One fish, two fish, red fish, blind fish

By Nour Bundogji (Pitzer College), Niti Nagar (Claremont McKenna College), and Sachin Shah (Claremont McKenna College) [Edited by Lars Schmitz, as part of BIOL 167 “Sensory Evolution”, an upper division class at the W.M. Keck Science Department. Written for educational purposes only].

Eyes are essential organs for detecting predators, foraging for food, finding shelter, mating, and other visually guided behaviors. But, how is it possible that evolution sometimes selects for the loss of eyes? More specifically, in what situations would this occur? The eyeless cavefish, Astyanax mexicanus, develop eyes as embryos but lose them as they mature (Jeffery, 2001). They serve as an ideal species to study because they are one of the few cave animals that have living surface-dwelling ancestors, which allows for a natural and direct comparison. The river surface provides a light environment where bigger eyes are more helpful than smaller eyes. However, in the cave, big eyes have no benefit because it is completely dark. For cave-dwelling fish, not having eyes may have a number of advantages. Energy spent on maintaining eyes can instead be conserved and invested in traits, such as expanded taste buds and cranial neuromast modules that are useful in a dark environment (Franz-Odendaal & Hall, 2003). Thus, it was adaptively advantageous for fish with smaller eyes to be selected.

Figure 1.  Blind Mexican cavefish [image from "Grand-Duc, Wikipedia, http://en.wikipedia.org/wiki/User:Grand-Duc"]

Figure 1. Blind Mexican cavefish [image from “Grand-Duc, Wikipedia, http://en.wikipedia.org/wiki/User:Grand-Duc”%5D

To answer how this evolutionary change occurred, it is required to take a closer look into the extent to which our biological programming can be altered by environmental influences. Unlike Darwin’s emphasis on mutation and subsequent natural selection, Conrad Waddington proposed evolution by pre-existing genes that could be turned on (or off) depending on environmental conditions. Although these pre-existing genes were usually hidden, they could be activated by particularly stressful environmental factors. In a landmark study, Waddington exposed fruit fly pupae to stress using bursts of heat or chemicals which resulted in an array of abnormal features, such as extra body segments in adult flies (Waddington, 1953). By applying heat or chemicals, Waddington lifted a repression mechanism that allowed for hidden genetic variation to manifest as physical variations.

Figure 2.  Waddington showed how developmental mechanisms could be studied through the analysis of mutations of the Drosophila wing [Photograph from Robertson, A. Conrad Hal Waddington 8 November 1905–26 September 1975. Biogr. Mem. Fellows R. Soc. 23, 575–622 (1977) © The Royal Society]

Figure 2. Waddington showed how developmental mechanisms could be studied through the analysis of mutations of the Drosophila wing [Photograph from Robertson, A. Conrad Hal Waddington 8 November 1905–26 September 1975. Biogr. Mem. Fellows R. Soc. 23, 575–622 (1977) © The Royal Society]

Under certain stressful environmental conditions, biological processes that mask genes can be destabilized, allowing for variance in genes and traits. When exposed to stress, variants that improve the animal’s ability to adapt to a new environment can then be selected for, and passed on to future generations. Thus, repression mechanisms allow certain genes to be masked resulting in unvarying traits within a population despite underlying genetic variation.

In accordance with Waddington’s idea, Rohner and his team of researchers may have found evidence for a mechanism of evolutionary change that is not due to natural selection of a spontaneous mutation for eye loss in A. mexicanus. The team demonstrated how “cryptic” or existing genetic variations in cavefish, which have been inherited from prior generations without causing any physical changes, can be “unmasked” by the shock of entering a new environment. Scientists hypothesized the environmental shock that induced the loss of eyes in cavefish occurred when a group of cavefish colonized deep and dark underwater caves. The cave presented an unfamiliar environment, where the water was purer and less conductive of electricity than the river water inhabited by surface fish.

It has been previously shown that the “heat shock” protein called HSP90 functions to buffer genetic variation and morphological changes. HSP90 helps other proteins fold properly, stabilizes proteins against heat stress, and aids in protein degradation.

Figure 3. HSP90 is activated under stressful conditions [Rohner et al., 2013]

Figure 3. HSP90 is activated under stressful conditions [Rohner et al., 2013]

Under stressful conditions, HSP90 can be depleted which allows for changes in protein folding that could ultimately lead to changes in phenotypes (Taipale et al., 2012). Under typical circumstances, cavefish show little variation in eye size. However, when treated with radicicol, a chemical that mimics stress by inhibiting HSP90, increased variation in eye size was seen in both surface and cavefish (Figure 4). HSP90 inhibition unmasked cryptic genetic variations, allowing for a larger range of eye sizes to be expressed in individuals. Interestingly, cavefish showed a significant decrease in orbit size (Figure 5). This difference suggests cavefish may have eliminated the upper range genetic variation for eye size and that genetic variation for eye loss is sensitive to HSP90.

