- Email: [email protected]
Restoring sight in blind cavefish
Magazine R23
which sleep uni-hemispherically or bi-hemispherically depending on the context [3], captive cetaceans almost never sleep bilaterally [1,8,9] even when they are motionless [10]. Unraveling how marine mammals manage the inherent tradeoff [3] between wakefulness and sleep will require the development of procedures for observing eye closure and other physiological variables, including EEG, in free-ranging animals. Supplemental data Supplemental data are available at http:// www.current-biology.com/cgi/content/ full/18/1/R21/DC1 References 1. Mukhametov, L.M., Supin, A.Y., and Polyakova, I.G. (1977). Interhemispheric asymmetry of the electrocephalographic sleep patterns in dolphins. Brain Res. 134, 581–584. 2. Siegel, J.M. (2005). Clues to the functions of mammalian sleep. Nature 437, 1264–1271. 3. Rattenborg, N.C., Amlaner, C.J., and Lima, S.L. (2000). Behavioral, neurophysiological and evolutionary perspectives on unihemispheric sleep. Neurosci. Biobehav. Rev. 24, 817–842. 4. Miller, P.J.O., Johnson, M.P., Tyack, P.L., and Terray, E.A. (2004). Swimming gaits, passive drag and buoyancy of diving sperm whales Physeter macrocephalus. J. Exp. Biol. 207, 1953–1967. 5. Amano, M., and Yoshioka, M. (2003). Sperm whale diving behavior monitored using a suction-cup-attached TDR tag. Marine Ecol. Prog. Ser. 258, 291–295. 6. Campbell, S.S., and Tobler, I. (1984). Animal sleep: a review of sleep duration across phylogeny. Neurosci. Biobehav. Rev. 8, 269–300. 7. Crocker, D.E., LeBoeuf, B.J., and Costa, D.P. (1997). Drift diving in female northern elephant seals: implications for food processing. Can. J. Zool. 75, 27–39. 8. Lyamin, O.I., Mukhametov, L.M., Siegel, J.M., Nazarenko, E.A., Polyakova, I.G., and Shpak, O.V. (2002). Unihemispheric slow wave sleep and the state of the eyes in a white whale. Behav. Brain Res. 129, 125–129. 9. Ridgway, S.H. (2002). Asymmetry and symmetry in brain waves from dolphin left and right hemispheres: some observations after anesthesia, during quiescent hanging behaviour, and during visual obstruction. Brain Behav. Evol. 60, 265–274. 10. Lyamin, O.I., Pryaslova, J., Kosenko, P., and Siegel, J. (2007). Behavioural aspects of sleep in bottlenose dolphin mothers and their calves. Physiol. Behav. doi:10.1016/j.physbeh.2007.05.064 1Sea
Mammal Research Unit, School of Biology, University of St. Andrews, Fife, KY16 8LB, UK. 2Ocean Research Institute, The University of Tokyo, 1-15-1 Minamidai, Nakano, Tokyo 164-8639, Japan. 3Department of Animal Sciences, Teikyo University of Science and Technology, 2525 Yatsuzawa, Uenohara, Yamanashi 409-0193, Japan. E-mail: [email protected]
Restoring sight in blind cavefish Richard Borowsky Twenty-nine populations of the blind cavefish, Astyanax mexicanus, are known from different caves in North-Eastern Mexico (Figure 1). They evolved from eyed, surface-dwelling forms which only reached the area in the mid-Pleistocene [1]. Quantitative genetic analyses have shown that the evolutionary impairment of eye development — as well as the loss of pigmentation and other cave-related changes — results from mutations at multiple gene sites (‘eye loci’) [2,3]. Eye loss has evolved independently at least three times [4,5] and at least some of the eye loci involved differ between the different cave populations [3]. Hybrids between blind cavefish from different caves have larger and better developed eye rudiments than their parents (Figure 2) [6], reflecting these independent origins and complementation [3,7,8]. Given the large number of mutations at different loci that have accumulated in these populations, we reasoned that hybridization among independently evolved populations might restore visual function. Here we demonstrate restoration of vision in cavefish Figure 1. Map of NorthEastern Mexico indicating locations of the cave populations studied. Molino and Pachón are 53 km distant and located in different drainages. Pachón and Tinaja are over 60 km distant and also in different drainages, while the Tinaja and Curva caves are only 12 km distant and in the same drainage.
