The Regulation of Sleep and Circadian Rhythms: The Role of Melatonin and Adenosine in Zebrafish

Sleep is a highly conserved behavioral state whose regulation is still unclear. In this thesis I initially briefly introduce the known sleep circuitry and regulation in vertebrates, and why zebrafish is seen as a good model to study sleep-regulation. I describe the existing two-process model of sleep regulation, which posits that the two processes C (circadian) and S (homeostatic) control timing of sleep-wake behavior. I then study the role melatonin plays in the circadian regulation of sleep using zebrafish. Firstly, we find that the absence of melatonin results in a reduction of sleep at night, establishing that endogenous melatonin is required for sleep at night. Secondly, melatonin mutants show a reduction in sleep in animals with no functional behavioral rhythms suggesting that melatonin does not require intact circadian rhythms for its effect on sleep. Thirdly, melatonin mutants do not exhibit any changes in circadian rhythms, suggesting that the circadian clock does not require melatonin for its function. Fourthly, we find that in the absence of melatonin, there is no rhythmic expression of sleep, suggesting that melatonin is the output molecule of process C. Lastly, we describe a connection between adenosine signaling (output molecules of process S), and melatonin. Following this we proceed to study the role adenosine signaling plays in sleep-wake behavior. We find that firstly, adenosine receptor A1 and A2 are involved in sleep- wake behavior in zebrafish, based on agonist/antagonist behavioral results. Secondly, we find that several brain regions such as PACAP cells in the rostral midbrain, GABAergic cells in the forebrain and hindbrain, Dopamine and serotonin cells in the caudal hypothalamus and sox2 cells lining the hindbrain ventricle are activated in response to the A1 antagonist and VMAT positive cells are activated in response to the A2A agonist, suggesting these areas are involved in adenosine signaling in zebrafish. Thirdly, we find that knocking out the zebrafish adenosine receptors has no effect on sleep architecture. Lastly, we find that while the A1 agonist phenotype requires the zfAdora1a receptor, the antagonist and the A2A agonist behavioral phenotypes are not mediated by the zfAdora1a, zfAdora1b and zfAdoraA2Aa, zfAdora2Ab receptors respectively.

Epigenetic regulation in neural crest development : role of DNA methyltransferases 3A and 3B

The neural crest is a group of migratory, multipotent stem cells that play a crucial role in many aspects of embryonic development. This uniquely vertebrate cell population forms within the dorsal neural tube but then emigrates out and migrates long distances to different regions of the body. These cells contribute to formation of many structures such as the peripheral nervous system, craniofacial skeleton, and pigmentation of the skin. Why some neural tube cells undergo a change from neural to neural crest cell fate is unknown as is the timing of both onset and cessation of their emigration from the neural tube. In recent years, growing evidence supports an important role for epigenetic regulation as a new mechanism for controlling aspects of neural crest development. In this thesis, I dissect the roles of the de novo DNA methyltransferases (DNMTs) 3A and 3B in neural crest specification, migration and differentiation. First, I show that DNMT3A limits the spatial boundary between neural crest versus neural tube progenitors within the neuroepithelium. DNMT3A promotes neural crest specification by directly mediating repression of neural genes, like Sox2 and Sox3. Its knockdown causes ectopic Sox2 and Sox3 expression at the expense of neural crest territory. Thus, DNMT3A functions as a molecular switch, repressing neural to favor neural crest cell fate. Second, I find that DNMT3B restricts the temporal window during which the neural crest cells emigrate from the dorsal neural tube. Knockdown of DNMT3B causes an excess of neural crest emigration, by extending the time that the neural tube is competent to generate emigrating neural crest cells. In older embryos, this resulted in premature neuronal differentiation. Thus, DNMT3B regulates the duration of neural crest production by the neural tube and the timing of their differentiation. My results in avian embryos suggest that de novo DNA methylation, exerted by both DNMT3A and DNMT3B, plays a dual role in neural crest development, with each individual paralogue apparently functioning during a distinct temporal window. The results suggest that de novo DNA methylation is a critical epigenetic mark used for cell fate restriction of progenitor cells during neural crest cell fate specification. Our discovery provides important insights into the mechanisms that determine whether a cell becomes part of the central nervous system or peripheral cell lineages.