Angelman Syndrome and the Roller Mutant of
Caenorhabditis elegans
Sarah Zannoni
Teacher: Mary Ladage
Due by November 11, 2012
Table of Contents
ABSTRACT……………………………………………………………………………...........................................1
INTRODUCTION…………………………………………………………………………………………………...2
Caenorhabditis elegans and Angelman syndrome………………………………...……….2
Neuron’s in Caenorhabditis elegans……………………………………………………………..3
Furthering Studies………………………………………………………………………………..….…4
METHOD/SPECIFIC AIMS…………………………………………………………………………………….5
Research Design…………………………………………………………………………………….......5
Instruments…………………………………………………………………………………………….…6
Procedure………………………………………………………………………………………………….7
EXPECTED RESULTS…………………………………………………………………………………………….8
DISCUSION…………………………………………………………………………………………………………..8
BIBLIOGRAPHY…………………………………………………………………………………………….……10
Abstract:
Angelman syndrome (AS) is a debilitating neurological disorder that affects the
nervous system. It causes severe speech impairment, severe delayed development,
intellectual disability, abnormal gait, small head size, scoliosis, epilepsy and
tremulousness of the limbs (Dagli, Williams). In Caenorhabditis elegans, there is a
mutant with a few similar symptoms to that of Angelman syndrome. This mutant is
known as a roller mutant (Brenner). C. elegans have been used in various studies to
further research human diseases as well as biological processes (Padilla…et al). In this
study, we will use the model organism C. elegans to research developmental processes
related to movement by doing a forward genetic screen. We are specifically interested in
relating the neuronal issues of the roller mutant to the neuronal issues of patients with
Angelman syndrome. Different genes cause the different neurological symptoms of
Angelman syndrome (Dagli, Williams). We can use C. elegans to study different
biological pathways (Brenner). More specifically we can use C. elegans to further study
the genes that play a role in it’s nervous system and what happens to it’s movement when
those genes are mutated. We want to find a gene that regulates the movement of C.
elegans to help us better understand the biological process of the nervous system and the
role it plays in movement. My hypothesis is that I expect to find roller mutants in the F2
generation that have specific mutated genes, which are involved with the nervous system
and neurological processes of the worm.
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Introduction
Caenorhabditis elegans and Angelman syndrome:
Sydney Brenner first used Caenorhabditis elegans during his research to study
genetic pathways that influence complex processes (Padilla…et al). In his scientific paper
titled The Genetics of Caenorhabditis elegans, Brenner talks about how very little is
known about which sets of genes control the development of certain complex structures
in multicellular organisms (Brenner). Brenner explains that there
must be genes that control the development of the nervous system.
All of the neurons that make up the nervous system resemble each
other, but are very different cells placed in specific areas
throughout a C. elegans body (Brenner). Specific neurons may
target different parts of a C. elegans but, are definitely connected
Figure 1: Neurons in a
Caenorhabditis elegans
brain
making up the nervous system (Brenner). This fact raises a lot of
questions for scientists and is the reason why we are doing this
study. C. elegans are a great model organism and have been used in many research
studies, which is why we will be using this organism in our study.
We want to find out more about the connection between the nervous system in C.
elegans and how it is related to movement. By studying mutants we will be able to see
how genetic mutations affect the nervous system and in return affects the organism’s
movement (Brenner). In people with Angelman syndrome, the nervous system is affected
by a genetic mutation (Greer…et al). This syndrome causes a lot of neurological
problems and especially movement issues (Genetics Home Reference). We think that the
nervous system in C. elegans can be studied in depth in order to find out about the
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relationship between genetic mutations and movement. With this knowledge we want to
find a connection between the mutations that affect the nervous system and movement in
C. elegans and that of Angelman syndrome patients.
Neuron’s in Caenorhabditis elegans:
A Caenorhabditis elegans nervous
system functions with specialized structures
known as a synapse. A synapse allows
neurons to send an electrical or chemical
signal to a specific targeted cell
(Richmond). As discussed in Brenner’s
paper, neurons are not connected all
together so they must send signals to
targeted cells to communicate what
functions to perform such as movement. C.
