February | 2015 | Musings of a BIOL463 Student

Monthly Archives: February 2015

Final Project Outline

TOPIC CHOSEN:

Effects of Paternal Alcohol Consumption on Fetal Development

SPECIFIC QUESTION:

Are Stfmb2, Snrpn-Ube3a, Dlk-Dio3 differentially methylated in the sperm DNA of CD1 mice treated with ethanol compared to saline?

HYPOTHESIS:

Stfmb2, Snrpn-Ube3a, Dlk-Dio3 are differentially methylated in the sperm DNA of CD1 mice treated with ethanol compared to saline.

EVIDENCE ON WHICH THE HYPOTHESIS IS BASED (INCLUDE REFERENCES):

It has been observed by Lee et al.(2013) that preconception consumption of ethanol by male mice results in skull malformation. Knezovich & Ramsay (2012) found that transgenerational toxic effects are due to epigenetic mutations in sperm DNA caused by alcohol. Laufer et al. (2013) found three imprinting control regions (ICRs), Sfmbt2, Snrpn-Ube3a, Dlk-Dio3, that are differentially methylated when the fetus is exposed to alcohol. These three ICRs are related to the development of the brain (Laufer and Singh, 2012).

PREDICTION(S):

If these imprinting control regions are differentially methylated, then there would be a possibility that these epigenetic mutations will be passed on to the offspring. This may be a reason why Lee et al. (2013) see skull malformations in the fetuses.

EXPERIMENTAL APPROACH TO TEST PREDICTION (INCLUDE ANY DETAILS THAT YOU HAVE WORKED OUT SO FAR):

Treat CD1 mice (postnatal day 49) with a 4 g/kg EtOH with 0.9% saline oral administration once in the morning and once in the evening, for a 7 week period. Control mice would be fed a saline solution. (adapted from Lee et al. 2013)

Mature sperm from the experimental and control group is harvested and DNA is extracted. Using oxidative bisulfite sequencing (OxBS-seq), methylation in the three loci will be analysed. This method is able to distinguish between 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) (Booth et al., 2013). Knezovich and Ramsay (2012) used a method that could not differentiate between these two bases and proposed that this led to the observation of very little demethylation of the sperm DNA. Look for differential methylation (only 5-mC) in the 3 loci between the control group and the experimental group.

LIST OF RELEVANT PRIMARY AND REVIEW ARTICLES READ, AND SUMMARY OF RELEVANT INFORMATION FROM EACH (this is the start of an annotated bibliography):

Laufer et al. 2013 – found three differentially methylated ICRs when the fetus were treated with alcohol that are related to development of the brain

Laufer & Singh 2012 – more in depth review of Sfmbt2, Snrpn-Ube3a, and Dlk-Dio3

Knezovich & Ramsay 2012 – found epigenetic mutations in sperm DNA, will be using an adapted experimental from this paper

Lee et al. 2013 – found skull malformations in mice fetuses when the fathers are fed alcohol

Liu et al. 2009 – found alcohol to changes the DNA methylation in mouse embryos during early development of neurons

Booth et al. 2013 – paper about oxidative bisulfite sequencing

HOW DOES THE QUESTION FIT INTO THE BROADER PICTURE, AND WHAT IS ITS IMPACT?

If we can find a direct link in between how transgenetic toxic effects of alcohol are passed on to offsprings, such as epigenetic mutations in the sperm DNA, it will no longer be just a correlation and people will pay more attention to the consumption of alcohol if they are planning to have offsprings.

POTENTIAL WAYS TO MAKE YOUR QUESTION KNOWN TO THE PUBLIC AT LARGE (OR TO YOUR NON-BIOLOGIST FAMILY AND FRIENDS):

Blog posts, posters, word of mouth

ANY OTHER PARTS OF THE PROJECT COMPLETED SO FAR:

None yet! Sorry!

ANYTHING YOU WOULD LIKE SPECIFIC FEEDBACK ON:

Does the topic need to be more specific? Would “Effect of Paternal Alcohol Consumption on the Epigenome of Sperm” be better?

