c. elegans primary literature

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Shiva Mozaffarian The effect of silencing genes pdl-1 and F26A10.1 on the chemosensory function of the organism Caenorhabditis elegans. Abstract The ability of the Caenorhabditis elegans to respond to chemicals in the surrounding environment is an indispensible asset to its survival. In this experiment, the chemosensory implications of suppressing the genes pdl-1 and F26A10.1 in the C. elegans’ neural sensory pathway were tested. The hypothesis was tested by performing an RNAi experiment over the course of one week during which gene specific dsRNA was incorporated into the DNA of C. elegans progeny through ingestion of Escherichia coli. A subsequent chemotaxis assay revealed inconclusive evidence that the pdl-1 gene may play a role in inhibition of chemosensory processes. The results also indicated that the F26A10.1 gene was not involved in the chemosensory ability of C. elegans. Introduction The purpose of this experiment was to determine what physiological factors are involved in the ability of the organism Caenorhabditis elegans to recognize and respond to odors in its environment. Specifically, this experiment addresses the role of “transcription factors and signaling molecules” on the nervous system function of chemotaxis (Bush, et al., 2012). The affect of physiological factors on the function of the nervous system can be observed effectively due to the many physiognomies that make the C. elegans an optimal model organism. In 1963, Sydney Brenner discovered that the organism was Figure 1. On the

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Page 1: C. Elegans Primary Literature

Shiva Mozaffarian

The effect of silencing genes pdl-1 and F26A10.1 on the chemosensory function of the organism Caenorhabditis elegans.

AbstractThe ability of the Caenorhabditis elegans to respond to chemicals in the surrounding

environment is an indispensible asset to its survival. In this experiment, the chemosensory

implications of suppressing the genes pdl-1 and F26A10.1 in the C. elegans’ neural sensory

pathway were tested.  The hypothesis was tested by performing an RNAi experiment over the

course of one week during which gene specific dsRNA was incorporated into the DNA of C.

elegans progeny through ingestion of Escherichia coli.  A subsequent chemotaxis assay

revealed inconclusive evidence that the pdl-1 gene may play a role in inhibition of

chemosensory processes. The results also indicated that the F26A10.1 gene was not involved

in the chemosensory ability of C. elegans.

IntroductionThe purpose of this experiment was to determine what physiological factors are

involved in the ability of the organism Caenorhabditis elegans to recognize and respond to

odors in its environment. Specifically, this experiment addresses the role of “transcription

factors and signaling molecules” on the nervous system function of chemotaxis (Bush, et al.,

2012).

The affect of physiological factors on the function of the nervous system can be

observed effectively due to the many physiognomies that make the C. elegans an optimal

model organism. In 1963, Sydney Brenner discovered that the organism was particularly

suitable for genetic and physiological studies (Chao and Hart, 2009). The 1-millimeter,

transparent nematode is an ideal specimen to be observed and maintained in the laboratory.

Many features of the C. elegans’ cellular anatomy can be seen using an inverted fluorescent

microscope (Figure 1). Its short life cycle and large number of offspring also allow for the

organisms rapid growth and reproduction on agar plates in the laboratory (Bush, et al., 2012).

Figure 1. On the left is a fluorescent image of the C. elegans, on the right is a bright field image of the C. elegans. These still images were taken from an inverted fluorescent microscope (Bush, et al., 2012).

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Shiva Mozaffarian

The model organism is comprised of only 959 somatic cells. This simplicity in the

structure of the C. elegans in comparison to the complex functionality of its systems and

internal processes is quite advantageous in research. The nervous system is the most complex

organ of the C. elegans, consisting of 302 neurons and 56 glial cells (Brenner, 1986). Among

these neurons, 32 are chemosensory neurons that control the complex behavior of chemo-

perception. Each of these neurons expresses certain G proteins and transmembrane receptors

that detect and respond to different odorants (Bargmann, 2006). The chemosensory neurons

AWC and AWA are involved in chemo-attraction, and the chemosensory neuron AWB is

involved in chemo-repulsion. The ciliated endings of the chemosensory cells (shown in Figure

2) recognize odorant molecules, which transduce the signal to motor neurons and ultimately to

muscles that move animal towards or away from stimuli (Bush, et al., 2012).

