Introduction:
The early stages of zebrafish, Danio rerio development are characterized by meroblastic dicoidal cleavage. Initially cell division only occurs in the blastodisc, which is a thin region on the animal cap of the egg. This blasotdisc contains all of the egg's cytoplasm and organelles, the rest of the egg is filled with yolk. After about 10 cell divisions the midblastula transition begins and the cells differentiate into three groups. The yolk syncytial layer (YSL) is made up of the cells on the vegital end of the blastula which fuse with the yolk. The second population of cells is the enveloping layer cells (EVL) which are positioned on the outer edges of the embryo. The deep layer cells make up the third popultaion and are the cells that actually give rise to the embryo itself. During gastrulation the YSL cells migrate down the edges of the embryo towards the vegital pole in a process called ebiboly that envelops the yolk. At the edge of this migration the deep cells involute/ingress under themselves and form the mesodermal layer. Of these new mesoderm cells, some of the ones that migrate anteriorly become the prechordal plate mesoderm.
When zebrafish are treated with ethanol during early gastrula stages a condition known as cyclopia can be induced (Blader and Strähle, 1998). This is a condition in which there is one narrow eye slit and usually there are no eyes at all. It is believed that exposure to ethanol causes the prechordal plate mesoderm to migrate abnormally (Blader and Strähle, 1998). This abnormal migration causes the prechordal plate mesoderm cells to end up in an incorrect location, and it is this that is believed to cause cyclopia. The prechordal cells express the genesgoosecoid and islet-1 which code for proteins that help control cell differentiation in the anterior region of the embryo, and their release in the incorrect location is the cause of deformations (Blader and Strähle, 1998). Lack of sonic hedgehog expression is also known to cause cyclopia in mice and chicks, and ethanol may act to disrupt its expression too (Gilbert, 2000).
Procedure:
1. Select embryos that are at the dome/30% epiboly stage stage (Figure 1).
2. Prepare three separate petri dishes that contain normal zebrafish embryo medium, a 2.5% ethanol zebrafish embryo medium solution, and a 1% ethanol embryo solution.
3. Place at least 10 of the selected embryos into each of the petri dishes and let them sit for 3 hours.
4. After 3 hours has passed, place all 3 sets of embryos into petri dishes with normal zebrafish embryo solution.
5. 24 hours later observe embryos for abnormalities and photograph them.
Figure 1: Zebrafish embryo in epiboly stage
Results & Conclusions:
The zebrafish in the control group developed normally as was expected. At the stage that they were observed, they showed normal eye development. (figure 2). The embryos in the 1% ethanol solution also showed normal development in the region of the head (figure 3). Apparenty this concentration of ethanol was not sufficient to cause cyclopia.
The 2.5% ethanol experimental group showed a limited number of deformed embryos. Out of the ten original embryos two failed to survive, though this was consistent with the survival rate of the control group. Out of the eight that survived, six developed normally and two were slightly deformed. It appears that under these experimental conditions a higher concentration of ethanol was needed to produce a higher frequency of teratogenic results. It was difficult to determine whether the two deformed emrbyos had full blown cyclopia but they did not have visible eyes (figure 4).
Figure 2: An embryo from the control group, eye pigment can be clearly seen.
Figure 3: Embryo from the 1% ethanol group showing normal eye development.
Figure 4: Embryo from 2.5% ethanol group showing underdeveloped or absent eyes.
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