Is the length of this experiment adequate? Should I include looking for differentially methylated at these 3 loci in genome of the fetus similar to Knezovich and Ramsay (2012) looked at in the offspring?

Learning Journal 2 Week 5

A. Factual knowledge

Please describe, briefly, one new piece of factual knowledge that you acquired or developed so far in the BIOL463.
I learned that a morphogen is a diffusible substance (ligand, mRNA, protein) that changes the fate of cells in a concentration dependent manner.

Please describe how you know that you have acquired or developed this piece of factual knowledge, and provide some evidence for it.
I can now identify what a morphogen is when given a scenario. For an example, in the terminal system in Drosophila, active trunk is a morphogen. Trunk is activated by torsolike, protein localized at the terminals of the embryo, which means there will be a gradient of active trunk with a higher concentration at the ends of the embryo and little to none in the middle of the embryo. This gradient of active trunk will allow for asymmetrical development of the embryo.

B. Conceptual knowledge

Please describe, briefly, one new piece of conceptual knowledge that you acquired or developed so far in BIOL463.
I learned about maternal effect genes are which are genes that are expressed by the mother in her nurse cell and passed on as mRNA to the oocyte. This means for these set of genes, the mother’s genotype will be the offspring’s phenotype.
Please describe how you know that you have acquired or developed this piece of conceptual knowledge, and provide some evidence for it.
I now understand the mechanism for how some flies that are homozygous for a recessive lethal gene can still survive; however, her offspring’s will not survive. I can now apply this knowledge for genetic screening of flies.

C. Skills

Please describe, briefly, one skill that you acquired or developed/are developing so far in BIOL463.
I am continuing to develop my abilities to develop a novel and interesting research question. This is for the final project of this class and that assignment gave me an opportunity to further develop this skill. In my other classes and in the lab, I rarely get an opportunity to develop a research question I would like to answer. In this class, I can finally exercise this skill so in the future, I will be easier for me in graduate school.

Please describe how you know that you have acquired or developed this skill, and provide some evidence for it.
It took me a very long time to read many articles to come up with my research question for my final project. From the articles, I have to piece together what is already known in the field and find the missing piece that I can use to develop my research question. I was able to find a few articles that were somewhat related; however, direct links were not made. From these articles, I came up with my question to see if there is a true link between the findings of these related paper. Perhaps in the future, it will not take me as long to develop a novel and interesting research question.

D. What is factual knowledge useful for?

Think about a piece of factual knowledge that you developed/acquired. Briefly describe what you think it is useful for.
I learned that eve has different enhancers that cause it to be expressed in different segments. For an example, there is an enhancer just for the expression in stripe 2 (eve2). This is useful in understanding how the expression of eve results in the pattern seen in the embryo. Then we can apply it to other genes with a similar expression pattern. By knowing these facts, we are able to come up with models on how other things work.

Draft Question

It has been observed by Lee et al.(2013) that preconception consumption of ethanol by male mice results in skull malformation. Knezovich & Ramsay (2012) found that transgenerational toxic effects are due to epigenetic mutations in sperm DNA caused by alcohol. Laufer et al. (2013) found three imprinting control regions (ICRs), Sfmbt2, Snrpn-Ube3a, Dlk-Dio3, that are differentially methylated when the fetus is exposed to alcohol. These three ICRs are related to the development of the brain (Laufer and Singh, 2012).

My question is are these 3 imprinted regions differentially methylated in the sperm when the mice is fed ethanol compared to a no ethanol diet regime and then passed on to their offsprings to produce abnormal phenotypes such as skull malformations observed by Lee et al. (2013)?

Pam’s Feedback:

Hi Ryan,

your question is excellent! One thing to consider: Knezovich & Ramsay
mention two loci that were differentially methylated in the progeny of
males exposed to alcohol (but were not differentially methylated in the
sperm of the fathers).
You will want to take into consideration that something similar might also
be the case for your three selected loci.

Proceed with your question, it’s a good one!

Cheers

Pam

Musings of a BIOL463 Student