whose immediate ancestors were blind and whose separate lineages may not have been exposed to light for the last one million years. We generated numerous F1 and F2 hybrid crosses among cave populations to test whether hybrids could see, and to elucidate the underlying genetics. Purebred cave and surface fish served as controls. We also created compound hybrids by crossing F1 surface/caveA hybrids with F1 surface/caveB hybrids. Eye regression progresses with age [7], so we studied fry (9 to 40 days old), to have the best chance of detecting vision. We measured body length and eye size and tested for vision using the optokinetic response: the reflexive following of moving stripes by the subject’s eyes [9]. Purebred cavefish from Molino, Pachón and Tinaja caves were tested for their optokinetic response (Supplemental data) and/or evidence of a dorsal light reflex, visual orientation to food items or response to a looming dark object. All cavefish were blind, consistent with the observation of serious defects in both lens and retinal structure during ‘Stage III’ of eye development [7]. Quantitative trait loci (QTL) studies identified at least twelve unlinked eye loci in Pachón cavefish [2]. For purposes of discussion only, we take this number to be typical of
Current Biology Vol 18 No 1 R24
Figure 2. Eye restoration in Astyanax. Cave, intercave F1 hybrids, and surface Astyanax mexicanus fry illustrating relative eye sizes. The Pachón individual is albino and its eye size is indicated by the paired pipe marks in the top panel. Intercave hybrids have larger eyes than pure cave fish but significantly smaller eyes than surface fish.
A. mexicanus cave populations. The average additive effect of each QTL was 64% of its total effect [2]. Thus, the genetics of eye size variation is largely additive. F1 hybrids between surface fish and cave fish have smaller eyes than surface fish, but are fully visual [7], even into adulthood (data not shown). Thus, one surface allele at each of the population-specific eye loci is sufficient for restoring vision. In contrast, 54% of the Pachón/surface and 24% of the Tinaja/surface F2 hybrids were blind. Estimating from the Poisson distribution, these correspond
approximately to the proportion of progeny being homozygous for ‘cave alleles’ at three or four of the twelve eye loci. This suggests that loss of function at three or four eye loci suffices to abolish vision. F1 hybrid progeny between different cave populations all had some individuals that exhibited clear optokinetic responses (Supplemental data). On average, 39% of Molino/Tinaja, 8% of Pachón/Tinaja, and 1% of Molino/Pachón F1 hybrids could see. (The Molino/Pachón fry were albino, which might account for the low percentage.) Significantly fewer of the F2 than F1 Molino/ Tinaja hybrids could see (8% vs. 39%; Fisher’s exact p << .001), reflecting the increased probability of homozygosity in the F2. To estimate the extent of non- overlap between sets of eye loci between cave populations, we tested compound hybrids for vision. Offspring of Tinaja/surface crossed to Pachón/surface hybrids (ET/EP) and offspring of Molino/surface crossed to Pachón/ surface hybrids (EM/EP) had high proportions of seeing individuals (0.940 ± 0.026 and 0.950 ± 0.022, respectively); a significantly lower proportion of offspring from Curva/ surface crossed to Tinaja/surface hybrids (EC/ET) could see (0.409 ± 0.074; Fisher’s exact p << 0.001). In their proportions of visual competence, ET/EP and EM/EP progeny resemble cave/surface F1 hybrids, while EC/ET progeny resemble cave/surface F2 hybrids. Based on complementation, our interpretation of the high proportions of seeing individuals in ET/EP and EM/EP crosses is that the Pachón population is genetically distinct from both the Molino and Tinaja populations. While there are alternative explanations for the non-complementation and low proportion of seeing ET/EC individuals, the most parsimonious is that the geographically proximate Curva and Tinaja populations have a shared cave ancestry and shared eye loci. These interpretations are consistent with geography (Figure 1): the Molino and Pachón caves as well as Pachón and Tinaja are distant, while Tinaja and Curva are close [1]. Thus,