Figure 2: Synapse structure and transfer of signals
between neurons
elegans in their hermaphrodite form have a
total of 302 neurons forming 7000 synapses (Richmond). C. elegans are one of the best
model organisms to use when studying synaptic function, because when a C. elegans has
a mutation that affects synaptic function it will pass on this mutation by self-fertilization
(Brenner). This allows us to see the effect of the loss of proteins on synaptic function, the
nervous system and movement of the organism. Previous genetic screens have showed
that there are key proteins, which are required for synaptic transmission (Richmond). In
normally functioning C. elegans acetylcholine is released causing muscle contraction and
γ -aminobutyric acid is released to cause muscle relaxation (Richmond). Mutant C.
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elegans have an odd locomotory phenotype, which is caused by disrupted signaling in the
nervous system. How the signals are disrupted by a mutation could be further studied to
make a connection with how the signals in the nervous system of a person with
Angelman syndrome affect their movement.
Furthering Studies:
Many studies have been conducted to find out more information about Angelman
syndrome. It is obvious that the nervous system of those with Angelman syndrome is
affected by the genetic mutation (Genetic Home Reference). A lot of the symptoms are
neurologically controlled such as epilepsy and tremulousness of the limbs. However, it is
not fully understood at the synaptic level how the neurons are affected by the genetic
mutation (Greer…et al). In other words we want to find out what specifically is
happening at the synaptic level that causes those with Angelman syndrome to have these
symptoms. We can do this by studying the nervous system in C. elegans. Some studies
have been conducted on Angelman syndrome patients to discover what is happening at
the synaptic level, but very few studies have been conducted. A study was done involving
the gene Ube3A on humans with both Angelman syndrome and Autism (Greer…et al).
The findings of this study showed that this gene plays a big role in cognitive function.
However, the researchers of this study weren’t able to discover the specific role that this
gene plays in the nervous system or what exactly it does that causes cognitive
impairment. All they found out was that there was a connection between a mutation in the
Ube3A gene and cognitive impairment. We want to take this idea and use it for our study.
There must be a gene that is mutated in both Roller mutants and those with Angelman
syndrome that causes locomotive impairments. We know that movement is controlled by
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the nervous system in both organisms (Farabee). With the conformation that mutated
genes are in fact tied to specific Angelman syndrome symptoms we know that we are on
the right pathway for figuring out how these mutated genes play a role in the nervous
system, which controls movement (Greer…et al).
In this study we will isolate mutagenized C. elegans and grow an F1 generation
offspring. We will selectively choose 10 different worms to produce the F2 generation
offspring. From the F2 generation we will isolate mutant roller Caenorhabditis elegans.
We will be able to compare the movement of the roller C. elegans to the movement of
people with Angelman syndrome, such as abnormal gait and tremulousness of the limbs.
Method/Specific Aim
Research Design:
In this experiment we will do a forward genetic screen and look specifically at
roller mutants in the F2 population of an original P0 generation of C. elegans. The
purpose of this experiment is to find a specific gene that plays a role in the nervous
system and affects movement when mutated. We want to find a connection between this
gene in C. elegans and in people with Angelman syndrome. One question we have is; is
there a specific gene that when mutated causes dysfunction of the nervous system in C.
elegans? If so, does this gene affect the movement of the C. elegans and cause it to have
the “roller” movement? Is there a connection between this gene in both C. elegans and
people with Angelman syndrome? We plan to combine research methods done in
previous studies in order to answer these questions.
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We know from Brenner’s research article that all neurons are similar and different
in many ways. They are placed in specific places in the body, but remain connected by
the ability to send signals to each other (Richmond). The nervous system does control
motor and sensory information in organisms (Farabee). One way we can answer
questions about the nervous system in C. elegans is to study the effects of mutations on
the nervous system (Brenner). By looking at roller mutants in our F2 population, we will
be able to study the effects of mutations on the nervous system by observing how
movement was affected by a genetic mutation.
Instruments:
All Petri plates that we use in this experiment will have Nematode Growth
Medium. The plates also have a strain of Escherichia coli on them known as OP50. This
strain of E. coli is non-pathogenic, but gloves will still be worn when handling the plates
as a precaution. Our lab instructor gave us our original P0 plate. This plate has a
mutagenized P0 population of C. elegans. The P0 C. elegans were treated with a
chemical called ethyl methanesulfonate, which is a mutagen (Padilla…et al). This
chemical is capable of generating point mutations, making it dangerous to work with. We
were also supplied with a package of new Nematode Growth Medium with an E. coli
lawn on it. Before ever placing worms onto the plate, we must make sure that the plate is
free of bacterial growths. Throughout the experiment the plates must be continuously
checked in order to prevent starvation of the C. elegans. An important tool that is used
during this experiment is known as a pick. A pick is a made from a glass Pasture pipet
and a 1-inch piece of 32-gauge platinum (Padilla…et al). The pick is used to pick up a C.
