Frog legs: they’re smarter than they look!

By Sera Moon Busse

Studying limb regeneration in model organisms is important for the advancement of regenerative medicine in humans. We set out to study regeneration in the hind limbs of the African Clawed Frog Xenopus laevis – this animal is able to regenerate its hind limbs very early in development, but it loses this ability during metamorphosis. Additionally, there is a large and still growing body of evidence suggesting that charged particles called ions (for example, sodium, potassium, and chloride) are important for regulating pathways that control growth and regeneration. Though a relatively small number of biologists’ work is centered around this field, called bioelectricity, its implications thus far in the fields of developmental biology and regenerative medicine have been compelling. As an undergraduate, I had the privilege to be trained and to perform research in a lab that is at the forefront of this field, which had many unanswered and yet unresearched questions.

We – being myself and my team of mentors, Dr. Patrick McMillen and Professor Michael Levin, who were both Tufts undergraduates ten and twenty years before me, respectively – married the concept of bioelectrics and Xenopus hind limb regeneration to ask the question: what bioelectric changes do younger, regenerating froglets exhibit in response to amputation that older, non-regenerative tadpoles do not? This is part of the lab’s mission to discover regenerative therapies, based on manipulating bioelectric signaling, for various biomedical applications. We amputated the limbs of both regenerating tadpoles and non-regenerating tadpoles, and soaked them in a solution containing a molecule that glows in response to changing ion concentrations across cell membranes, also called membrane potential. By the time we were ready to analyze the results of this work, the product of observation and the scientific method led us to a bigger, more perplexing question to answer.

In performing these initial experiments, I did what I believed was my due diligence as a researcher: I needed controls! So I used the contralateral, uncut limbs of the froglets as controls, a common method used across many fields, to make sure the dye wasn’t being randomly soaked up by cells and to establish a baseline background for what the fluorescence would look like in un-injured, intact tissues. In fact, I quickly found that the dye was being taken up by cells in the contralateral limb, but only in the intact limbs of frogs that had the other limb amputated. Un-amputated froglets did not exhibit staining from the dye in either limb. I took this information to my mentors, sure that they would already have an explanation prepared, as professors and teachers before them always had when a question arose in lecture. For the first time in my academic career, nobody had an answer for me. This phenomenon had never been observed before, therefore nobody had asked the question; there was truly no one with an answer.

It is at this point in many young scientists’ careers that the potentially interesting project is swooped out from under them, their mentors realizing the value of the new findings, hungry for the credit. For others, their mentors do support intellectual curiosity and give them the freedom to pursue their own projects, but their projects do not see the light of day because there simply are not enough resources. For these reasons, the Tufts biology department, and more specifically, the Levin lab, are extremely unique. I could not have been more fortunate, because I was given both the freedom to head my own project and the resources to do so.

Ultimately, we found that the contralateral limbs of regenerating tadpoles glowed (in the presence of that molecule I mentioned earlier) in a region on the contralateral limb that very closely mirrors the plane of amputation. Our data revealed that the un-injured limb somehow knows the relative location (and even type) of injury, within about 30 seconds (Busse et al. Development 2018; doi:10.1242/dev.164210). This phenomenon has interesting implications for regenerative medicine. What we found is a distal region that not only recognizes, but also encodes information about an injury incurred by the body. If similar signaling phenomena can be found in mammalian systems, there is potential for the development of surrogate site diagnostics – looking at one site to decipher information about the health of another. This information is also evidence that contralateral limbs are not an appropriate control for experiments, and hopefully encourages everyone to think twice before using them as controls!

There are many more questions that we intend to answer in the future; for example, how does one part of the body sense that another part of the body has been injured? In the meantime, the context of this research is extremely important for anyone in any field of research to understand. I asked a question that nobody at the time could answer, and was given the opportunity to find an answer. The result was that I learned more collaborating with Dr. Patrick McMillen, Professor Michael Levin, and many others in the Levin lab than any biology course could have taught me. Many students are prompted to spend time answering questions throughout the duration of their degree, but I was encouraged to ask them, and it has fundamentally changed the way I think about learning. I had teachers who were not eager to prove what they knew, but rather who were eager to teach how they knew it.

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