Blog #3: Progress with Research on PTEN Impact on Synapse Morphology
I have continued to developed the various techniques that I have learned in the lab. I am now more comfortable utilizing the confocal microscope with its proper settings. Furthermore, I am learning more about image analysis as well as the use of preliminary data as shown below.
WT – Tg(mpeg1:gfp)
Wild type at 2.5 days within the tectum
In the images above have wild type zebra fish showing the normal number of microglia. The reason we are using the time of 2.5 days is because this is around the time that microglia migrate to the brain. In addition, this is also the time that the axons are entering into the tectum and making more connections with other neurons. These connections that are being made will then become part of the visual circuit. We see what a normal level of microglia looks like in the zebra fish, but then we analyzed what the levels of microglia will when we had pten-b heterozygous zebra fish in the images below.
Ptenb – Heterozygote – Tg(mpeg1:gfp) – Scale Bar: 30 µm
As we can see in the images above, there are significantly fewer microglia in the brain of the zebra fish. We know that zebra fish have “genome duplication”, therefore, they have pten-a and pten-b. Without the pten-b the microglia seem to be low in number which can lead to the excessive axonal cell growth. If pten-a and pten-b are both knocked out it proves to be lethal, but if only pten-b is knocked out than the zebra fish get retinal cancer at around 9 months while knocking out pten-a results in no retinal cancer. However, from this data we do have some questions that come to the surface. One question is whether the microglia is just not developing or if the microglia’s development is just delayed. If the development of these microglia are delayed then we can start imaging zebra fish at 3-4 days and see if there is a difference in microglia development.
gal4:brn3c, uas:mcherry Left is WT and right is Ptenb Heterozygote
In the images above, we see the development of the axons reaching the tectum. However, the axons of the pten-b heterozygote have already reached the tectum which signifies that the microglia are not doing the axonal pruning that they should. On the other hand, on the right we can see that the axons are still growing and moving towards the tectum.
In terms of progress, I have been able to learn different and valuable techniques in the lab. These techniques include micromanipulation of larval fish by utilizing specific instruments such as a dissecting microscope, preparation of fish for in vivo imaging as well as screening for larval phenotypes (fluorescent reporting on the epi-fluorescent microscope). I have even been taught how to use a confocal microscope and an epi-fluorescent microscope. Dr. Marik and I have imaged preliminary data and have concluded on a time interval that we will be using for in our time-lapse experiments. It took some time to figure out that the optimal time points to collect data, but we have found that 2.5 days post fertilization at 15-minute intervals for microglia imaging was showing the best results. The next step is to analyze the wild-type and mutant fish and learn how to collect data based on the analyses we make in the lab.
For this research, I need to manage enough time to allow myself enough to image because it does take time for imaging and preparing fish. The Resident Assistant positions has taken time away from research time but when this happens I have to schedule a type of “research make-up” later in the week. In addition, there is a lot of information that must be taken in by both the actual experiments and from Dr. Marik teaching me laboratory techniques and I have to make sure that I don’t only keep up but grow from all of this information. To be sure that I am up to date with all the information I should know and to be sure that the time I am scheduled to be in the lab, Dr. Marik at least knows that I will be attending, Dr. Marik and I communicate through email and from text message from time to time when it is an emergency. Dr. Marik usually lets me know what I will be doing in the lab if I am not only continuing what I did the previous week.
Dr. Marik is also extremely understanding and works with me to create a schedule that considers the Resident Assistant position as well as other responsibilities I have on campus. I also know that Dr. Marik has many other responsibilities, therefore, I try to be as accommodating as possible when it comes to scheduling times we will both be in the lab simultaneously and when I must be conducting research when she is teaching or handling other responsibilities. I look forward to learning more about the effects of PTEN on the brain as well as other techniques that will prove to be helpful in any other laboratory setting.
Dr. Marik and I are working on research revolving around The Impact of PTEN on Synapse Morphology. We are focusing on the PTEN gene because out of the 500+ genes that affects where on the autism spectrum an individual is, PTEN is mutated in approximately 25% of people with Autism. This is the highest percentage for any gene associated with Autism; this mutation causes PTEN to be reduced (called PTEN-ASD). What the PTEN gene does is send a signal to the mTorr pathway to stop cell growth; what happens in individuals with Autsim is that this PTEN gene is not functioning properly and cells start growing more than they should. Neurons start to have axons that are abnormally long, causing Macrocephaly within individuals and creating inappropriate connections during development.
Dr. Marik and I wish to know if you can improve the removal of connections (axons) after initial development by reintroducing PTEN in later development. We will perform this experiment by using morpholino to modify the expression of the PTEN gene in zebra fish to simulate the development of a brain that has reduced PTEN. After a few days of development, the morpholino will be diluted and the PTEN gene should be reintroduced in the zebra fish. We will ascertain these effects by imaging fluorescently labelled excitatory neurons in zebra fish embryos over several days – when PTEN is reduced in early development and restored in later development. Throughout the experiment we will be using the wild type zebra fish with normal PTEN as our control group. Dr. Marik and I hope to see improved pruning when PTEN is restored.