Zebrafish/Cavefish Clock Polar Regions Flashcards
Which genes are light responsive in zebrafish?
Cry1a and Per2 are light-regulated genes in zebrafish
What did (Tamai et al., 2004) show?
It is well known that in zebrafish spawning is tightly timed, and that the fish lay eggs just after dawn. This may sound illogical, becuase this means the embryo will undergo DNA replication and rapid cell division at the peak of diurnal UV light exposure, consequently increasing the chance of DNA damage dramatically at this sensitive early stage of development.
However it has been demonstrated that early exposure to light is actually beneficial and, in fact, essential for survival in early embryos. Embryos on the first day of development, raised in DD, show only a 20% survival rate when exposed to a 5 s UV-pulse compared to 85% survival for sibling embryos raised on LD cycles (Tamai et al., 2004). This increase in survival rate in embryos raised on LD is due to the light-induced expression of DNA-repair enzymes, such as, but not exclusively, 6-4 photolyase, which is expressed and transcriptionally light-regulated from 6 hpf. During the first 6 h of development, however, zebrafish rely on maternally deposited 6-4 photolyase transcript. The large quantities of maternally deposited 6-4 photolyase is not only found in zebrafish, but also in cavefish embryos, and undoubtedly in many other species of fish.
Why do we study random fish like the Mexican blind cavefish (Astyanax mexicanus)?
An alternative to exploring the clock and its downstream biology by “knocking out” key circadian or light-regulated genes, or performing forward genetic mutant screens, is to study “naturally occurring mutants”—especially animals that have adapted to a life in the dark.
Beale et al., 2013
They showed that surface fish had a robust Per1 circadian rhythm in L-D cycle in the lab as well as in the field. In contrast, the Mexican blind cavefish show no molecular or behavioural circadian rhythms when studied in the wild. However, several of the Mexican blind cavefish strains were able to entrain to LD cycles under lab conditions, although the phase and timing of per1 gene expression was altered. Per 1 oscillations had a lower amplitude and peak expression was 6hrs later.
Furthermore, they found that in cavefish light sensitivity is altered, with higher basal levels of light-inducible genes cry1a and per2a compared to the surface strains. These results support the idea that cavefish do not use a clock underground, but instead, possess tonically activated light-dependent processes, including enhanced DNA repair. Therefore from a molecular standpoint, cavefish appear as if they experience ‘constant light’ rather than perpetual darkness.
As per2 is a light-induced clock repressor and is thought to be important for clock entrainment in fish systems, this level of increased tonic expression could explain, at least in part, the reduced amplitude and altered phase of molecular clock rhythms in cave populations. Furthemore, it has been found that overexpression of per2 in zebrafish cell lines has a significant damping effect on per1 rhythms, which is also true for overexpression of cry1a (Tamai et al., 2007).
However, it was demonstarted that when surface and cave strain was crossed to produce a F1 hybrid offspring, the light induction of Cry1a and Per 2 was restored (same as in surface fish).
Beale et al., (2013) also found that in cavefish populations, two genes involved in DNA repair, CPD phr and ddb2, show raised basal levels of expression. Quantification of DNA repair within animals kept in the lab revealed that cavefish are indeed more efficient than surface fish at repairing damaged DNA. It is therefore plausible to suggest that the greater expression of DNA repair genes in the wild cave populations would also lead to enhanced enzyme activity.
Interestingly, recent studies have shown that CPD photolyase can interact directly with the CLOCK protein to reduce CLOCK/BMAL-dependent transactivation and dramatically repress the mammalian circadian oscillator (Chaves et al., 2011). It is possible that the high levels of CPD photolyase we observe in the cave could have the same effect and be, in part, responsible for the highly repressed levels of clock function we observe in the wild.
How do you predict the clock would function in Polar regions, where animals experience the constant lightof summer, and the constant darkness of winter?
They are unlikely to have circadian rhythms that are entrained to 24 hour LD cycles as it is not beneficial to them. It possible that they could become seasonally arrhythmic:
summer: altered clock, reduced amplitude, due to tonic higher levels of DNA repair genes which repress the clock??
winter: normal clock oscillations and period as less requirement for DNA repair genes to be tonically elevated, however, also no light cues for entrainment, therefore could show arrhythmicity and free-running
Or some animals could respond to non-light zeitgeber such as temperature, or light intensity variability
It has been suggested that changes in temperature and light colour through the day could be important zeitgebers. Several studies have shown that, even with bright sunshine, between 9 pm and midnight was often the coldest and dimmest part of the day in the Arctic.
