The evolutionary and developmental mechanisms responsible for the loss of vision in cave animals are not understood. This proposal continues Dr. Jeffery's research on eye degeneration in the teleost Astyanax mexicanus, which has an eyed surface-dwelling form (surface fish, SF) and many eyeless cave-dwelling forms (cavefish, CF). CF form eye primordia but they arrest in growth and degenerate due to lens apoptosis. The results of his previous grant showed that: (1) Hedgehog (Hh) signaling is enhanced along the CF anterior midline and controls eye degeneration by inducing lens apoptosis, (2) the molecular chaperone Hsp90< is specifically activated in the CF lens and required for lens apoptosis, (3) Hh expression along the embryonic midline is gradually restricted to developing taste buds, and (4) expression of genes positively regulated by Hh signals (e. g. pax2.1a in the optic vesicles and nkx2.1a/b in the neural plate) are expanded in CF. Constructive traits that may compensate for vision, including increases in taste buds, olfactory nerve tracts and bulbs, and the ventral telencephalon and hypothalamus, have also evolved in CF. This continuation proposal explores the hypothesis that eyes have degenerated due to enhanced Hh midline signaling though pleiotrophic positive and negative effects on downstream target genes resulting in developmental tradeoffs between constructive and regressive traits. The specific objectives are: (1) to determine whether Hh signaling controls lens apoptosis by affecting an antagonism between the Dlx5 (and/or Pitx3) and Pax6 transcription factors and/or by regulating Hsp90< expression and coordinately increasing olfactory development, (2 and 3) to determine whether enhanced Hh signaling is responsible for (2) the increased number of taste buds in CF and (3) for enlargement of the ventral telencephalon and hypothalamus in CF, and (4) to determine by genetic analysis whether gustatory, olfactory, and hypothalamic enhancement are coupled to eye degeneration as developmental tradeoffs. Most of the methodologies and reagents to be used in the continued work, including a panel of more than 100 Astyanax cDNAs, have been acquired in the previous grants, and Dr. Jeffery has preliminary results showing that his hypothesis is plausible and ready for more intensive investigation. The results of this study are expected to provide new insights into the mechanisms responsible for CF eye degeneration.
Broader Impacts:
Studies of cave animals advance discovery and understanding because they provide unique perspectives to research and education in evolutionary developmental biology. Two hypotheses, neutral mutation and natural selection, have been advanced to explain regressive changes such as eye loss in cave animals, but until recently there was little or no experimental support for either of them. These experiments on Astyanax CF are the first to suggest that eye loss is an indirect effect of natural selection on adaptive traits via the positive and negative effects of pleiotropic Hh signals. Pleiotropic effects may have general implications in the evolution of development. In addition to the Hh pathway, other critical developmental signaling pathways have negative and positive regulated targets and the potential to generate evolutionary changes by developmental tradeoffs. The generation of evolutionary changes via pleiotropy is largely underappreciated as a mechanism for directing microevolutionary changes in development, and these studies are designed to shed more light on this process. Another broad impact of this research is the continued integration of research and education, which is accomplished by involving undergraduate students in original research, by training pre-doctoral and post-doctoral investigators, and by the laboratory's outreach to high school science programs. All of these activities include different genders and underrepresented minorities. The cavefish laboratory and colony will continue to be a useful and highly appreciated local resource for teaching and understanding evolutionary biology to high school students, and will also continue to serve as a resource for their short supervised science projects. The degeneration of the cavefish eye is an excellent example of a morphological change that is experimentally tractable, has occurred very recently, and is subject to interpretation through evolutionary theory. Finally, the cavefish colony has an impact beyond this laboratory, serving to support other researchers and springboard the generation of similar colonies internationally.
