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The Effect of Ephrin B3 on the Development of

Rohon Beard Neurons

Steven Forsythe

Zoology 555

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Abstract

Rohon Beard neurons are sensory neurons present in the development of

zebrafish and some vertebrates. They grow from the developing spinal cord and

innervate the skin in order to provide sensory input for the developing organism.

EphrinB3 is present in the midline of the spinal cord and for is an avoidance cue

developing neurons. In the present study, transgenic NGN:GFP zebrafish were

injected with EphrinB3 morpholino and the resulting Rohon Beard neuron growth

was quantified. Analysis of our results could not conclude a link between the growth

of Rohon Beard neurons and the knockdown of Ephrin B3 guidance cues. Although

our research did not conclude there was a link between EphrinB3 and the

development of Rohon Beard neurons, the creation and overall standardization of

our process could lead to further research that may provide a conclusive answer.

Introduction The Eph/Ephrin signaling system is one of the most widely utilized signaling

pathways amongst developing organisms. The Ephrin signaling system consists of

Eph receptors and Ephrin ligands. Eph receptors are tyrosine kinases that

phosphorylate their targets upon binding with ephrin ligands. However,

bidirectional signaling in which the ligand is instead phosphorylated also

occurs.1Unlike other axon guidance cues, neurons must physically contact one

another in order for the Eph/ephrin repulsive cue to function. The effects of ephrin-

B3 are concentration-dependent, in that a larger amount of ligand binding will

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produce a greater repulsion. Ephrin-B3 contains a transmembrane domain that acts

to anchor the ligand. Axon guidance requires precise signaling, and the

transmembrane domain may act to further localize the repulsive cues of ephrin-B3.

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Ephrin-B3 has been shown to mediate various developmental events,

particularly in the nervous system. Ephrin-B3 is expressed in the floor plate at the

ventral midline of the zebrafish embryo. 2 It provides repulsive cues towards

commissural axons preventing crossing of axon projections at the midline of the

spine.3 In particular, ephrin-B3 acts as a midline barrier that prevents corticospinal

projections from recrossing into the spinal gray matter. In a study involving

knockout mice of ephrin-B3, it was found that the mice had a loss of unilateral

motor control. These mice showed a peculiar hopping gait in which they could only

move both limbs simultaneously. The recrossing of these axons into the spinal gray

matter results in bilateral input to the motor cortex, leading to a loss in unilateral

motor control. 4

The particular cells of interest to us, Rohon Beard neurons, do not cross the

midline of the spinal cord.5 Rohon Beard neurons are specialized neurons that serve

as sensory neurons in the dorsal part of the spinal cord of developing fish and

amphibian embryos. The neurons innervate the skin of the trunk and tail projecting

from the spinal cord longitudinally and respond to both light touch and more

noxious stimuli.6 RB axons are found in the embryo ranging from near otocyst in the

hindbrain to the caudal end of spinal cord.7 In zebrafish, Rohon Beard neurons

generally develop around 10 hours post fertilization and most undergo apoptosis

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between days 2-5 post fertilization; they are functionally replaced by dorsal root

ganglion neurons.8

There may be a connection between ephrin signaling and the formation of

the dorsal root ganglion (DRG) in mammals, which is mediated by the Runx1

transcription factor. Runx1 is expressed in both Rohon beard neurons and their DRG

successors.8 Therefore, if a relationship between ephrin-B3 and Rohon Beard

neurons in zebrafish can be determined, there may also be a connection between

ephrin-B3 signals and the formation of mammalian dorsal root ganglion.

The zebrafish transgenic line utilized in this experiment is Ngn:GFP (Figure

1). It expresses a fluorescence pattern noticeable in the Rohon Beard neurons of the

posterior half of the animal’s spinal cord. Ephrin-B3 is expressed at the ventral

midline of the spinal cord.9 In this experiment, a mopholino was used to knockdown

ephrin-B3 expression to allow our group to determine whether it plays a role in the

growth of Rohon Beard neurons. The potential relationship between ephrin-B3 and

Rohon beard neurons has not been previously researched, making this a novel

experiment. Our group expects to see a decrease in longitudinal neural projections

in the Ephrin-B3 knockdown group. The reason for the decreased projections

relates to Ephrin B3 being a repulsive cue; without it, the Rohon beard neurons will

not being negatively reinforced to move to the ventral side of the embryo and as a

result the injected will exhibit decreased Rohon Beard neuron projection density as

compared to the control group.

