While we are no strangers to the role of epigenetics in environmental adaption, the lab of Brant Weinstein at the National Institute of Health (Maryland, USA) has brought forth an example of environmental adaption so extreme that it provides profound insight into eye degeneration and blindness. The talented team chose to examine the Mexican tetra Astyanax mexicanus, a tropical cavefish, as the entrapment of a group of these fish in dark caves a few million years gave rise to the Pachón blind cave morph.
Cave morphs begin eye development, but then undergo eye degeneration a few days after being born. Cave morphs eventually become fully blind at adulthood to conserve food in their nutrient-poor environment. Interestingly, cave and surface morphs can interbreed, but genomics has not uncovered any inactivating mutations in crucial eye genes.
Previously, while examining a zebrafish knockout of DNA methyltransferase 3bb.1 (dnmt3bb.1) for its role in hematopoiesis, the Weinstein lab also noticed an enlarged eye phenotype. Strikingly, in their current cavefish study, the team observed an up-regulation of dnmt3bb.1 and a down-regulation of crucial eye-genes via quantitative RT-PCR. This inverse relationship between dnmt3bb.1 and eye-size suggested that DNA methylation represented the missing link.
Here’s what the talented team discovered when they applied a differential comparison of RNA-seq and whole-genome bisulfite sequencing (WGBS) to RNA and DNA co-simultaneously isolated from the eyes of cave and surface fish larvae:
- A large number of eye-development genes show decreased expression
- Increased expression of dnmt3bb.1 was confirmed
- 128 genes exhibit promoter hypermethylation and decreases in expression
- 39 and 26 of these genes show expression in the mouse and human eye, respectively
- 19 of these of these genes are implicated in human eye disease
- Of the genes implicated in human eye disease, the group focused in on opn1lw1, gnb3a, and crx
- They confirmed promoter hypermethylation via targeted bisulfite sequencing and decreases in expression by whole-mount in situ hybridization
The team then turned to zebrafish double mutants deficient for tet2 and tet3, as the lack of these key players in DNA demethylation leads to genome-wide hypermethylation. They found that not only do these mutants have smaller eyes but also that targeted bisulfite sequencing and qRT-PCR revealed hypermethylation and decreased expression of gnb3aand crx in larval eyes.
Finally, the team tested whether they could rescue the eye degeneration phenotype by injecting the DNA methylation inhibitor 5-azacytidine (5-aza) into the eye of blind cavefish larvae. Upon injection, they found that 5-aza leads to a significantly larger and better-organized eye than either the uninjected control eye or a DMSO-injected vehicle control fish. They also observed that 5-aza injection caused gnb3a promoter hypomethylation and increased expression of gnb3a and crx.
Senior author Brant Weinstein shares, “Subterranean animals provide a unique opportunity to study how animals thrive in extreme environments, some of which can mimic human disease conditions. Many of the cavefish genes identified in our study are also linked to human eye disorders, suggesting these genes are conserved across evolution and may be similarly regulated in people.”