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Effect of Thalidomide on Axolotl Limb Regeneration
Gene Expression
BIO399
Bethel University
Fall 2014
Background and Significance
In the animal kingdom, it is well established that organisms evolve and adapt according
to their surroundings. These changes occur due to mutations in the animal’s gene sequence,
leading to altered gene expression and phenotypic variation. While normally detrimental to the
organism’s ability to survive, occasionally a mutation may offer an animal a substantial
advantage over its competitors. Having increased the organism’s ability to survive and
reproduce, these mutations are generally passed down to future generations. One of the most
fascinating manifestations of this phenomenon is the tendency of certain animals to regenerate
their limbs and other appendages after amputation. This ability can occur in relatively archaic
organisms, such as flatworms and sponges, as well as in complex animals, including axolotls and
lizards (Kimball, 1994). The biological method by which each animal regenerates body cells is
often unique and tailored to the organism’s specific circumstances.
Mechanisms of Limb Regeneration
The specific mechanism in which limb regeneration occurs differs from organism to
organism. Most of these processes, however, may be divided into one of two categories: the
presence and activation of pluripotent stem cells or the differentiation of specialized cells in the
appendage that is to be regenerated (Kimball, 1994). In the method utilizing pluripotent stem
cells, the cells are present in the organism from its beginning and have the potential to direct the
differentiation of cells at the site of amputation. Cell differentiation, on the other hand, occurs
when cells lose their characteristic protein makeup, and are essentially reprogrammed, allowing
for differentiation (Kimball, 1994). For many years, there was considerable debate within the
scientific community concerning which mechanism limb regeneration uses (Kimball, 1994).
However, today it is now known that both methods play a role, with certain organisms relying on
one type or the other, or in some cases, a hybrid of both types. In salamander tail regeneration,
for example, both methods are utilized. Stem cells in the spinal cord translocate into the tail
stump where they facilitate the differentiation of a local mass of undifferentiated cells, known as
blastema. These blastemal cells will then differentiate into all of the components necessary for
re-growth of the tail - nerve, muscle, skin, cartilage (Kimball, 1994).
Axolotls, a certain species of salamander, exclusively utilize blastema to regenerate
limbs. When a limb is lost, the wound is quickly covered by an epidermal layer (Wu et. al.
2013). After this layer is in place, the axolotl’s method of healing diverges from the normal path
of recovery that is observed in other organisms. If the epidermal layer makes contact with nerve
tissue, it will differentiate into an apical epithelial cap (Wu et. al. 2013). The formation of this
cap triggers fibroblasts to congregate en masse under the epidermal layer, creating a blastema.
This blastema grows and divides, forming the new tissue of the lost appendage (Wu et. al. 2013).
It is important to note that blastemal cells are generally found in the embryos of many organisms.
The axolotl, however, is able to utilize these growth cells outside of embryonic development for
the purpose of limb regeneration.
A notable example of an animal that utilizes stem cells for regeneration rather than
blastema is a mouse. In mice, various tissues, including all the ectoderm and mesoderm that
participate in the regeneration of an amputated digit, originate from a population of adult stem
cells in the stump that possesses developmental potential (Kimball 1994). When amputated, the
limb of a neonatal mouse experiences hypertrophy and subsequently, regenerates to some extent
(Masaki, Ide, 2007). It has also been found that this regeneration occurs regardless of the
presence or absence of nerves, indicating that the mechanism is distinct from that of axolotl limb
regeneration (Masaki, Ide, 2007). Due to the fact that regeneration in neonatal mouse limbs
decreases as age increases, it can be concluded that stem cells are responsible for this form of
regeneration (Masaki, Ide, 2007). Thus, stem cells are an essential part of embryonic and
infantile growth, but cease to be produced and utilized fully by adult organisms.
