by three factors; synthesis, diffusion, and degradation of morphogen. So, they attempt to spatiotemporally regulate the gradient shape by regulating three factors. They are planning to regulate these three factors using light stimulation method.
All the embryos start with one cell that it will divide into millions of different ones. Nevertheless, each cell will become part
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The dsDNA was transcribed with the mMESSAGE mMACHINE kit (life technologies) using the SP6. The mRNA was also extracted with the phenol/chloroform and precipitated with the isopropanol protocol. The samples were stored at -80ºC until their use.
Zebrafish
Wild-type embryos were obtained by natural spawning11. The evening before, the pairs are set up separated by a mating container (Figure 2), a smaller box with a mesh bottom. These boxes fit with the aquarium tanks allowing the female and male to be separated; still sharing the same water allowing them to fill it with hormones12. In the wild, zebrafishes spawn at dawn; so more eggs will be gather if the recovery of the eggs is made in the morning. So first thing in the morning the pair are transfer inside the matting container (Figure 2), the grid will allow the eggs to fall to the bottom of the tank and they will be protected from being eaten. After 10 minutes the embryos are collected and kept in E3 medium in 21ºC for slow growth or 28ºC for normal growth until they are