In the present study, we were unable to conclude a link between the

longitudinal growth of the Rohon Beard axon projections and the knockdown of

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Ephrin B3. Our results showed that the neuron projection density for both the

control and the injected embryos was not different enough statistically to be

relevant. Ephrin B3 repulsive cues are not a factor in the axon growth of Rohon

Beard neurons away from the dorsal towards the ventral side of the developing

zebrafish.

Figure 1

Figure 1 shows the Ngn:GFP transgenic fish. The fluorescence of the Rohon Beard neurons can be seen located primarily in the posterior spine. This image was taken at 24 hpf with low magnification camera. The area circled is representative of the area selected for imaging. Methods

Animals

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Ngn:GFP transgenic zebrafish embryos were used for this experiment. Half of the

embryos were used for microinjection and the other half served as noninjected

controls.

Microinjection of Ephrin B3 morpholinos/ Fixing

We knocked down Ephrin B3 expression using morpholino injection containing

Ephrin B3 morpholino oligomers. This was done to observe Ephrin’s effect on

Rohon Beard axon development from the spinal cord. The injections were

performed at the one cell stage and embryos were allowed to develop for 24 hours.

The injected and control embryos were fixed using paraformeldahyde at 24 hpf in

order to observe similar stages of development between embryos.

Imaging

Fixed embryos were washed and mounted for imaging. The head and yolk sac were

removed prior to mounting the embryos. Images were taken at 200x and 400x on

high magnification confocal scope.

Analysis

Images were processed using Photoshop. To quantify results, we selected sections

from the posterior spinal column between the yolk sac and the yolk tube and

manually counted Rohon Beard axon projections from the spinal cord that could be

linked to a single cell body (Figure 2). Areas outside of this were determined to

have too much background fluorescence to image properly. All branches coming

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from the same cell body were counted as one projection. Once images were

reviewed, a student’s t-test was used to determine any significant difference

between axon projection of wild type and Ephrinb3 morphants. Alongside looking at

the number of axon projections, we also studied the pattern of projection. Since

Ephrin has been shown to be a guidance cue, particularly a repulsive cue, we

thought there would be an observable difference in projection pattern between

contol embryos and Ephrin B3 morphants. Since Rohon Beard axons generally

project longitudinally and laterally from the spinal cord, we expected to see more

sporadic or horizontal projections due to a lack of guidance from the spinal column.

Lateral projections should not be affected. After we qualitatively analyzed the

images for sporadic branching, no noticeable difference was noted and we did not

pursue it further.

Figure 2

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Figure 2 is a 400x control shot with the neurons that were counted for analysis circled with red. Note that although branching makes the center neuron appear to be two, it originates from the same cell body. The total projection count for this image is three. Results

Of the 249 embryos injected, 130 survived for 24hpf. Of the 215 control

embryos, 164 survived. The percentage of survival was 52.2 and 76.3 accordingly.

The experiment provided 64 viable images to analyze, a breakdown of 44 control

and 20 injected. The neurons imaged at 200x and 400x were analyzed separately.

The control images from 200x had mean number of 15.86 Rohon Beard neuron

projections and the injected had a mean number of 10.75 (Figures 3a and 3b).

Although the numbers seem far apart, the images had a statistically insignificant p-

value of 0.18. The control neurons imaged at 400x had a mean number of 7.43 and

the injected had a mean number of 8.25 (Figure 4a and 4b). The neurons imaged at

40x had a p-value of 0.50, which is also not statistically significant.