Chemosensation is fundamental to the survival of the C. elegans and it is a component

of their biology to which over 5% of its genome is devoted. The entire genome of the

organism, consisting of 100 million base pairs, was completely sequenced, which allowed us

to study the function of specific genes in the organism (Bargmann, 2006). The affect of

specific genes on the chemosensory function of C. elegans can be tested using the ribonucleic

acid interference (RNAi) technique. In 1998, Andrew Fire and Craig Mello discovered a way

to inhibit specific genes in the organism C. elegans. The technique of RNAi utilizes specific

gene sequences of double stranded RNA (dsRNA) for post-transcriptional gene silencing

(Driver, 1998). A larval C. elegans can ingest double stranded bacterial plasmid dsRNA from

its food source, Escherichia coli, and incorporate the dsRNA into the next generation’s

genome. The interfering RNA represses a specific gene and therefore allows genes of interest

to be studied independently (Bush, et al., 2012).

Figure 2. The structure of a chemosensory organ. A sensory cell with its ciliated endings exposed to the outside environment. Specific chemosensory neurons AWA, AWB, and AWC are shown. These neurons are involved in chemosensation (Bargmann, 2006).

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Shiva Mozaffarian

A chemotactic assay reveals whether the removal of the specific gene affects the

chemo-perception of the C. elegans by blocking translation of proteins they encode. The

ODR-10 gene is known to code for a G-protein-coupled receptor involved in chemotaxis. It

was used as a positive control in the experiment to ensure that when the silencing of a gene

results in a malfunction of chemotaxis, the gene is involved in chemoreception. L4440 is a

non-nematode gene used as the negative control in the experiment to ensure that the methods

of the experiment did not have any effect on the chemotaxis ability of C. elegans (Bush, et al.,

2012). The experimental genes pdl-1 and F26A10.1 will be statistically tested against this

negative control.

No previous testing had been done on the F26A10.1 gene. Therefore, the gene

sequence, genetic position, and its coding protein are unknown (Worm base: Gene F26A10.1).

In 1995, pdl-1 in C. elegans was discovered to encode for a

phosphodiesterase protein, PDE6D, responsible for cGMP

breakdown (Maduro and Pilgrim, 1995). In 2006, mutations in pdl-

1 were tested to identify its role in the gustatory plasticity of C.

elegans. The worms containing mutated pdl-1 were pre-exposed to

NaCl and a subsequent chemotaxis assay to NaCl revealed, with

99% confidence, that the gene “regulates the gustatory adaptability

of an organism to changes in its environment or differences

between its various habitats”. These results are graphed in Figure 3

(Burghoorn, et al., 2006).

After the results of the experiment were collected, the experimenters presented a

plausible pathway in which PDE6D could affect gustatory plasticity (Figure 4). They

proposed that once odorant molecules bind to the G protein-coupled receptors (GPCRs), the

signal transduces into the cell where it activates G proteins. As a result, PDE6D is activated

and hydrolyzes cGMP to GMP (Burghoorn, et al., 2006).

Figure 3. A graphical representation of the experimental results. The mutation of pdl-1 (P<0.01) affected gustatory plasticity. (Burghoorn, et al., 2006).

Figure 4. Pathway of phosphodiesterase in regulating gustatory plasticity. The protein signals cGMP molecules which can then be involved in many functions (Burghoorn, et al., 2006).

Page 4: C. Elegans Primary Literature

Shiva Mozaffarian

Many neurons involved in chemotaxis, such as AWC and AWB, utilize cGMPs to

open cGMP-gated channels required for the function of chemotaxis (Maduro and Pilgrim

1995). As such, a protein responsible for the breakdown of cGMP could potentially affect the

function of chemotaxis. Although this experiment did not offer conclusive evidence for the

role of the pdl-1 gene in the chemosensation of diacetyl, the experiment offers a plausible

cascade of signaling events that could interfere in the chemotactic processes of the C. elegans.

All aforementioned factors of both the experimental genes and the model organism

propelled us to investigate and discover the function of the genes pdl-1 and F26A10.1 in the

chemosensory system of the C. elegans.

MethodsThe RNAi experiment was conducted by setting up four agar plates corresponding to

the four genes being tested. The plates contain the bacteria, E.coli, transformed with gene-

specific RNAi constructs. A 4l suspension of C. elegans’ larvae was placed at the edge of

the bacterial lawn in the middle of the agar plate. The plates were then sealed and incubated at

15℃ for 7 days, during which the worms were able to reproduce in the presence of the RNAi

construct. After one week, a chemotaxis assay was conducted by setting up four large agar

plates shown in Figure 5.

Sodium azide (2l) was dispensed in the circles on both the “DA” and “O” sides in

order to paralyze the worms once they reached either side. While the sodium azide absorbed

into the plates for 10 minutes, the RNAi treated worms were harvested from the plates using

0.75 ml water. The worm suspension was then pipetted onto a filter screen with kimwipe

underneath. The filters were rinsed with 200l of water and subsequently pressed into the

center of the chemotaxis plates. The diacetyl (2l) was dispensed in the circle to the right,

Figure 5. An example of a completed chemotaxis assay plate. A line is drawn down the middle of the plates. Three circles are drawn: one on the centerline and two smaller ones on the left and right hand sides of the plate. The left side is labeled “DA” and the right side is labeled “O” (Bush, et al., 2012).