Figure 4. HSP90 inhibition in F2 hybrids treated with radicicol reveal significant increase in SD of eye size, but not average eye size. Here are examples of eye size variation in F2 population hybrid  [Rohner et al., 2013]

Figure 4. HSP90 inhibition in F2 hybrids treated with radicicol reveal significant increase in SD of eye size, but not average eye size. Here are examples of eye size variation in F2 population hybrid [Rohner et al., 2013]

Figure 5. HSP90 inhibition in cavefish show increased variation and a lower average eye size [Rohner et al., 2013]

Figure 5. HSP90 inhibition in cavefish show increased variation and a lower average eye size [Rohner et al., 2013]

Secondly, experimenters found that the reduced conductivity of the cave environment can elicit a stress response in fish that causes the up-regulation of the “heat shock” protein called HSP90. Surface fish embryos raised in low-conductivity conditions of the cave up-regulated HSP90, showing that they are indeed in a state of physiological stress response. Moreover, they activated the same heat shock response gene with HSP90 inhibition by radicicol and similarly showed a significant increase in eye variation (Figure 6). Thus, the environment encountered by these fish during their evolutionary transition from surface to cave stressed the protein homeostasis mechanisms of the organism in a manner similar to the inhibition of HSP90’s chaperone activities. This demonstrates that a cave-specific environmental stress can inhibit biological processes resulting in the activation of genes for smaller eyes. This means genetic material that coded for proteins that produce smaller eyes already existed but wasn’t activated until stress was present.

Figure 6. Environmentally stressful conditions inhibit HSP90 and unmask genes which allows for greater genetic variation. [Rohner et al., 2013]

Figure 6. Environmentally stressful conditions inhibit HSP90 and unmask genes which allows for greater genetic variation. [Rohner et al., 2013]

Finally, the team investigated whether these developmentally induced changes could be genetically assimilated, meaning could the phenotype for eye loss become genetically encoded via natural selection. To test for genetic assimilation, Rohner and his team treated surface fish with radicicol and interbred individuals with the smallest eyes. Even when raised without radicicol, the stress stimuli, offspring had significantly smaller eyes than the untreated adult population (Figure 7). Therefore, once genes for small eyes were activated and manifested as physical traits, they could be passed down to future generations despite the presence of environmental stress.

Figure 7. F2 generations of radicicol treated offspring have significantly smaller eye sizes [Rohner, et al., 2013]

Figure 7. F2 generations of radicicol treated offspring have significantly smaller eye sizes [Rohner, et al., 2013]

Although there is convincing evidence that eye development is buffered by HSP90 and stress responses result in increased heritable eye size variation, we think the argument of genetic assimilation is somewhat lacking. The experiment conducted by Rohner and his team does not have a control group of untreated surface fish. If the F2 generation of untreated surface fish produced similar results, a more compelling explanation is needed. Alternatively, if there is very little response to selection, the results become much more convincing.

Nevertheless, Rohner and colleagues’ findings give us great insight on evolutionary mechanisms and confirms Waddington’s hypothesis that genetic variation can be exposed by disruptions in development mechanisms caused by environmental stress. Yet, these findings also open a door for further investigation such as determining if HSP90 controls the evolution of many species or a few. Furthermore, the process relies on sudden environmental changes that usually occur at microscopic scales whereas larger organisms experience evolution in gradually changing environments. So, are blind cavefish an exception to the norm or the first example of a common phenomenon? This poses the question if natural selection by pre-existing genetic variation is more common in smaller than larger organisms and to what extent does environmental stress play a role in dictating traits of other organisms. Additionally, taking a closer into the neuronal visual processing function in cavefish would be interesting. Do these cavefish essentially lose their ability to visually process and if so, what other type of processing replaces it?

This style of evolution is no different from what Darwin envisioned. Variation is still passed down to generations and produces traits that affect the fitness of individuals. The only difference is that the variation is not caused by spontaneous mutations, but already exists just in a hidden form. This variance still provides a platform on which natural selection can run its course–it’s just faster.

References

Franz-Odendaal, T., Hall B. K. 2006. Modularity and sense organs in the blind cavefish, Astyanax mexicanus. Evolution & Development 8: 94-100. (DOI: 10.1111/j.1525-142X.2006.05078.x)

Jeffery, W. R. 2001. Cavefish as a model system in evolutionary developmental biology. Developmental Biology 231: 1-12. (DOI: 10.1006/dbio.2000.0121)

Rohner H., Jarosz D. F., Kowalko J. E., Yoshizawa M., Jeffery W. R., Borowsky R. L., Lindquist, S., Tabin, C. J. 2013. Cryptic variation in morphological evolution: HSP90 as a    capacitor for loss of eyes in a cavefish. Science 342: 1372-1375. (DOI: 10.1126/science.1240276)

Taipale, M., Krykbaeva, I., Koeva, M., Kayatekin, C., Westover, K. D., Karras, G. I., Lindquist, S. 2012. Quantitative analysis of HSP90-client interactions reveals principles of substrate recognition. Cell 150: 987–1001. (DOI: 10.1016/j.cell.2012.06.047)

Waddington, C. H. 1953. Genetic assimilation of an acquired character. Evolution 7: 118.

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