Figure 3

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Figure 3a (Top) shows a 200x magnification control image of Rohon Beard neurons. Figure 3b (Bottom) shows a 200x magnification injected image of Rohon Beard neurons. The mean of 15.8 for control was well in excess of the injected mean of 10.75; however the small sample size of 20 made it difficult to get a significant p value. Figure 4

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Figure 4a (Top) shows a 400x magnification control image of Rohon Beard Neurons Figure 4b (Bottom) shows a 400x magnification injected image of Rohon Beard Neurons. Note the lateral projects of the Rohon Beard neurons along the spinal column. Our hypothesis would suggest these are from a lack of Ephrin expression.

The difference in means between the two, 7.43 for control and 8.25 for injected, as well as the large p-value of .5, shows that in this reference frame

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there is no difference in longitudinal Rohon Beard Neuron density. The larger sample size present for this number also collaborates the statistical analysis. Discussion

Based on the results we gathered throughout the course of the experiment,

we are unable to conclude a link between Ephrin B3 axon guidance and the

longitudinal Rohon Beard neuron axon density. Although extensive research has

shown the repulsive nature of Ephrins in neuron guidance, our results show that the

growth of Rohon Beard neurons towards the ventral side of the developing embryo

is not linked to Ephrin B3 expression. The p values gathered from the student T-test

were the most telling piece of date collected. The 400x images proved to be less

useful for determining the projection counts from each section as part of the neuron

projections were usually cropped from the image. The key was to get a large enough

section to capture enough Rohon Beard projections to get larger numbers. The 200x

images were optimal for this calculation because they included a large enough area

to get an accurate count and in addition they kept the branching in focus, allowing

for a branch analysis if one became available. The p value, although showing

insignificance, was most likely skewed due to the smaller sample size. Using the

mean, however, showed a 50% difference in projection density and had more

images been taken, there would be a clearer answer to the original study question.

As this was a novel experiment, our group constantly refined procedures

until ending with the final methods presented in this paper. Several unexpected

issues presented during the course of the experiment and the following discussion

reflects these issues and how our group found solutions to each of the problems

encountered. Some issues were not overcome during the course of this experiment

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and possible solutions to those problems are presented for the discretion of future

researchers.

The Ngn:GFP embryos used provided many interesting insights into the

growth of the Rohon Beard neurons. However, they did come with several issues of

their own. An unexpected problem encountered was the failure of embryos

contained in a 1:00PM sunrise environment to successfully spawn and produce

usable embryos, costing our study two periods of work. This resulted in our group

changing to an environment where the sunrise came at 10:00PM, solving this issue.

In addition, injecting the NGN:GFP embryos neutralized a large amount of the

embryos; a fact which our group was aware but still hindered our efforts to

accumulate results. Also an additional embryo type we planned to analyze for

neurological development proved to be unsuccessful. The Pax2a genotype our group

planned to use to determine if hindbrain development was impeded by Ephrin B3

knockdown showed extraordinarily high phenotypical defects for both control and

injected embryos. After analyzing these embryos, we resolved to focus on the

NGN:GFP embryos to strengthen our results.

Overall the imaging process contained several issues that were not apparent

until analysis of results began. Many of the surviving embryos that were mounted

onto slides to be analyzed showed too little fluorescence to be of use to the

experiment. Some of this can be accounted to expected failure of all embryos to

express fluorescence; however the group discovered an additional problem.

Embryos that had been fixed for over one week would result in severely reduced

fluorescence and almost invariably failed to produce images. Scheduling problems

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such as days between fixing and imaging embryos were accountable for these errors

and standardizing the schedule allowed this to be fixed. Antibody staining may also

assist in allowing for an extended viewing window. Additionally, human error must

be taken into account for the experiment as each counter may have used a slightly

different criteria for branched axon to cell body counting.

Branch analysis, a large focus point of the experiment, was proven

impossible due to a number of issues. A method of branching analysis in which the

group was interested, the Sholl analysis, was proven ineffective as it only focuses on

the connections of a single axon. This made the use of Sholl ineffective, as it was

desired to use a method that could count the total branches present in each cell

body. Also complicating issues, as shown by Figure 5, was the issue of getting the

entire axon body into the same focus plane. Fine tune focusing proved difficult to

include all of the branching present for each neuron as they innervated the skin of

the embryos. A solution presented is the removal of the head and yolk entirely to

create as flat of an image as possible, allowing for all of the axon branching being in

the same plane of view. Relating to branch analysis is the use of 400x magnification

as a way to make branch analysis easier. As projections were analyzed, it was

determined the magnification was too great to properly see many of the axons

terminate towards the ventral side. As a result, these images were impossible to use

for branch analysis. A future recommendation would be to focus only on 200x

magnification as this demonstrated the entirety of the axon projection while not

obscuring any branching.