Molino, Pachón, and Tinaja-Curva represent three independent multilocus convergences on eye regression. This observation underscores the power of a well defined environment to repeatedly direct the evolution of the same end phenotype, regardless of initial genotype. Supplemental data Supplemental data including experimental procedures are available at http://www.current-biology.com/cgi/ content/full/18/1/R23/DC1 Acknowledgments This study was supported by grants from the US National Science Foundation (IBN0217178; CT and RB) and US National Institutes of Health (1RO3EYE016783-01; RB). I thank M. Agyeiwaah, K. Capaccione, M. Conrad, C. Creange and L. Mekiou for technical help, H. Wilkens for supplying the Molino fry and B. Borowsky for thoughtful discussion of the manuscript. References 1. Mitchell, R.W., Russell, W.H., and Elliott, W.R. (1977). Mexican Eyeless Characin Fishes, Genus Astyanax: Environment, Distribution, and Evolution (Lubbock: Texas Tech Press). 2. Protas, M., Conrad, M., Gross, J.B., Tabin, C., and Borowsky, R. (2007). Regressive evolution in the Mexican cave tetra, Astyanax mexicanus. Curr. Biol. 17, 452–454. 3. Wilkens, H. (1988). Evolution and Genetics of epigean and cave AstyanaxFasciatus (Characidae, Pisces) - Support for the neutral mutation rheory. Evolutionary Biol. 23, 271–367. 4. Dowling, T.E., Martasian, D.P., and Jeffery, W.R. (2002). Evidence for multiple genetic forms with similar eyeless phenotypes in the blind cavefish, Astyanax mexicanus. Mol. Biol. Evol. 19, 446–455. 5. Strecker, U., Faundez, V.H., and Wilkens, H. (2004). Phylogeography of surface and cave Astyanax (Teleostei) from Central and North America based on cytochrome b sequence data. Mol. Phylogenet. Evol. 33, 469–481. 6. Wilkens, H. (1971). Genetic interpretation of regressive evolutionary processes Studies on hybrid eyes of 2 Astyanax cave populations (Characidae, Pisces). Evolution 25, 530–544. 7. Wilkens, H. (2007). Regressive evolution: ontogeny and genetics of cavefish eye rudimentation. Biol. J. Linn. Soc. 92, 287–296. 8. Wilkens, H., and Strecker, U. (2003). Convergent evolution of the cavefish Astyanax (Characidae, Teleostei): genetic evidence from reduced eye-size and pigmentation. Biol. J. Linn. Soc. 80, 545–554. 9. Easter, S.S., Jr., and Nicola, G.N. (1996). The development of vision in the zebrafish (Danio rerio). Dev. Biol. 180, 646–663.
Richard Borowsky Department of Biology, New York University, New York, New York 10003, USA. E-mail: [email protected]
which sleep uni-hemispherically or bi-hemispherically depending on the context [3], captive cetaceans almost never sleep bilaterally [1,8,9] even when they are motionless [10]. Unraveling how marine mammals manage the inherent tradeoff [3] between wakefulness and sleep will require the development of procedures for observing eye closure and other physiological variables, including EEG, in free-ranging animals. Supplemental data Supplemental data are available at http:// www.current-biology.com/cgi/content/ full/18/1/R21/DC1 References 1. Mukhametov, L.M., Supin, A.Y., and Polyakova, I.G. (1977). Interhemispheric asymmetry of the electrocephalographic sleep patterns in dolphins. Brain Res. 134, 581–584. 2. Siegel, J.M. (2005). Clues to the functions of mammalian sleep. Nature 437, 1264–1271. 3. Rattenborg, N.C., Amlaner, C.J., and Lima, S.L. (2000). Behavioral, neurophysiological and evolutionary perspectives on unihemispheric sleep. Neurosci. Biobehav. Rev. 24, 817–842. 4. Miller, P.J.O., Johnson, M.P., Tyack, P.L., and Terray, E.A. (2004). Swimming gaits, passive drag and buoyancy of diving sperm whales Physeter macrocephalus. J. Exp. Biol. 207, 1953–1967. 5. Amano, M., and Yoshioka, M. (2003). Sperm whale diving behavior monitored using a suction-cup-attached TDR tag. Marine Ecol. Prog. Ser. 258, 291–295. 6. Campbell, S.S., and Tobler, I. (1984). Animal sleep: a review of sleep duration across phylogeny. Neurosci. Biobehav. Rev. 8, 269–300. 7. Crocker, D.E., LeBoeuf, B.J., and Costa, D.P. (1997). Drift diving in female northern elephant seals: implications for food processing. Can. J. Zool. 75, 27–39. 