Page 5: C. Elegans Primary Literature

Shiva Mozaffarian

labeled “DA”. Once chemical attractant was added to the plates, they were sealed for a 60-

minute time period. After the hour, each plate was counted for worms present on the diacetyl

side (designated ‘A’) and the sodium azide side (designated ‘B’) not including the worms still

left inside the middle circle of the chemotaxis plate. A chemotaxis index for each plate and

corresponding gene was then calculated using the equation: (A)/(A+B) (Bush, et al., 2012).

Results The chemotaxis indices were calculated for each of the four plates and presented in

Table 1. The ODR-10 positive control plate had a chemotaxis index of 0.48, while the L4440

negative control plate had a chemotaxis index of 0.67. The experimental genes, pdl-1 and

F26A10.1 yielded a chemotaxis index of 0.44 and 0.63, respectively.

Plate Number of worms on RNAi plate

Number of worms on “DA” side (A)

Number of worms on “O”side (B)

Chemotaxis index, P,(A)/(A+B)

Class Average Chemotaxis Index

% Error compared to class average

ODR10 4 26 28 .48 .52 8%L4440 3 16 8 .67 .65 3%pdl-1 5 19 24 .44 .66 33%F26A10.1 3 20 12 .63 .66 5%

Table 1. A comparison of my individual and class average chemotaxis values which were determined from the number of worms on the “DA” side of the chemotaxis plate divided by the number of worms on the “DA” side of the plate plus the number of worms on the “O” side of the plate.

The class average chemotaxis indices were utilized to assess the precision of my own

data (Figure 6).

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Shiva Mozaffarian

_x0006_Odr-10 _x0005_L4440 _x0005_pdl-1 _x0008_F26A10.10

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

The Effect of RNAi on Chemotaxis in C. elegans

My Data

Average Data

Che

mot

axis

Ind

ex

Figure 6. A graph comparing my chemotaxis indices to that of the class averages.

The average chemotaxis index for the positive control of a sample of 513 students was

0.52. The average chemotaxis index for the negative control of a sample of 519 students was

0.65. The chemotaxis index average for the pdl-1 gene of a sample of 30 students was 0.66,

excluding outliers. The average chemotaxis index for the F26A10.1 gene of a sample of 29

students was 0.66, excluding outliers. The method of comparison between my data and the

data of my colleagues was to calculate percent error. My data for the chemotaxis index of the

ODR-10, L4440, pdl-1, and F26A10.1 yielded an 8%, 3%, 33%, and 3% error compared to

that of the class average.

A statistical approach using a Z-score was then applied to a normal curve, which is

used to determine the statistical significance of our data, enough to make a confident

conclusion as to whether or not the experimental genes play a role in chemosensory ability.

The statistical analysis tests the “null hypothesis”, the hypothesis that the difference between

the chemotaxis indices of the experimental and controls are due to sampling error (Pcontrol =

Pexperimental) by calculating the Z-scores of the two experimental genes using the equation:

(Pexperimental – Pcontrol) / [Pexperimental(1-Pexperimental)/n]. Where ‘P’ is the chemotaxis index and ‘n’ is

the standard deviation of the negative control (Bush, et al., 2012). The Z-score of the pdl-1

gene was calculated to be -1.84 with a P value of 0.0329 (Table 2). Since the corresponding P

value is lower than 0.05, there is 95% confidence that the difference between the chemotaxis

index of the pdl-1 gene and the control are significant, therefore, we reject the null hypothesis

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Shiva Mozaffarian

for the gene. The Z-score of the F26A10.1 gene was calculated to be -0.02 with a P value of

0.4920 (Table 2). Since the corresponding P value is higher than 0.05, there is low statistical

confidence that the difference between the chemotaxis index of the F26A10.1 gene and the

control are significant, therefore, we accept the null hypothesis for the gene.

Z-score ProbabilityPdl-1 -1.84 0.0329F26A10.1 -0.02 0.4920

Table 2. The Z-scores and corresponding P-values of the experimental genes. We accept the null hypothesis for F26A10.1 and reject the null hypothesis for pdl-1.

DiscussionOur experiment was designed to investigate effect of removing certain proteins on the

chemosensory function of C. elegans.

