1. Fall 2016 Research Summation: Allie Poles
This past semester, I was given the opportunity to work with Jack Parent, M.D., in the
Neurodevelopment and Regeneration Laboratory at the University of Michigan Medical
Center. The lab focuses on adult neurogenesis and regeneration using multiple model systems
such as, mouse, zebrafish and induced pluripotent stem cells (iPSCs). Its overall aim is to further
understand the underlying processes that occur with brain injury and other neural malfunctions in
order to combat these issues in the future. One group of projects in his laboratory analyzes
differences in the neural regeneration process between mammalian and teleost vertebrate brain
injury models. Within the Parent lab, zebrafish are used as a comparison against the mammalian
model in order to compare and contrast their responses to injury and regeneration. This
difference will allow a strategy to be created that will influence the mammalian nervous system
to promote more robust regeneration and overall survival. More specifically, the studies explore
the role of adult neurogenesis, the process where neural stem cells produce new neurons
throughout life, in brain repair. With regard to neurogenesis, mammals exhibit a low level of
this process in adulthood, but it fails to help with brain regeneration when injury or
neurodegenerative disease becomes a factor. In contrast, teleost fish are able to efficiently
regenerate damaged brain regions after acute injuries. We therefore aim to compare two model
systems to study brain repair: the zebrafish and the mouse. Our overarching hypothesis is that
understanding the mechanisms by which zebrafish regenerate damaged brain will provide
insights into brain restorative therapies to promote mammalian brain regeneration. As human
brain injury remains such a prevalent health issue in the US, our lab explores ways to enhance
brain repair so that patients are, in fact, able to recover from traumatic brain injury or other
acquired brain insults.
Within the Dr. Parent lab, I work alongside postdoctoral fellow Palsamy Kanagaraj
(Kanagu) specifically with the zebrafish model. While Kanagu and I work with zebrafish, other
members of the lab are studying the mouse model. When injury occurs, microglia immediately
react, however the way these cells communicate with progenitor cells (stem cell-like,
differentiable units), is currently unknown. Examining the zebrafish model more closely, the
migration of progenitor cells towards injured sites appears to be induced by inflammatory
signals. The new neurons survive and integrate in damaged regions to achieve
regeneration. When similar injury occurs in a mammalian model, progenitor cells only partially
migrate to damaged regions, and most fail to survive and integrate; due to this, adequate brain
regeneration does not occur. Kanagu is specifically exploring neural inflammatory pathways to
analyze what occurs within a fish telencephalon (a part of the forebrain) following induced brain
injury that allows successful regeneration. By first applying injury to the brain through
quinolinic acid, a drug that induces robust regeneration in zebrafish (Skaggs et al., 2014), or
through a vehicle injury, RNA sequencing is used to analyze the differentially regulated
genes. Through this process, we are using a candidate gene approach to try to identify the
significance of the genes using both loss of function and gain of function studies. Using
CRISPR, a genome editing tool, as well as other forms of transgenic technology, we are able to
make conditional mutations or generate overexpression models. One candidate inflammatory
mediator, STAT3, is being explored by injecting a chemical inhibitor both intraperitoneally and
into the telencephalon during injury to allows for pharmacological screening before the genetic
mutant study. Following injection, the brain was dissected out 1 day post lesion or 2 days post
lesion. In order to analyze the results, histochemistry is used to identify pre-determined proteins
through certain markers. Within this experiment, my job was to help Kanagu create the

2. construct that was going to be used to make the conditional allele. To do this, I performed
procedures including PCR, DNA/RNA purification, bacterial transformation, colony PCR, and
DNA sequencing.
While analyzing the inflammatory pathway through a candidate gene approach is a
significant portion of what we have been working on, we have also been trying to identify the
differences between the inflammatory pathways of mice and fish. It has been shown, in fish,
inflammation is necessary to promote regeneration; conversely, it creates a glial scar in
mice. Seeing the two very different responses between mice and fish, it is important to analyze
how exactly inflammation causes regeneration in zebrafish and not in mice during different time
points following injury. Pro-inflammatory pathways, which are immediately induced following
injury, act as an automatic signal in both mammalian and fish models that likely inhibits repair at
the injury site. At the opposite spectrum, anti-inflammatory pathways are meant to decrease
these signals, allowing for progenitor cells to enter the area. In the mammalian model, it is
thought that pro-inflammatory pathway activation overpowers the anti-inflammatory pathways,
preventing new cells from integrating into damaged areas. On the other hand, fish allow for anti-
inflammatory pathways to take control, which permits the entrance of new cells into the injury
site and subsequent differentiation and integration of mature neurons. This process allows the
replacement of degenerated tissue at the injured site and can thus “heal” the wound.