elegans from one plate and move it to a different plate. Any time we open a Petri plate or
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pick C. elegans we must work by a lit Bunsen burner. Prior to and after each transfer, the
platinum part of the pick should be held over the Bunsen burners flame for sterilization
purposes. We will use a dissecting microscope when picking worms. We must ensure that
the plate is not kept on the dissecting microscope to long. The light on the microscope
can heat up the plate and if exposed to the heat for to long, the worms may die. All of the
above statements are important to the procedure and success of this experiment.
Procedure:
We first start of with a mutagenized P0 plate of C. elegans. We will transfer 5
worms into 5 separate NGM plates labeled A-1 through A-5. Once eggs are laid we will
move the same 5 P0 worms to 5 separate NGM plates labeled B-1 through B-5. Once
eggs are laid on the B plates we will pick all 5 original P0 worms and flame them on the
Bunsen burner. The F1 generation is on the A and B plates. We will then wait until the
worms in all the A and B plates are big enough to pick. Once the worms from the A
plates are big enough, we will transfer 5-7 worms from each plate onto a new plate
labeled F1A-1 through F1A-5. We will do the same for the B plates, but the B plates will
be labeled F1B-1 through F1B-5. The F1 generation will lay eggs on these plates. These
eggs are the F2 generation. Once the eggs hatch we can screen for mutants. We will
screen for mutants from the F2 generation that have characteristics of roller mutants and
place each of these separately onto a mini NGM plate. We
will write down the characteristics of these mutants and
whether or not we observe the roller phenotype. If we do
observe the roller phenotype we can look more closely at
Figure 3: Roller mutant (Indicated by
arrow)
the mobility of the movement in comparison to other roller mutants and wild type C.
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elegans. If we do observe the roller phenotype in the F2 generation we can also observe
the offspring of the mutants from the F2 generation on the mini plates. By observing
whether of not all or some of the offspring carry the phenotype of the F2 mutant we can
calculate the penetrance of the gene.
Expected Results
As stated earlier, my hypothesis is that I expect to find roller mutants in the F2
generation that have specific mutated genes, which are involved with the nervous system
and neurological processes of the worm. The objective is to do a genetic screen on the
mutants of the F2 generation of mutaginized P0 worms that have a roller mutation. Our
goal is to find the specific gene that plays a role in the nervous system of C. elegans that
when mutated causes the roller phenotype. We want to find a connection between this
gene and a gene in the nervous system of those with Angelman syndrome that also causes
similar mobility issues.
Discussion
Our expected results can be rationalized by previous experiments that have been
discussed throughout this paper. Sydney Brenner explained that to understand how the
nervous system worked in C. elegans, the effects of mutated genes on the nervous system
could be studied (Brenner). By using a mutagenized P0 population whose DNA has been
altered by EMS, we know that genetic mutations will be passed on to the “clones” of the
self fertilizing hermaphrodite C. elegans. (Brenner). The nervous system does control the
movement of an organism (Farabee). A study done on humans with Angelman syndrome
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and Autism showed that a mutation in the Ube3A gene caused patients to have cognitive
impairments (Greer…et al). We can relate this study to ours, because it confirms that
specific genetic mutations can cause certain symptoms in different genetic disorders. In
order to figure out if specific genes do affect the nervous system in organisms, we can
study the effects that mutated genes have on the movement of an organism. From this
forward genetic screen we will gain knowledge about specific genes that when mutated
affect the nervous system. With this knowledge we can further study the effect of genes
that play a role in the symptoms controlled by the nervous system of those with
Angelman syndrome.
Bibliography
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7. Richmond, Janet. "Synaptic Function*." Synaptic Function. 7 Dec. 2007. Web. 09
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Image Sources
Figure 1: http://research.ua.edu/wp-content/uploads/2009/03/protein01.jpg
Figure 2: http://en.wikipedia.org/wiki/File:Chemical_synapse_schema_cropped.jpg
Figure 3: http://i2.ytimg.com/vi/YVpqyyRexlE/mqdefault.jpg
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