I’m often asked if I miss benchwork. On balance, the answer is usually no. Working as an editor allows one to come to grips with a wide range of fascinating science without the daily disappointments of actually doing the experiments. Of course, you also miss out on the thrill of a new discovery. If I had it do over again, I think the burgeoning intersection of genetics and evolution would be an area that might have held my interest for the longest period of time as an experimentalist. The ability to apply molecular genetic tools to organisms with interesting evolutionary histories, such as the cichlid and the stickleback (see here, here, and here, for example) seems like a uniquely appealing interdisciplinary development (for some of this year’s standout findings, see Science’s breakthrough of the year special). That fundamental new insights into the genetic basis of evolution are occurring against the backdrop of the unfortunately resilient distraction known as ‘intelligent design’ is one of the great ironies of our time (see the full text of the recent Kitzmiller decision, however, for good news on this front). This is all by way of introducing the second in our series of ‘Paper Trail’ reports from authors of NG papers. First author Meredith Protas explains the origins of her work generating a linkage map of the cavefish Astyanas mexicanus, and its application to the evolution of albinism, as reported in the journal.

Meredith Protas writes:

During my first year in graduate school, one of my professors mentioned that Dr. Cliff Tabin was starting to work on the genetic basis of beak size variation in the Galapagos finches. I was amazed that it was possible to look at the genetics behind classical systems in evolutionary biology and joined Cliff’s lab to work on this project. Eventually, this particular project became impossible to do, but after seeing the amazing variation in beak size and shape between the different Galapagos finch species I still wanted to pursue the genetic basis of morphological diversity. We searched for another system in which these types of questions would be possible to address. At this time, there were QTL analyses being performed on cichlids and sticklebacks, which already had provided interesting and useful information about how certain morphologies evolved. For many of the same reasons that cichlids and sticklebacks were chosen for QTL analyses, as well as many additional unique features, we were drawn to the system of Astyanax mexicanus, the Mexican cave tetra. We then set up a collaboration with Dr. Richard Borowsky at NYU, who was working with this species (see also here).

Cave animals are a fascinating group. Two very common characteristics in obligate subterranean species (ranging from salamanders to shrimp) are loss of eyes and loss of pigment. Because similar light-less environments cause or allow these same phenotypes to occur over and over again in vastly different organisms, it is a system well suited to the study of parallel evolution. Looking at fish alone, there are 86 known species of obligate cave dwelling fish that have some degree of eye and pigment loss. The mystery behind cave animals is why are eye size and pigmentation reduced? There are three main theories; the first is that eye size and pigment loss is advantageous in cave animals because of energy conservation. The second is that eye and pigment loss is advantageous in cave animals because the genetic changes that cause eye and pigment loss also cause adaptive changes that allow the animals to be better suited to life in the cave. The final theory is that neutral mutation causes regression of the eye development and pigmentation pathways because there is no selective advantage to maintaining these systems in the dark environment.

Astyanax mexicanus has two basic morphs, the surface or river dwelling morph which is eyed and pigmented and the cave-dwelling morph which has very regressed eyes, is albino, has an increased number of tastebuds, and has many other morphological and behavioral differences. The two morphs interbreed, both in the wild and in the laboratory. In addition, there are 30 known cave populations of A. mexicanus, some of which are thought to have evolved the distinguishing characteristics independently. Also, from a practical standpoint, this species can be bred in the laboratory, has relatively small sized adults, has a 4-6 month time to maturity, produces large numbers of offspring per spawn, and many studies, both genetic and developmental in nature have already been performed on these fish. Similarities between zebrafish and A. mexicanus have recently allowed for the techniques of morpholino injections and RNA overexpression to be performed in A. mexicanus further increasing the genetic potential of the system.

Of course, though the system is ideal in many ways, there are many hurdles to surmount when working on a non-model system, the first being obtaining the organism. I was lucky enough to go on a cavefish collecting expedition in Mexico with our collaborator Dr. Richard Borowsky. It is amazing to descend into these lightless caves, trudge through huge piles of bat guano, and then in pools, some of which contain very little water, see pink, eyeless fish. The biggest hurdles occurred after coming back to the lab and attempting to breed the fish; highlights included floods, cannibalism, and the near overturning of a 50 gallon vat of water in front of the medical school.

Despite these issues, finally we generated a microsatellite linkage map of A. mexicanus. We focused on the phenotype of albinism and found that albinism was linked in two different cave populations to the gene Oca2, Ocular and cutaneous albinism gene 2, which is the most common gene mutated in cases of human albinism. We found different deletions in the coding region of Oca2 in the two different cave populations and using a cell based system showed that these deletions cause loss of function of the Oca2 protein. Therefore, we showed that the two cave populations have evolved albinism independently by mutation in the same gene.

Further questions that need to be addressed are: Is Oca2 mutated in the other two albino populations of A. mexicanus? Are mutations in Oca2 also responsible for albinism in other albino cave-dwelling vertebrate species (fish and salamanders)? Could it be possible that the function of Oca2 is conserved even to invertebrates and that mutations in this gene are responsible for albinism in cave-dwelling invertebrates as well? If mutation in Oca2 is the most common way to cause albinism in cave animals, is this because the structure of the gene supports rapid genetic change or that Oca2 mutations have pleiotropic effects and confer some kind of adaptive function on the animal in the cave environment? By addressing these questions, we will have a better understanding of the specifics and breadth of parallel evolution as well as the knowledge of how traits that appear to provide no benefit to the organism, such as albinism, evolve.