Professor, students use chicken embryo eyes to research sight

By Amanda Phipps

phippsa@thejohnsonian.com

Eric Birgbauer looks at the retinas of chicken embryos to study axon guidance. Photo by Kathleen Brown • brownk@thejohnsonian.comUsing cells from a chicken embryo eye, one chemistry, two biology students and a professor studied the development of the visual system. Each student worked on a different aspect of the project.

Assistant professor of biology Eric Birgbauer worked with the students to study how the eye system works and how the signals are sent to the brain to allow sight.

Photoreceptors detect light and send it to cells in the visual system, Birgbauer said. Retinal ganglion cells (RGC) then transmit the messages from the photoreceptors to the brain. The ganglion cells are neurons in the eye that send out axons during development that grow and make connections to the brain.

“These axons need to be guided up to the brain and then to the correct position in the brain,” he said.

Several specific brain regions process vital information, Birgbauer said.

“These processing centers need to be connected to each other and in the right order,” he said.

The centers wire themselves, so they have to know how and where to connect in the brain, Birgbauer said. In the visual system in mammals, the first step in brain processing occurs in the LGN and superior colliculus. This means the visual input the eye sends to the brain along the axons of the RGC must go to these areas. In order for this to happen, the areas in the brain must be wired right during development so the axons can grow and connect in them.

“If they got wired to the wrong processing center, it would not work,” he said. “I am interested in learning how these correct connections are formed.”

Axons have “growth cones,” which are motile structures at the tip of the axon that sense the brain environment they are growing through and lead the axon to the right place, Birgbauer said. The growth cones read the molecules in the brain environment and determine if the axon should connect there, grow further or avoid that region and grow somewhere else.

“They act as the leaders of the axon,” he said.

Specifically, Birgbauer and the students are looking at what molecular signals are involved in guiding the axons to the brain. They are looking at the LPA (lysophospholipid acid) and S1P (sphingosine-1-phosphate) molecules to see if they are involved in this process.

“We suspect LPA and S1P might be involved as molecules in the brain that may guide axons away from the wrong places,” Birgbauer said. “They may also be involved in an injury response to prevent axons from growing if there is an injury.”

Birgbauer and the students are currently working with four receptor types of the axon used to detect LPA in the brain environment to see which one may be important to the development of the visual system.

“In experiments in the culture dish, when we add LPA, the growth cones of the retinal ganglion cell axons collapse and stop growing, at least temporarily,” he said.

This process may be able to guide the axons because if the axons grew to an area they didn’t normally grow that secreted LPA, they would collapse and avoid that region, going instead to the region they should grow, Birgbauer said. He compared this to the LPA molecule “smelling bad” to the axons, which they would then avoid.

The brain guides the axons using inhibitory molecules, which it places in regions the axon should not grow and uses molecules that attract the axons in regions they should grow, he said.

“What each axon detects and what it “smells like” depends on the receptors on the growth cone and how the cell interprets the signals from these receptors,” he said.

Different students are currently involved with the project and have been involved in the past.

Pathways Involved

Students previously established that LPA and S1P are involved in axon guidance. One of these students was senior chemistry and biology double-major Canaan Whiteneck, who worked with Birgbauer from the summer of 2008 to fall 2010.

Whiteneck worked with the LPA and S1P receptors to understand the signaling that occurs inside the cell that mediates the LPA/S1P induced growth cone collapse.

He used inhibitors to block the receptors and saw if blocking them caused growth cone collapse. Through his work, he discovered that both LPA and S1P were involved in this process.

S1P was originally going to be used as the control because it does not cause growth cone collapse in mice, Whiteneck said. However, when tested with chicken embryo RGC, it did cause growth cone collapse.

“It was not expected,” he said.

After he saw S1P had an effect, Whiteneck began to work on understanding if it had a similar effect that LPA does. He confirmed S1P did cause growth cone collapse, which is the effect LPA has in the chicken embryo retina.

Junior chemistry major Jarod Fincher confirmed Whiteneck’s data this year and is currently working on discovering the pathways LPA and S1P activate.