Today, while there is a high degree of scientific consensus concerning the process of limb
regeneration in amphibians and mice, the exact mechanisms for either form are not completely
clear. There are many factors to consider and many different genes that are expressed during
both processes. Pinpointing the key genes expressed during regeneration could potentially give
valuable insight into how complete limbs could be regenerated in humans. One method of
investigating this hypothesis involves utilizing a limb growth inhibitor and observing its effects
on gene expression, which will directly correlate to phenotypic limb regeneration. By examining
the genes that are expressed or repressed as a result of the inhibitor, the timing of differential
expression of both necessary and sufficient genes may be elucidated.
Thalidomide: An Inhibitor of Limb Development
One inhibitor that may work well for this purpose is the drug known as thalidomide.
Thalidomide first emerged in 1956 in Germany as a sedative/hypnotic to be used in surgeries
(von Moos et. al. 2003). It gained initial popularity due to its relative non-toxicity, and quickly
rose to affluence in Europe, and subsequently in America, where despite its lack of approval by
the FDA, it was used for a different purpose; treatment of morning sickness in pregnant women
(Kim, Schialli, 2012). In the mid-1960s, it became apparent that the drug was a major
contributor to birth defects, which will be discussed in more detail later, and resulted in as many
as ten thousand affected children worldwide (Franks, et. al, 2004). Naturally, its usage was
quickly curbed, and many countries banned the drug. Today, however, thalidomide is returning
to the medical discussion, as it has recently reemerged as a treatment for leprosy and multiple
myeloma (Kim, Schialli, 2012). With the use of thalidomide continuing today, a discussion of
the specific defects it causes, specifically those that affect growth of embryonic limbs, is in order
before the proposed research is discussed.
Effects of thalidomide on human development: A historical retrospective
One factor that contributes to the toxicity of thalidomide is its tendency to dissociate
when placed in an aqueous environment with a pH of 7.0 (Franks, et al. 2004). Upon
dissociation, the drug has a number of adverse physiological effects, including inhibition of
blood vessel formation and blockage of cellular endothelial proliferation (Franks, et al. 2004).
With endothelial cells unable to reproduce via mitosis, new tissue growth is severely inhibited.
The absence of new blood vessels only enhances this effect. Thus, inhibited tissue growth would
naturally lead to deformations in the developing fetus (Franks, et al. 2004).
One of the most obvious manifestations of this phenomenon can be observed in the limbs
of affected infants. Perhaps the most prominent deformities in the upper limbs are reductions.
Beginning with the shoulder, the ball and socket joint between the humerus and the scapula is
structurally compromised, leading to an increased risk of dislocation (Smithells, Newman, 1992).
In the humerus, inhibited development begins at the medial end and progresses a certain length
up the shaft of the bone. The exact length is determined by the severity of the inhibition of
endothelial proliferation (Smithells, Newman, 1992). The radius and ulna often become fused
together, causing the hand and wrist to develop with poor alignment. This, in turn, causes the
wrist to rotate laterally, leading to the formation of a radial club hand (Smithells, Newman,
1992).
The lower extremities also have a plethora of possible malformations, some of which are
unique, and others that are share with the upper. The articulation of the femur and the coxal
bones may suffer from a defect that is analogous to that of the humerus and scapula; increased
risk of dislocation due to the structural weakening of the joint (Smithells, Newman, 1992). The
tibia and fibula may express their own unique malformations. Due to the anatomically tandem
relationship of the two bones, if one becomes deformed, the other must adjust to correct for said
deformity. The most common manifestation of this trend occurs when one of the two bones is
shortened due to inhibited endothelial proliferation. To compensate for this defect, the other
bone will bow outward (Smithells, Newman, 1992). With these limb deformities in place, the
integrity of the articulations formed by affected bones will be severely compromised, resulting in
weak joints that function poorly and are prone to dislocation. In the most severe cases ankles
and wrists may fail to form at all (Smithells, Newman, 1992). Here it is worth noting that, in the
majority of recorded cases, either the upper or lower extremities are affected; not both.
All of these limb deformities are caused by the ability of thalidomide to alter gene
expression in embryonic development. By using a test organism that has limb regeneration
capabilities, exposing individual animals to thalidomide, and observing which genes change
expression levels, we can obtain valuable information about which genes are key regulators of
limb regeneration.
