January 2012

Why are cavefish a good example of evo-devo?

Jeffery: Scientists study all kinds of organisms in evolutionary developmental biology, but when I started working in the evo-devo field, I decided that in order to understand how development evolved, we would have to look at two closely related species that have diverged recently [developed in separate directions] or to look at the same species in the process of divergence. I looked around for models, and I found several of them. One of them happened to be in caves, and the species is called Astyanax mexicanus, the blind Mexican cavefish. This cave organism is one of the few in which the acknowledged ancestor is still present on the surface, and the descendent organism is still present in the caves. They are the same species but they are in the process of divergence. These species can breed with one another, which allows for the genetic study of interesting traits. This combination of special circumstances fits my criteria for ideal development models. We could determine what the ancestral situation looked like by studying today’s surface fish and examine what the derived species looked like by studying present cavefish.

After the divergence, how do the cavefish differ from their surface counterparts?

Jeffery: Well, actually, we can study this very nicely in caves, particularly in Mexico. Cavefish populations were founded independently of one another and at different times. Which means we have some populations to study that are relatively young, that were founded recently, in addition to some that were founded early on in their evolutionary history. We can look at both the early and the late populations, and we can get an idea of what happened almost immediately when some fish moved from the surface to live in caves.

Interestingly, two things that happened in the changed environment are that the cavefish started to lose its pigmentation [coloring], and it started to lose its eyes (fig. 1). This was not a sudden phenomenon; it took some time, and we know that because the intermediates still have a little bit of pigment, and they have bigger eye remnants than the older forms.

Figure 1. The teleost _Astyanax mexicanus_ has diverged into a surface form (left) and a cave form (right); the cavefish has lost pigmentation and eyes. Research and photos from the laboratory of William Jeffery.

To describe what happened, I would say that first there was a great separation by natural selection as to which organisms could survive and which could not. Second, those that could survive have exacerbated their chances for survival through inbreeding. Then selection acted upon the phenotypes [expressions of genetic traits] that were highly adaptive to their cave environment. This weeding out process is still going on. This is a classic example of survival of the fittest.

If evolution adapts for survival of the fittest, how is being eyeless a survival mechanism?

Jeffery: Well, eyelessness is not the survival mechanism—I think it is the by-product. When you first look at cave organisms, you are immediately aware of two features: lack of eyes and lack of pigment. But many other things are going on that are not obvious to the naked eye, and if you could look very closely, you would see that there are constructive features that have evolved as well. Some of these features are not found on the body surface—like changes to the brain or sensory organs. In some cases, the fish have a heightened olfactory sensitivity—the organisms can smell better, or they have evolved the ability to detect vibrations.

They detect vibrations along their lateral line, a characteristic unique to aquatic vertebrates that sense pressure changes and movement in the water.¹ It is similar to the antennae on insects in its function. These fish have an increased ability to conserve energy and an increased ability to store metabolic products for sparse times. All of these things are beneficial for living in caves.

Now, how is the loss of things like eyes and pigment related to survival? This question has been a big question mark. My personal opinion from doing many experiments is that the lost features are a by-product of those features that are gained. For example, in the case of eyes, eyes are connected in development through a pleiotropic gene [a gene that controls many seemingly unrelated traits²] called Sonic hedgehog [Sonic hedgehog gene (SHh)] with the development of feeding structures, such as the jaws and the taste buds. These traits are enhanced in cavefish; for example, the jaw gets bigger, and perhaps, its function is improved as well. The improvements in this particular case could not have happened without a reduction in the size of the eye because that is just the way pleiotropy works. The evolution of cavefish has happened in a relatively short period during evolutionary time, and because it was so short, it is likely that the easiest changes were made. But there are by-products of those changes, and one of them happens to be the loss of the eyes. Of course, this could only happen in the dark, where eyes are useless anyway.

Did the enhanced features come first, and therefore, lead to the degenerating effects, or was it the other way around?

Jeffery: We cannot know for sure but I would say that based upon the theory of pleiotropy, it would be those features that were selected for constructive uses that would adapt a fish or any other animal to the cave. Then, as a by-product, other things were lost, or space was needed to put those other features into action.