First we determine whether the silencing of the unknown gene, F26A10.1, has any

effect on chemo-perception. As graphed in Figure 6, our results show that F26A10.1 has a

chemotaxis index of 0.63, which is lower than that of the negative control (0.65). In order to

gauge the precision of my data, it was compared to the class average data (Figure 6) and

yielded a 3% error (Table 1). This low percent error affirms the precision of my data.

Statistical analysis of the data lead us to accept the null hypothesis with low confidence (Table

2). In other words, the statistical procedure did not provide us with enough confidence to

accept that the removal of F26A10.1 had a significant effect on the chemo-attraction of the

worm to diacetyl. We conclude that the unknown gene F26A10.1 does not play a role in

chemotactic function.

Subsequently, we determined whether the silencing of the gene, pdl-1, has any effect

on chemosensation. As graphed in Figure 6, our results show that pdl-1 has a chemotaxis

index of 0.44, which is lower than that of the negative control (0.65). Statistical analysis of

this data lead us to reject the null hypothesis with 95% confidence (Table 2). In other words,

the statistical procedure supplied us with enough confidence to accept that the removal of the

pdl-1 gene had a significant effect on the chemo-attraction of the worms to diacetyl. The pdl-1

gene encodes for PDE6D, the phosphodiesterase responsible for hydrolyzing cGMP into

GMP. The use of cGMP to open cGMP-gated channels plays a crucial role in the function of

chemosensory neurons. As such, it is not surprising that blocking the translation of PDE6D

altered chemosensory function in C. elegans (Burghoorn, et al., 2006).

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Shiva Mozaffarian

My results confirmed the hypothesis that silencing network proteins responsible for

chemotaxis alter the organisms’ functionality. Although my datum is conclusive of this, the

class average leads to a different conclusion. A calculated 33% error to the class average

undermined the precision of my data. The class averages do not support my data or

conclusions based on my data, however, there are many factors that destabilize the reliability

of the class average chemotaxis index for the pdl-1 gene such as small sample size and a wide

range of chemotaxis values. The pdl-1 gene was silenced in only 30 experiments that

contributed to the class average, and the values of chemotaxis indices for pdl-1 ranged from

0.27-1.00.

Although statistical analysis of my data confirms that pdl-1 affects chemo-perception,

the overall data is inconclusive because of discrepancies when compared to the data of my

peers. In future experiments, in order to prevent such discrepancies, it may be beneficial to

increase the sample size of the experimental genes in order to increase the reliability of the

averages. In order to further ensure that the differences in the chemotaxis indices are

significant, the incubation time of the RNAi plates could be extended. This could help to

increase the number of worms on the chemotaxis plates and decrease the chance that the

distribution of the worms are due to random sampling. Further experimentation of pdl-1 with

these improvements set in place would assist in building a more reliable database that can be

used to conclusively prove this genes role in chemosensory function.

ConclusionThe experiment revealed conclusive evidence that the unknown gene F26A10.1 was

not involved in the chemosensory function of C. elegans. The experiment also revealed

inconclusive evidence to support predictions that pdl-1 is involved in these chemosensory

functions. The class average data did not support the statistical analysis of my results. Further

research of pdl-1 is warranted to settle these discrepancies.

ReferencesBargmann, C. I. (2006). Chemosensation in C. elegans. Wormbook, 2-16.

Burghoorn, J., Dekkers, M. P., Hukema, R. K., Jansen, G., and Rademakers, S. (2006). Antagonistic sensory cues generate gustatory plasticity in Caenorhabditis elegans. EMBO Journal 25, 313-318.

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Shiva Mozaffarian

Brenner S., Southgate E., Thomson J.N., and White J. (1986). The structure of the nervous system of the nematode Caenorhabditis elegans. Philosophical Transactions of the Royal Society 34, 212–240.

Bush, D., Kaska, D., and Paulsen, K. (2012). Exercise 4: Introduction to Research: The Nervous System of C. elegans. In Introductory Biology II Laboratory Manual (Santa Barbara: University of California), pp. 66.

Chao, Y. M., and Hart, C. A. (2009). From Odors to Behaviors in Caenorhabditis elegans. Neurobiology of Olfaction 1, 4-19.

Driver S.E., Fire A., Kostas S.A., Mello C.C., Montgomery M.K., Xu S. (1998).Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, pp. 806-811.

Maduro, M. F., and Pilgrim, D. B. (1995). Identification and cloning of unc-119, a gene expressed in the Caenorhabditis elegans nervous system. Genetics 141, 980-988.

Wormbase: Gene F26A10.1. (N.d.).Web. Retrieved from http://www.wormbase.org/db/get?name=WBGene000178;class=Gene