The overall goal of this procedure is to study the effects of ethanol exposure on the developing zebra fish. This is accomplished by first exposing zebra fish to ethanol doses in normal embryo water during early development. Next, the ethanol is removed and the fish are allowed to develop to the desired time point.
Then both gross physical changes as well as alterations in gene expression are assay in treated compared to control embryos. Finally, the embryos are manipulated with mRNA, morphoses, or drugs to determine if the ethanol induced developmental defects can be altered. Ultimately, a combination of quantitative PCR and in C two hybridization show that ethanol effects on developing zebrafish embryos are linked to gene expression change.
Subsequent manipulation of gene expression levels may improve or prevent ethanol induced developmental defects. The techniques we're demonstrating today extend our knowledge of the nature of fetal alcohol syndrome, as well as conserve developmental processes in vertebrates. Demonstrating today will be Steph Erhardt, a technician in my lab To treat embryos with ethanol.
Raise them from wild type matings to dome stage at 28 degrees Celsius in embryo water. Split the embryos from a clutch into the desired number of dishes with between 10 and 50 embryos per dish. Add a solution of ethanol between 0.2 to 2.5%volume to volume in fish water to the embryos, and return to the incubator for up to 24 hours after the desired length of exposure.
Replace the ethanol containing water with fresh embryo water for a specific period of development to collect mRNA for quantitative PCR. Place H matched embryos in einor tubes. Remove the fish water and add lysis buffer with TCEP.
Using a motorized pulverizer, physically dissociate the embryos and then place in the centrifuge for a slow spin. Transfer the solute to a pre-clear column, then centrifuge the column. To remove disassociated large pieces.
Use an RNA isolation kit to extract the RNA. Then synthesize CDNA using standard methods for QPCR or microarray analysis to prepare embryos for in C two hybridization and antibody stating. Fix six to 24 hours post fertilization embryos in 4%para formaldehyde overnight at four degrees Celsius, following three washes and PBS plus 0.1%tween.
Remove the choon using two sharp forceps under the microscope. Then move the embryos into methanol using a series of liquid change of increasing methanol concentration and decreasing PBT concentration. After transferring the embryos into 100%methanol, store them at negative 20 degrees Celsius to stain cartilage.
Raise embryos to five or six days post fertilization. Fix them overnight in 4%paraform aldehyde at four degrees Celsius. Then wash them three times in PBT To determine what gene expression levels have changed from ethanol exposure.
Perform QPCR by first designing primers to validate the primers. Test them on three biological samples. These control biological samples can be of any age as long as the gene of interest is expressed at that particular age.
Once the primers have been verified, use the previously prepared CDNA to perform QPCR running samples in triplicate and including a control sample for a zebrafish control, we use the primers to gap dh. Normalize the results to gap DH levels. Then calculate the relative level of gene expression using the powerful method.
Variation on two delta delta CT displaying difference in experimental relative to wild type. For in C two hybridization construct. Ditch labeled ribo probes to the gene of interest.
Then incubate them with age-matched embryos using standard conditions and detect the hybridization pattern using alkaline phosphatase image the samples using a dissecting microscope to assess morphogenic development, such as so might shape image live embryos to assess interocular distance and body length. First, fix embryos in para formaldehyde. Then collect images to examine the effects of alcohol treatment on the developing cartilage structures in the larva stain.
Fixed zebrafish using lc and blue for several hours to overnight detain and clear the embryos before imaging. Analyze cell death in living embryos by incubating them in five milligram per milliliter of aine orange in PBS for one hour. Wash the embryos three times in PBS, then mount the embryos on slides, image the samples using a confocal microscope and use Z series to create composites.
After identifying genes whose expression has decreased due to ethanol exposure, inject 25 to 200 picograms per nanoliters of capped mRNA for a gene of interest into one to two cell stage. Allow the eggs to recover in a 28 degree Celsius heated incubator until they reach dome stage and then treat them with ethanol as before. For genes whose expression increased as a result of ethanol exposure, knock them down by injecting picogram to nanogram amounts of antisense morphos into one to two cell stage embryos.
This protocol is similar to that for capped mRNA. If injecting an mRNA, splicing morpho, measure the efficiency using R-T-P-C-R to compare the amounts of spliced and unsliced transcripts to analyze dose dependent gene activity, titrate the injected morino and allow the embryos to develop to a desired stage before treating them with ethanol. Zebra fish embryo exposure to ethanol results in a number of developmental and genetic defects that are related to the phenotypes found in other vertebrates.
Abnormal development of axial tissues, including Noor, leads to a shortened and occasionally disrupted noord, which in part leads to the later abnormalities in somites as shown here. While the untreated controls have a strong chevron shape, the ethanol treated somites take on the U-shaped appearance seen with reduced sonic hedgehog signaling. As seen here.
Another consequence of ethanol exposure, possibly downstream of early developmental delay and subsequent Noor defects is shortened embryo length. This finding is similar to the persistence of pre puberty, short stature found in children exposed to ethanol during development, suggesting that the zebrafish model is relevant for the understanding of this human birth defect. It has been demonstrated in animal models that severe ethanol exposure leads to pronounced fetal alcohol syndrome phenotypes, including synthia and opia.
In zebrafish, the intraocular distance or IOD decreases in an ethanol dose dependent fashion consistent with the nature of the human birth defect. Opia is only found with very high doses, but lower doses do produce a significant reduction in the IOD suggesting that this animal model has a similar defect in midline facial development. In this figure, the early gene expression levels of six three B and glee one are compared in untreated and ethanol treated embryos.
Both genes demonstrated reduced levels of expression in the ethanol treated samples. Using NC two hybridization, the patterns of expression for six three B and gooID were analyzed as seen. Here, both genes showed a decrease in spatial expression in ethanol treated embryos compared untreated controls.
Because both genes are expressed in tissues destined to become C craniofacial midline tissues, their reductions at eight hours post fertilization is consistent with the reduction in IOD later in development. Once genes that are affected by ethanol are identified, there are mechanisms by which their expression can be increased or decreased. In this example, mRNA was injected for sonic hedgehog, a ligand that increases glee one levels, and it rescued the gross defects due to reductions in glee one and ethanol treated embryos.
Injection of six three B however, was unable to rescue the ethanol exposed phenotype. After watching this video, you should have a good understanding of how to explore the genetic and developmental defects produced by ethanol, a developing zebrafish embryo. The techniques used here can also be applied to those who want to study the toxicity of other water soluble compounds of the developing vertebrate embryo.