When I refer to space, I mean morphological [structural] space. So, for example, to make bigger neuromasts, sensory organs in the skin of the head that are part of the lateral line system and detect water movements, you need more surface space, and with having large eyes there, it does not give you enough surface space. If you are going to have more taste buds, and more sensory structures, a better olfactory system, and a better gustatory system (that is the taste system), you need places in the brain that can integrate the information and convert it into behaviors. Organisms, therefore, need to create space in the brain that controls these places, as well. The space being used for visual input is now defunct, so that space can be taken over; there is only so much space that can fit in a head, and it can be taken over by some sensory functions that are more important now. This is an old idea; in fact, Theodosius Dobzhansky, an evolutionary biologist from the first part of the last century, had this idea but experiments have now been done to support it.

Are the cavefish becoming genetically different from their surface relatives as quickly as the morphological changes are happening?

Jeffery: Genetic changes would happen before morphological changes. So yes, they are changing genetically. The gene Sonic hedgehog has not mutated in cavefish—as far as we can tell, the sequences are the same in cavefish and surface fish. Therefore, an upstream gene must control Sonic hedgehog to make it expand its role in cavefish, and therefore, result in more taste buds and bigger jaws. Scientists are searching for these genes but they have not found them yet. When you do genetic crosses with cavefish, you can detect genes and you can study them. When you do crosses with cavefish and surface fish, you get progeny that are intermediate, as far as eyes are concerned—they have eyes, but they are smaller. Then, if you breed the progeny, you get a broad distribution, all the way from animals lacking sight to ones with big eyes. This tells you there are many genes involved in the process and that they segregate independently in that lineage. So, it is a genetic principle that allows you to find this. If there are many genes, there are probably many ways to destroy the eye, and we know a few of them. One example is a pleiotropic interaction with taste buds, but there are probably other examples to illustrate the selection pressure for this phenomenon. A mechanism probably affects eye development; although, in the case of space, we do not know what the exact mechanism is.

Without eyes, how do they sense food, movement, or predators?

Jeffery: This is the natural situation in caves. Without eyes, they probably sense food by using novel behaviors; for example, the behavior in which they are attracted to vibrations in the water. Vibrations in the water can result from many things, but often, they indicate food, wiggling crustaceans, which is the case for fish. Additionally, being able to get there quickly, before your competitor does, would be a good thing to do because that food is sparser in the cave than it is on the surface. This evolved behavior is risky for the cavefish because if you are guided to every movement, that is risky—you could be surprised by a moving predator rather than your prey. That would not work on the surface, but it works in the cave because there are very few organisms high in the pyramid of life that could serve as predators. Cavefish do not usually have macroscopic predators in the cave, so they can evolve these risky behaviors. Scientists use this behavior to their advantage. When they go to collect cavefish, they put a net in the water and the vibration alone will attract the fish. That is a risky behavior to have in any other place, and if I were a predator that would be the end of that cavefish.

Is vibration the same as the shadow effect?

Jeffery: No, the shadow effect is something entirely different. The shadow effect is something that is present in surface-dwelling fish [which typically flee from a shadow], as well as cave-dwelling fish. The effect is caused by a temporary shading of light, so it would not happen in a cave. When I talk about that shadow effect, which cavefish have retained, the question is, “Why have they retained it?” Selection is relaxed for anything like that—they have not seen the light for a million years. But those phenotypes are not completely destroyed during life in a cave—even if they are not used, they are kept. As for why they are kept, it may be that these traits are involved in some other phenomenon that we do not know about yet.

The shadow response is controlled by the pineal organ [an endocrine gland in the vertebrate brain, also known as “the third eye”³], which in fishes is responsive to light, particularly in photoperiods. The pineal gland produces melatonin, and as a result, organisms undergo circadian rhythms. As far as we know, this does not happen in a cave; although we are not so sure about that because all of the circadian rhythms we know about are dependent upon light. Other types of circadian rhythms that are dependent on things other than light may exist; for example, bats go in and out of caves according to light. When they go back into the cave in the morning, they go back to the roost, and they shake and things fall out, such as ectoparasites [parasites that subsist in or on the skin but not inside the body]. That would be a good time for a cavefish to be swimming right underneath the bat in a pool because of the food provided; so, cavefish can be in tune to things like that. There may be ways other than light in which circadian rhythms are controlled.

