Ultraviolet irradiation impairs epiboly via microtubules in Zebrafish embryos

 

Introduction
Zebrafish have transparentembryos that develop outside the mother. They develop rapidly, so that at 24 hours after fertilization, the embryo has formed most of its tissue and organ primordia and displays the characteristic tadpole-like form. Also, the cell division occur only in the animal pole blastodisc. These divisions are rapid, with a periodicity of about 15 minutes each. The first 12 divisions occur synchronously, forming a mound of cells that sits at the animal pole of a large yolk cell.
Three kind of movements (epiboly, involution and convergence) can be distinguished in the early development of zebrafish embryo. Involution and convergence result of migration of deep cells moving presumably using fibroblasts, but the molecular nature of the mechanism driving the epiboly is not known yet.
Previous experiments showed that irradiation of zebrafish zygotes with ultraviolet light selectively impairs epiboly resulting in embryos with open blastopores but well-formed anterior axes. The ultraviolet light effect is not restricted to the zygote stage as irradiation of later embryonic stages also impairs epiboly. The ultraviolet-sensitive target may thus be maternally encoded components of the machinery driving epiboly.

Reference: U. Strahle and S. Jesuthasan, Development 119, 909-919 (1993)

Objective
The objective of this experiment was to expose several embryos at different level of UV irradiation and observe the potential malformations .

Selection of embryos
1)Embryos were fertilized on the morning.

2) After fertilization, we selected several newly fertilized zygotes which consist of a large region rich with yolk vesicles and a smaller yolk-free blastodisc. We used the embryos just before the 2- cell stage (first cleavage).

 

 

Procedure for UV irradiation

1) We put 15 embryos, randomly oriented in their chorion in 3 different petri dishes.
To produce the UV irradiation we used a 254 nm CL-1000 UV crosslinker.
To change the UV doses we can change the exposure times. Nevertheless, in this
experimentwe increased the irradiation without change the time of exposure.

2.) Exposure I ( 1.8 mJ/cm2)
We placed 15 embryos in a petri dish and then we put the petri dishes in the CL-1000 UV
crosslinker as explained previously.

3)Exposure II ( 3.6 mJ/cm2)
Same thing but the level of irradiation was doubled.

4)We also put 15 embryos in a petri dish and did not irradiated them. Theyserved
as reference during the observation (the next day).

5)We placed the 3 petri dishes to incubate at 28ºC in embryo media.