Seeing the light using blind cavefish
Aniket V Gore and Brant M Weinstein
The paper in Nature Ecology & Evolution is here: https://go.nature.com/2L7tDLJ
It was 2015 and we had just submitted the revised version of a study on the role of DNA methylation in zebrafish hematopoiesis. Our laboratory primarily studiesvascular development using the zebrafish as a model organism, but we had discovered that one particular DNA methyltransferase, dnmt3bb.1, was expressed in hematopoietic stem and progenitor cells (HSPC) and carried out a series of studies demonstrating that it was required for HSPC maintenance1. We decided to write a review on the role of DNA methylation during development and in the course of doing a lot of literature research on DNA methylation in different species we came across some very interesting papers on the Astyanax mexicanus (Mexican cavefish) and how its two morphologically and metabolically quite different but genetically very similar cave and surface morphs offer an excellent opportunity to study evolution and developmental genetics. Interestingly, no obvious inactivating mutations in the coding sequences of eye genes had been linked to eye loss in this species, despite numerous null mutations in key eye genes in other eyeless subterranean species such as the naked mole rat2. This suggested the possibility that epigenetic mechanisms might be contributing to eye loss in the relatively recently evolved Mexican cavefish morph. We were therefore particularly intrigued by a report showing that one of the QTLs for eye loss in the cave morph of Astyanax mexicanus maps close to DNA methyltransferase gene, dnmt3bb.13, an ortholog of zebrafish dnmt3bb.1. This was especially interesting to us because developing zebrafish also express dnmt3bb.1 in the eyes in addition to HSPCs, and during our earlier HSPC studies we had observed that the zebrafish dnmt3bb.1 mutants we generated also had enlarged eyes!
We decided to test the idea that Dnmt3bb.1 might be globally regulating eye gene expression and eye development and that eye loss in cavefish might be at least partly the result of excess Dnmt3bb.1-based eye gene repression. Our first problem was that our lab did not work on or keep cavefish. Luckily for us, one of the world’s foremost cavefish researchers was located only a few miles away at the University of Maryland in College Park, Dr. William Jeffrey. After receiving our first set of Astyanax mexicanus surface and cavefish embryos from the Jeffery lab and getting over our initial fascination with their interesting morphological differences, we set to work on a long series of studies involving numerous tissue dissections, DNA/RNA extractions, whole genome bisulfite/RNA sequencing runs, validations of the targets, whole mount in situ hybridization staining, histological sections, and eye “rescue” experiments. While all this work was going on in our cave and surface Astyanax mexicanus morphs, we were also carrying out a parallel analysis in DNA methylation- and DNA demethylation-deficient zebrafish mutants. As the results came in we were very excited to see that the pieces of the cavefish eye degeneration puzzle seemed to indeed be fitting together as we had hypothesized, with DNA methylation based eye gene repression playing an important role in cavefish eye loss. We presented our preliminary results at the biannualwhere it generated quite a bit of interest from the cavefish research community.
Completion of our study showed that DNA methylation-mediated combined silencing of eye genes contributes to eye degeneration in cave morphs of A. mexicanus. The increased expression of dnmt3bb.1 in A. mexicanus cave morphs is most likely due to a non-coding mutation in the gene or in regulatory sequences leading to its increased mRNA synthesis or increased mRNA stability. As we note in our paper, our results show that discrete changes in DNA methylation-based gene repression can have significantly amplified global effects and serve as an important molecular mechanism generating phenotypic diversity during development and evolution. This study would have been impossible without the help and support of many talented people including Daniel Castranova, Kelly Tomins, Andrew E. Davis, Li Ma, James Iben, and we benefitted from the advice and encouragement of a number of different members of the cavefish and zebrafish communities. Our experience on this study illustrates how a little “light reading” about unusual model organisms can lead to unexpectedly interesting and important new findings.
1 Gore, A. V. et al. Epigenetic regulation of hematopoiesis by DNA methylation. Elife 5, e11813, doi:10.7554/eLife.11813 (2016).
2 Kim, E. B. et al. Genome sequencing reveals insights into physiology and longevity of the naked mole rat. Nature 479, 223-227, doi:10.1038/nature10533 (2011).
3 McGaugh, S. E. et al. The cavefish genome reveals candidate genes for eye loss. Nat Commun 5, 5307, doi:10.1038/ncomms6307 (2014).