Experimental Methods and Design
In this investigation, the test organism that will be used is Ambystoma mexicanum, or the
axolotl. This is an aquatic species of salamander that retains gills throughout its lifespan due to
the fact that it never goes through complete metamorphosis in its life cycle. Furthermore, it has
well-known regenerative abilities, as well as a small stature. These factors, coupled with the fact
that the axolotl is relatively easy to care for, makes this animal an ideal test organism. The
regeneration method, utilized by both adults and juveniles, involves producing blastema at the
site of amputation.
Before the individuals are randomly assigned into groups, each axolotl will be tagged and
its tail length will be measured, in centimeters, from the base to the tip. The test sample will
consist of three groups of sixty axolotls, each containing thirty males and thirty females; each
group of sixty will constitute one trial of the experiment, with the second and third trials
beginning one month after the previous respective trial. Within each group of sixty axolotls, ten
will be assigned as controls and given no treatments, while the remaining axolotls will be split up
into five groups of ten individuals. Each test group (sixty axolotls) will be housed in two tanks;
one holding the males, while the other houses the females. Separation of males and females
prevents any possible aggression due to mating behaviors. Furthermore, reducing the number of
individuals in a single enclosure reduces the likelihood of disease, crowding, fighting, and stress.
Each trial will begin by sedating the axolotls and amputating the tails at the base using surgical
tools.
Fig. 1. Flow chart depicting the concentrations of thalidomide to be received by each test group of axolotls.
As previously mentioned, the remaining salamanders not assigned to the control group
will be divided randomly into five equal test groups, each of which will receive a different
dosage of thalidomide. The dosages to be administered are as follows: 2 milligrams of
thalidomide per kilogram of body weight, 5 mg/kg, 10 mg/kg, 20 mg/kg, and 50 mg/kg (Fig. 1).
The dosage will be administered each morning by injection for a 60 day period. After this time
has elapsed, the tail stubs of each salamander will be measured, and a percentage of regrowth
will be calculated by dividing the length of regrowth by the initial length. This entire process will
be repeated two more times, each subsequent trial beginning one month after the previous trial
has begun. Thus, there will be three total trials, with each trial containing ten salamanders in
each test group, as well as ten in the control.
As the overarching goal of this research is to identify gene expression, and inhibition
thereof, in limb regeneration in the axolotls, there are several genes which we will focus on.
These genes have been confirmed by the biological literature to play significant roles in the
process of limb regeneration. The first gene we will focus on is retinoblastoma (RB) gene. The
protein coded for by this gene, (the retinoblastoma protein) has two primary functions; first, it
regulates cell division by inhibiting progression within the cell cycle until the cell is mature and
ready to divide. It also plays a role in chromatin rearrangement (Brockes, 1997). The CDK4
gene, another gene we will examine, codes for the cyclin-dependant kinase 4 protein. This
protein, only active in G1-S phase, is responsible for the phosphorylation of the retinoblastomal
protein (Brockes, 1997). Another gene that we will investigate is the fgf-8 gene, which codes for
fibroblast growth factor. This growth factor plays a role in limb regeneration by causing
embryonic endothelial tissue to proliferate (Wu, Et. Al. 2013). One last gene that will be
examined is actually a group of genes; the PAX genes. These genes play a role in the
differentiation of blastemal tissue into different specific tissues (Wu, Et. Al. 2013).
In order to monitor the expression of the previously described genes, real time reverse
transcriptase PCR will be used. This method involves bursting open cells collected from the
regenerating tail stub, and harvesting the messenger RNA. A specific RNA primer (poly T) will
bind to each strand of RNA that codes for the gene product of the genes we are monitoring, and
DNA polymerase will synthesize a new, complementary strand of DNA (cDNA). This DNA
strand will, in turn, be used to synthesize another complementary strand, this one having the
same polarity as the messenger RNA originally used. The end result will be a double-stranded
DNA molecule that is identical to the gene in the original genome. In order to identify the
degree of gene expression, fluorescent markers will be used. Utilizing complementary base
pairings, the copies of the genes of interest that are created through the PCR reaction will be
fluorescently tagged. This fluorescent tagging will allow us to monitor varied levels of gene
expression, as higher levels of fluorescence translates into elevated copies of the gene being
tested. The higher levels of fluorescence correlates with elevated gene expression.