8. Lyamin, O.I., Mukhametov, L.M., Siegel, J.M., Nazarenko, E.A., Polyakova, I.G., and Shpak, O.V. (2002). Unihemispheric slow wave sleep and the state of the eyes in a white whale. Behav. Brain Res. 129, 125–129. 9. Ridgway, S.H. (2002). Asymmetry and symmetry in brain waves from dolphin left and right hemispheres: some observations after anesthesia, during quiescent hanging behaviour, and during visual obstruction. Brain Behav. Evol. 60, 265–274. 10. Lyamin, O.I., Pryaslova, J., Kosenko, P., and Siegel, J. (2007). Behavioural aspects of sleep in bottlenose dolphin mothers and their calves. Physiol. Behav. doi:10.1016/j.physbeh.2007.05.064 1Sea
Mammal Research Unit, School of Biology, University of St. Andrews, Fife, KY16 8LB, UK. 2Ocean Research Institute, The University of Tokyo, 1-15-1 Minamidai, Nakano, Tokyo 164-8639, Japan. 3Department of Animal Sciences, Teikyo University of Science and Technology, 2525 Yatsuzawa, Uenohara, Yamanashi 409-0193, Japan. E-mail: [email protected]
Restoring sight in blind cavefish Richard Borowsky Twenty-nine populations of the blind cavefish, Astyanax mexicanus, are known from different caves in North-Eastern Mexico (Figure 1). They evolved from eyed, surface-dwelling forms which only reached the area in the mid-Pleistocene [1]. Quantitative genetic analyses have shown that the evolutionary impairment of eye development — as well as the loss of pigmentation and other cave-related changes — results from mutations at multiple gene sites (‘eye loci’) [2,3]. Eye loss has evolved independently at least three times [4,5] and at least some of the eye loci involved differ between the different cave populations [3]. Hybrids between blind cavefish from different caves have larger and better developed eye rudiments than their parents (Figure 2) [6], reflecting these independent origins and complementation [3,7,8]. Given the large number of mutations at different loci that have accumulated in these populations, we reasoned that hybridization among independently evolved populations might restore visual function. Here we demonstrate restoration of vision in cavefish Figure 1. Map of NorthEastern Mexico indicating locations of the cave populations studied. Molino and Pachón are 53 km distant and located in different drainages. Pachón and Tinaja are over 60 km distant and also in different drainages, while the Tinaja and Curva caves are only 12 km distant and in the same drainage.
whose immediate ancestors were blind and whose separate lineages may not have been exposed to light for the last one million years. We generated numerous F1 and F2 hybrid crosses among cave populations to test whether hybrids could see, and to elucidate the underlying genetics. Purebred cave and surface fish served as controls. We also created compound hybrids by crossing F1 surface/caveA hybrids with F1 surface/caveB hybrids. Eye regression progresses with age [7], so we studied fry (9 to 40 days old), to have the best chance of detecting vision. We measured body length and eye size and tested for vision using the optokinetic response: the reflexive following of moving stripes by the subject’s eyes [9]. Purebred cavefish from Molino, Pachón and Tinaja caves were tested for their optokinetic response (Supplemental data) and/or evidence of a dorsal light reflex, visual orientation to food items or response to a looming dark object. All cavefish were blind, consistent with the observation of serious defects in both lens and retinal structure during ‘Stage III’ of eye development [7]. Quantitative trait loci (QTL) studies identified at least twelve unlinked eye loci in Pachón cavefish [2]. For purposes of discussion only, we take this number to be typical of
Current Biology Vol 18 No 1 R24
Figure 2. Eye restoration in Astyanax. Cave, intercave F1 hybrids, and surface Astyanax mexicanus fry illustrating relative eye sizes. The Pachón individual is albino and its eye size is indicated by the paired pipe marks in the top panel. Intercave hybrids have larger eyes than pure cave fish but significantly smaller eyes than surface fish.