Is this loss of pigment a universal feature in cave creatures? And if so, why?

Jeffery: Well, loss of eyes is fairly universal too; but before we go on, I should make the point that some cave dwellers have not lost their eyes. Those animals are just as interesting as the ones that have lost their eyes, and examining them would help explain the loss of the eye too; but they are not studied very often. The situation with pigment is that there is a very broad convergence among different organisms that have lost their pigment in caves. Additionally, this happens not only in caves but also deep in the soil or manmade tunnels, or in parasites existing in the body of other animals. It is very common that pigmentation is lost in all sorts of groups of animals after lack of exposure to light.

Let me digress for a minute—this also happens in humans—there are many different types of albinos [people who lack of pigmentation] in humans. We can study them effectively because there is no natural selection against them, and so there is something that produces albinos frequently in nature, and in environments where it is not adaptive, albino organisms are filtered out. Now, why would they be filtered out? Well, one reason is pigmentation protects organisms from ultraviolet (UV) radiation, so in lighted environments, they could be filtered out because they have less offspring—because they have melanomas, for example. This is probably not so in caves, where UV radiation does not penetrate. So albinism can persist without dire consequences in the dark cave environment, and there may be something good about this trait too. Many bad phenotypes can have good features—sickle cell anemia and so forth. Anyway the question is, “What are these good features?” Well, we will get back to that in a second, but let us just start out with: What is the cause of melanism [dark coloration of skin]? We do not know the cause of albinism in every cave animal, and it is something I am very interested in, and trying to study.

We know a lot about it in cavefish thanks to the work of Meredith Protas, who was a graduate student in Cliff Tabin’s lab at Harvard University,⁴ several years ago, and she was able, through genetic analysis, to find out what gene was involved in the loss of pigmentation in cavefish. It is actually a longer story than that because perhaps twenty years before, genetic analysis, such as the type that I described to you for loss of eyes, was also done for loss of pigment in cavefish, and actually, a different result was obtained: a single mutated gene was found. To make the story short though, Protas did some crosses of phenotypes like in the earlier experiment. The pathway leading to melanin is very well known throughout the animal kingdom because there are not that many genes involved. It is a very simple, linear path, so Protas worked with just a few candidate genes. She mapped the candidate genes onto the place in the cavefish genome, and she discovered one candidate gene that mapped very closely with the genetic defect. This turned out to be a gene called, Oculocutaneous albinism type 2 (the reason for the long name is that it is an albinism gene that was already known), and it caused a loss of pigmentation in both the eyes and the skin of humans. Cavefish have naturally developed albinism using the same gene as humans have.

We know this gene is causing albinism in cavefish at least, but we do not know if it is the same gene in other cave animals; I suspect it might be. Maybe this is just a gene that has a high frequency of mutation. So it keeps appearing, keeps appearing, and keeps appearing. On the lighted surface, it is a deleterious feature [having harmful effect(s)] because UV radiation can kill; but in a cave, it is not. It is possible there is something else good about this the mutant form of this gene, but we do not know what it is, although there are a couple of possibilities.

Has any of your research been used to support human medical applications?

Jeffery: Yes, some of it does get used. I have a grant from the National Eye Institute [part of the National Institutes of Health], and they are very interested in the causes of cataracts, for example, and it turns out this happens to be a downregulated gene. Some genes are downregulated in the cavefish lens—one is called alpha-A-crystallin, and that gene is involved in cataract formation in humans.⁵ So studying the deficiency in alpha-A-crystallin could help us understand how cataracts are formed in humans.

The lens is an often neglected, but it is a very important organizer in the formation of the eye itself. The lens not only controls itself, it also controls other eye tissues around it, so for those tissues to prosper and develop normally, normal lenses are probably required. This has implications for human lens development. We are working on experiments to take a lens out of a surface fish to see what happens, We expect there would be some deficiencies in the retina when the lens is taken out. There may be clues here to understanding more about eye diseases because many eye diseases in humans involve the retina.