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There is another study that has been used before could also be used to test

our hypothesis in this study. A motility test could be created to determine if the

injected embryos have reduced sensitivity to touch.10This could be accomplished by

dechorionating an embryo and performing a light touch to see if it reacts in a

manner similar to a kinesis movement. The angle of movement of the tail could also

be recorded to determine if has reduced stimulation in avoiding the touch. The

experiment would have to be performed within 24-48 hours post fertilization in

order to avoid the natural Rohon Beard apoptosis. An additional study that we could

potentially test is the effect on varying amounts of morpholino that would be

injected into the developing embryo. As we only tested for one dose and knowing

that Ephrin signaling can be dose dependent, it would be an appropriate question to

ask if the changing doses cause a difference in neuron projection density.

This study relates to several current issues that are in the midst of being

researched today. Research has shown there is a common gene responsible for the

growth of both Rohon Beard neurons and the mature Dorsal Root Ganglion neurons

found in vertebrates: Runx1. Runx1 is crucial for the development of both neurons

and the lack of the transcription factor could have serious consequences for the

developing axons.8 This research could help to gain an understanding of disorders

where the neurons do not fully form and if a relation to the gene is found it could

result in a treatment being found. Another research path involves the pdrm1 gene, a

gene found in both zebrafish and mammals. For zebrafish, the gene is involved in

the differentiation of the Rohon Beard neuron.11 However, in humans it plays a large

role in the creation of B-lymphocytes and is largely involved in the proliferation and

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survival of these cells. Mutations in the pdrm1 gene have been linked with the

creation of lymphomas.12 If the mechanism behind the cause of the mutation of this

gene can be linked to a model zebrafish Rohon Beard deformity, it could help our

understanding of why the mutation causes lymphomas in humans and a treatment

method may be discovered for those found to be afflicted.

There are also several current research projects involving Ephrin B3. One

future research avenue involves another member of the Ephrin family, Ephrin A4

and its receptor Eph A4. Ephrin B3 also binds to the receptor Eph A4.13 Current

research has shown that the loss of the Eph A4 receptor can result in the recovery of

zebrafish with the mutant phenotype SOD1 and improves the survival of mice

suffering from ALS.14 In humans, the over activity of Eph A4 results in decreased

survival and its repression can lead to a longer life. If future research looked into the

reason why Eph A4 receptor inactivity can prolong life, it may be important to

developing drug treatments and possibly lifestyle changes that increase the average

lifetime of humanity dramatically. Another possibility involves studying the effects

of knockout EphrinB3 in the perspective of NT-3 signaling. NT-3, or neurotrophin 3,

is involved in the proliferation of neurogenic stem cells, and a lack of the NT-3

signaling results in the planned termination of Rohon Beard neurons.15 Other

research has shown Ephrins can lead to loss of NT3, possibly leading to cell death.16

Further research could look to see if Ephrin signaling affects the process of Rohon

Beard neurons planned apoptosis, and whether the lack of Ephrin caused by the

knockdown injection could cause Rohon Beard neurons to have a longer survival

rate than the controls.

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Although our research did not provide the solution that our group was

pursuing, we believe that our improved research process along with the refined

techniques developed can still salvage our hypothesis. With more trials, stricter

guidelines and a clear goal ahead, it is our opinion that a difference can be found in

the Ephrin B3 knockdown and control embryos’ Rohon Beard neuron longitudinal

density and hopefully can also determine an effect on branching of neurons.

Figure 5

Figure 5 is a 200x image and shows the lack of clarity often presented in images captured by confocal microscope. The area towards the developing spine (red) on the left is obscured, making this image unusable for our counting analysis. Also note the poor definition of the branching (Blue) would also prove this image unusable for any branching analysis. - References

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