The relationship between pro and anti-inflammatory processes is vital to understanding
why the two drastically different processes occur, and this can be further analyzed by looking at
the sequential expression and regulation of cytokines (proteins important for inflammatory cell
signaling). Looking into this expression, we are performing a time course analysis for 5 pro-
inflammatory, 5 anti-inflammatory, and two both pro/anti-inflammatory cytokines at different
points after injury. We are comparing uninjured brain to brains damaged with quinolinic acid, in
the presence or absence of dexamethasone (an inflammation suppressor) or zymosen A (an
inflammation inducer). While we are currently performing this experiment with fish, a similar
study will subsequently be performed in mice in order to compare the inflammatory reactions
and regenerative processes.
While most of our research is primarily concentrated on telencephalic injury, Kanagu and
I are also working on an epilepsy model in fish. One model mirrors epileptic encephalopathy,
which is a severe form of childhood epilepsy. Our laboratory is currently studying iPSCs
derived from patients with this disorder caused by gain of function mutations in the SCN8A
voltage-gated sodium channel gene. In order to introduce this mutation into the zebrafish to
model the disease, we are using CRISPR gene editing. I have learned how to use PCR to
generate an adequate amount of DNA copies, and perform gel electrophoresis to determine the
presence of the DNA mutation. With another model, we try to mirror the human mutation
SPNA2 that causes epilepsy. After using CRISPR and homologous recombination to induce this
mutation, we used an EEG to analyze brain activity. With this experiment, I helped make the
donor template carrying the mutation that will be inserted into the fish genome. To do this, I
used PCR, gel electrophoresis, DNA/RNA purification, plasmid isolation, sequencing, and DNA
concentration analysis. Overall, the purpose of this experiment is to study the mechanisms of
these genes in epilepsy and to screen for drugs that can reduce or inhibit any seizure activity seen
within the zebrafish model.
Serving as Kanagu’s apprentice, I have spent a significant amount of time training on a
myriad of protocols, methodology, and practices within the lab. Over the past few months, both
Kanagu and my undergraduate research partner, Vineeth, have taught me a wide variety of

3. common experiments and setups practiced within the Parent lab. Two of the most common
procedures that I perform on a day-to-day basis are processes revolving around gel
electrophoresis and bacterial transformation. Gel electrophoresis is used as a method to identify
a newly inserted gene by looking at the presence of mutations within the genome. If a mutation
is identified, we then use gel elution as a way of extracting the specific sections (bands) of DNA
from the gel electrophoresis. This band of DNA is purified using gel extraction and, finally, its
DNA concentration is analyzed using a spectrophotometer (NanoDrop2000). With bacterial
transformation, bacterial cultures grow with different gene insertions. This creates “knockout
genes,” in an attempt to figure out exactly what causes vertebrates’ migration of progenitor cells
to lead to brain regeneration. Using a micropipette to isolate colonies in different PCR tubes, we
add polymerase, primers, and water in specific ratios to ensure accurate amplification.
While those protocols revolve around the zebrafish DNA itself, I work directly with the
fish as well. In my first few months, I was trained on performing IP injections, brain dissections,
brain sectioning, cryoprotecting, and brain embedding. Most recently, in regards to the anti-
inflammatory pathway research, I injected chemicals that modulate different inflammatory
mediators into zebrafishes’ right telencephalons. By analyzing what occurs after 1, 2, 3, 4, 5,
and 6 hours in the brains of these fish, Kanagu and I aim to ascertain exactly which signals
influence the induction of the anti-inflammatory pathway.
While determining whether our experiments yield statistically significant differences that
address our question is ultimately our goal in research, the quality care of the animal subjects we
work with are extremely important as well. Before performing any procedure on the zebrafish,
we use the anesthetic tricane mesylate to ensure that they do not feel any pain. Additionally,
only certain numbers of fish are able to reside in each tank, which helps with cleanliness and
overall quality of life. Along with working directly in the lab, the other undergraduate
researchers and I alternate on cleaning the fish room. While the process of hand-cleaning each
tank might seem tedious, the health of the fish models we work with is vital in order to
accurately see any potential results. If any of the fish are subjected to conditions that are a
detrimental to their wellbeing, the effort that the researchers go through to perform certain
experiments on these vertebrates is nullified.
I am honored to have been one of two undergraduates selected to work under Kanagu. I
have enjoyed my work in the Parent Lab immensely, and have learned a tremendous amount
through weekly lab meetings in addition to my work in the laboratory. Not only are the
procedures I perform incredibly interesting, but I find the environment of the lab extremely
collaborative. As the current semester comes to a close, I embrace this priceless experience
while looking forward to continuing my essential work upon my return next semester.