Thus, we will have two separate sets of data. One will focus on the percentage of the tail
that is regrown, while the other will monitor increased or decreased expression of the four genes
we are focusing on. The two sets of data will be correlatively compared to each other, giving us
insight into how genotype (gene expression) correlates with phenotype (tail regrowth). Lastly,
an Anova test will be used to mathematically assess any correlation in the data. This will give us
a firm idea of how thalidomide affects gene expression, and how this expression affects limb
growth. Collaboratively, this will provide valuable insight into the biological process of
regeneration and potentially offer indispensable information that may pave the way for future
research in this field.
Budget
Item
Quantity
Price per Unit
Total Cost
Axolotls
180
$30
$5,400
20 gal Long Tanks
24
$33
$792
Thalidomide
0.6 grams
$75 (per mg)
$450
Soft-Sinking
20 kg
$2.25/kg
$45
Bedding (rocks)
120 lb
$5.49
$131.76
Filters
24 (one per tank)
$10
$240
Syringes
9,700
$0.20
$1,940
Micrometer Caliper
1
$70
$70
Analytical Balance
1
$995
$995
Plants in Tank
---
---
$100
Scalpel Handle
4
$1.70
$6.80
Salmon Pellets
(food)
Scalpel Blades
190
$3.00 (Per 10)
$57
Real time PCR
1
$12,000
$12,000
1
$471
$471
Hood
1
$6,000
$6,000
Pipettes
4
$70
$280
Test tubes
1,000
$0.09
$93.12
Pipette Tips
2,000
$60 per 1,000
$120
Test Tube Racks
2
$15
$30
Novacaine
18
12
$216
Scientist
1
$40,000/year
$40,000
machine w/
computer
Real Time PCR Kit
(1000 uses per kit)
Total Cost: $69,437.68
References
Alibardi, L. 2010. Morphological and Cellular Aspects of Tail and Limb Regeneration in Lizards
Bologna (Italy). 1-95.
Brockes, J. P. 1997. Amphibian limb regeneration: Rebuilding a complex structure. Science,
276(5309), 81-7.
Wu, C.H., Tsai, M.H., Ho, C.C., Chen, C.Y, Lee, H.S. 2013. De novo transcriptome sequencing
of axolotl blastema for identification of differentially expressed genes during limb regeneration.
BMC Genomics, 6:14-434.
Franks, M. E., Macpherson, G. R., & Figg, W. D. 2004. Thalidomide. The Lancet, 363(9423),
1802-11.
Gilbert, SF. 2000. Developmental Biology.
Godwin, J.W., Pinto, A. R., and Rosenthala, N. A. 2013. Macrophages are required for adult
salamander limb regeneration. Proceedings of the National Academy of Sciences, 110(23): 8-19.
Kim JH, Scialli AR. 2011. Thalidomide: The Tragedy of Birth Defects and the Effective
Treatment of Disease. Toxicological Sciences. 122(1):1-6.
Masaki, M and Ide, H. 2007. Regeneration potency of mouse limbs. Development, Growth and
Differentiation, 49.
Smithells, R.W. Newman, C.G.H. 1992. Recognition of Thalidomide Defects. Journal of
Medical Genetics, 29(10), 716-723.
Tsonis, PA. 1996. Limb Regeneration. Melbourne (Australia).
Von Moos, Roger. Stolz, R. Cerny, T. Gillessen, S. 2003. Thalidomide: From tragedy to promise
133(5): 77 -87.
Yokoyama, H. 2007. Initiation of Limb Regeneration: The Critical Steps for Regenerative
Capacity. Development, Growth, and Differentiation. 50(1):13-22.