A. mexicanus cave populations. The average additive effect of each QTL was 64% of its total effect [2]. Thus, the genetics of eye size variation is largely additive. F1 hybrids between surface fish and cave fish have smaller eyes than surface fish, but are fully visual [7], even into adulthood (data not shown). Thus, one surface allele at each of the population-specific eye loci is sufficient for restoring vision. In contrast, 54% of the Pachón/surface and 24% of the Tinaja/surface F2 hybrids were blind. Estimating from the Poisson distribution, these correspond
approximately to the proportion of progeny being homozygous for ‘cave alleles’ at three or four of the twelve eye loci. This suggests that loss of function at three or four eye loci suffices to abolish vision. F1 hybrid progeny between different cave populations all had some individuals that exhibited clear optokinetic responses (Supplemental data). On average, 39% of Molino/Tinaja, 8% of Pachón/Tinaja, and 1% of Molino/Pachón F1 hybrids could see. (The Molino/Pachón fry were albino, which might account for the low percentage.) Significantly fewer of the F2 than F1 Molino/ Tinaja hybrids could see (8% vs. 39%; Fisher’s exact p << .001), reflecting the increased probability of homozygosity in the F2. To estimate the extent of non- overlap between sets of eye loci between cave populations, we tested compound hybrids for vision. Offspring of Tinaja/surface crossed to Pachón/surface hybrids (ET/EP) and offspring of Molino/surface crossed to Pachón/ surface hybrids (EM/EP) had high proportions of seeing individuals (0.940 ± 0.026 and 0.950 ± 0.022, respectively); a significantly lower proportion of offspring from Curva/ surface crossed to Tinaja/surface hybrids (EC/ET) could see (0.409 ± 0.074; Fisher’s exact p << 0.001). In their proportions of visual competence, ET/EP and EM/EP progeny resemble cave/surface F1 hybrids, while EC/ET progeny resemble cave/surface F2 hybrids. Based on complementation, our interpretation of the high proportions of seeing individuals in ET/EP and EM/EP crosses is that the Pachón population is genetically distinct from both the Molino and Tinaja populations. While there are alternative explanations for the non-complementation and low proportion of seeing ET/EC individuals, the most parsimonious is that the geographically proximate Curva and Tinaja populations have a shared cave ancestry and shared eye loci. These interpretations are consistent with geography (Figure 1): the Molino and Pachón caves as well as Pachón and Tinaja are distant, while Tinaja and Curva are close [1]. Thus,
Molino, Pachón, and Tinaja-Curva represent three independent multilocus convergences on eye regression. This observation underscores the power of a well defined environment to repeatedly direct the evolution of the same end phenotype, regardless of initial genotype. Supplemental data Supplemental data including experimental procedures are available at http://www.current-biology.com/cgi/ content/full/18/1/R23/DC1 Acknowledgments This study was supported by grants from the US National Science Foundation (IBN0217178; CT and RB) and US National Institutes of Health (1RO3EYE016783-01; RB). I thank M. Agyeiwaah, K. Capaccione, M. Conrad, C. Creange and L. Mekiou for technical help, H. Wilkens for supplying the Molino fry and B. Borowsky for thoughtful discussion of the manuscript. References 1. Mitchell, R.W., Russell, W.H., and Elliott, W.R. (1977). Mexican Eyeless Characin Fishes, Genus Astyanax: Environment, Distribution, and Evolution (Lubbock: Texas Tech Press). 2. Protas, M., Conrad, M., Gross, J.B., Tabin, C., and Borowsky, R. (2007). Regressive evolution in the Mexican cave tetra, Astyanax mexicanus. Curr. Biol. 17, 452–454. 3. Wilkens, H. (1988). Evolution and Genetics of epigean and cave AstyanaxFasciatus (Characidae, Pisces) - Support for the neutral mutation rheory. Evolutionary Biol. 23, 271–367. 4. Dowling, T.E., Martasian, D.P., and Jeffery, W.R. (2002). Evidence for multiple genetic forms with similar eyeless phenotypes in the blind cavefish, Astyanax mexicanus. Mol. Biol. Evol. 19, 446–455. 5. Strecker, U., Faundez, V.H., and Wilkens, H. (2004). Phylogeography of surface and cave Astyanax (Teleostei) from Central and North America based on cytochrome b sequence data. Mol. Phylogenet. Evol. 33, 469–481. 6. Wilkens, H. (1971). Genetic interpretation of regressive evolutionary processes Studies on hybrid eyes of 2 Astyanax cave populations (Characidae, Pisces). Evolution 25, 530–544. 7. Wilkens, H. (2007). Regressive evolution: ontogeny and genetics of cavefish eye rudimentation. Biol. J. Linn. Soc. 92, 287–296. 8. Wilkens, H., and Strecker, U. (2003). Convergent evolution of the cavefish Astyanax (Characidae, Teleostei): genetic evidence from reduced eye-size and pigmentation. Biol. J. Linn. Soc. 80, 545–554. 9. Easter, S.S., Jr., and Nicola, G.N. (1996). The development of vision in the zebrafish (Danio rerio). Dev. Biol. 180, 646–663.
Richard Borowsky Department of Biology, New York University, New York, New York 10003, USA. E-mail: [email protected]