The axon guidance molecules guide the RGC to grow from the retina of the eye to form the optic nerve, according to chemistry major Jarod Fincher’s lab report.

Fincher worked on understanding which molecules are involved in axon guidance. The molecules LAP and S1P have been determined to cause growth cone collapse in the chicken embryo, according to the report.

Fincher’s report states: “These molecules LPA and S1P bind to G-protein coupled receptors which activate one of four possible intracellular pathways that, when activated, can lead to growth cone collapse.” He is currently working with the molecules to determine which of the four pathways they use.

Fincher does this by inhibiting one of the G-protein coupled receptor pathways that LPA and S1P activate.

He used the Gi pathway inhibitor Pertussis Toxin (PTX) and the G12/13 pathway inhibitor “Rock” to block two of the possible GPCR pathways that LPA and S1P use, he said.

“The results indicated that the Rock inhibitor prevented growth cone collapse in LPA, but remains inconclusive with S1P,” Fincher said. “There is also data that suggest LPA uses the Gi pathway from the PTX studies. More studies are being done with the PTX inhibitor to gain more conclusive results.”

These results can help explain which pathways are important in the guidance of axons, Fincher said.

“If the pathway is inhibited and there is no growth cone collapse, we know the molecules use that pathway,” he said.

The future may consist of more possible inhibitor studies, Fincher said.

“After we have enough data on those, we can look at other inhibitors and get more inclusive results,” he said.
    
Fincher heard about Birgbauer’s biomedical research experience through the biomedical director Kim Wilson, who helped him get into the lab.

“I was amazed at how academically successful he was, and I just couldn’t think of a greater opportunity ” he said. “To have the opportunity to work with someone who has achieved what Dr. Birgbauer has is an honor.”

Whiteneck said he enjoyed his experience when he worked in Birgbauer’s lab.

“Undergraduate research is amazing,” he said. “It gives you an edge (for graduate school.)”

Virus construction

To test the role LPA receptors play in axon guidance, biology major Josh Owens worked on constructing a virus that will knock out the LPA. There are five types of LPA receptors the LPA chemical binds to, he said. These are involved in the visual system. Owens is working with the LPA4 receptor.

The virus siRNA infects embryonic chicken retinas, but is harmless to humans, he said. Viruses work by modifying the genetic sequence of what it is attacking. His goal is to modify the virus so it will knock out the LPA4, a process known as “gene knockout.”

“We are making (the virus) do what we want it to do,” Owens said.

He has not modified the virus yet, but once it is completed, Owens will place it into the chicken embryo retina and study the affects, he said. This will help them determine the role LPA4 plays in axon guidance.

“If LPA4 is knocked out and something goes wrong (in the retina), we know the LPA was important for axon guidance,” he said. “A number of things could happen when it is knocked out.”

Owens said he will keep working with it until the virus is modified.

“There is a reason it is called research instead of just search,” he said. “We will know something when we do the experiment, which makes it worth doing.”

Each student worked on different aspects of the same project, Owens said. Eventually they will need to be finished with their part before the project can continue.

“Right now it looks as if we will all be finished at the same time,” he said. “That is when the cool stuff will happen.”

The Purpose

Once each aspect of the project is completed, it will be tested in the chicken embryo, Birgbauer said.

“(The research) is more complicated in the embryo, but it is more real,” he said.

LPA occurs in high concentration around injuries, Birgbauer said. The hope is that understanding axon guidance can help scientists use this knowledge for therapeutic processes.

If the inhibitors were removed, injured retinal cells may be able to regenerate nerves.

“If the optic nerve is damaged, it is permanent,” he said, “but if we could induce regeneration of this nerve and reconnection to the right place in the brain, we could restore sight for people with this injury.”

Other scientists have been researching this.

“There are many things going on that show some promise,” Birgbauer said.

The hope is that the receptor for LPA may become a druggable target, he said.

“We would have to validate that it is worth making a drug against,” Birgbauer said. “There is a way to go before it can be therapeutic for patients.”