The Regeneration Potential Of Zebrafish Caudal Fin And The Circadian Rhythm Of Brain, Heart And Brown Adipose Tissue In Mice

The regenerative capacities of vertebrate tissues/organs tend to decrease afterrepeated injury or when the animals become older. The zebrafish （Danio rerio） haverecently emerged as a new vertebrate model for genetic studies of tissue/organregeneration. Zebrafish exhibit an enhanced capability of regenerating adult tissues,which include retina, spinal cord, kidney, heart and fin. In this study, we examinedthe effect of repeated injuries （fin amputation） in zebrafish on caudal fin regenerationand the capability of regeneration in zebrafish with different ages. In zebrafish thatreceived repeated injuries, the potential for caudal fin regeneration, such as tissuegrowth and the expression of regeneration marker genes （msxb, fgf20a, bmp2b）, wasnot significant difference in comparison to zebrafish that received only oneamputation surgery. It shows that repeated injury has no effect on caudal finregeneration. During the process of initial fin regeneration （7days post-amputation,dpa）, there were not significant differences in tissue outgrowth and the expression ofregeneration marker gene （bmp2b） between different aged zebrafish. This suggeststhat caudal fin regeneration did not seem to correlate with age. In summary, by usingmorphological and gene expression analyses, the data suggest that zebrafish hasunlimited regenerative potential in the injured caudal fin.In mammals, many processes in physiology show daily variation under normalconditions. When these daily patterns persist under constant conditions, they aredescribed as circadian （²4hours） rhythms, and are driven by an endogenous clock.Glucocortcoids （GCs） have been implicated as being important in synchronizingclocks in peripheral tissues in vivo and in vitro. Circadian rhythms are apparent incardiac tissue at the molecular and functional level. It has been shown previously thatdexamethasone （DEX） which is a glucocortcoid receptor agonist can affect clockgene expression in the heart, although this has been tested only at one particular timeof day. Because of the reported susceptibility of clock genes to GCs, we set out to study the response of atrial tissue to dexamethasone （DEX） in a time informedmanner. Using in vitro atrial tissue cultures of mice carrying bioluminescencereporters for PER1and PER2activity, the circadian clock in this tissue and itsresponse to DEX treatment was monitored. Because DEX reportedly does not shiftthe rhythm of per1expression in the suprachiasmatic nucleus （SCN）, but has a knownresponse in liver, the SCN and liver of the same individuals were also cultured. Theresults show that the clock in atrial tissue shows a strong phase shifting effect inresponse to time of culturing. Together with the phase shift observed after mediumtreatment, this could suggest a time-dependent sensitivity of the atrial clock tomechanical treatment, which is highly relevant for a tissue that shows a dailyvariation in mechanical （stretch and contractile） activity. Atrial tissue also showsphase shifting responses to DEX, which is markedly different from that of the liver.DEX treated liver and atria show tissue specific response for all time points exceptbetween6-12hours after the last peak in bioluminescence. Moreover, in atrial tissuePER1LUCexpression is not rhythmic, but can be induced by DEX, while PER2LUCisrhythmic and shows a strong amplitude response at certain times within the circadiancycle. These data indicate that the clock in the atrium has defined glucocorticoidsensitivity and circadian clock characteristics in vitro.Positron Emission Tomography （PET） is a non-invasive and quantitative nuclearimaging modality used for a range of clinical diagnostic and pre-clinical experimentalapplications. PET often employs^18F-fluorodeoxyglucose （FDG） as the nuclear probefor imaging. This imaging technique provides functional information in detectingtissues with high glucose demands such as the heart, the brain, and many types ofcancers. In this study, we measured in vivo uptake of FDG in the brain, heart andinterscapular brown adipose tissue （iBAT） of C57BL/6mice at intervals across a24-hour light-dark cycle by PET. Our data describe a significant, high amplituderhythm in FDG uptake throughout the whole brain, peaking at the mid-dark phase ofthe light-dark cycle, which is the active phase for nocturnal mice. In addition, ourdata show24-hour patterns in glucose uptake in most of the brain regions examined,including several regions that do not show a difference in glucose utilization in theprevious studies. Under these conditions, heart FDG uptake did not vary with time-of-day, but did show biological variation throughout the24-hour period formeasurements within the same mice. Our data also emphasizes a methodologicalrequirement of controlling for the time-of-day of scanning FDG uptake in the brain inboth clinical and pre-clinical settings, and suggests waveform normalization of FDGmeasurements at different times of the day. Nervous and endocrine control of iBATpotentially exhibit circadian variation. Our data reveals a strong24-hour profile ofglucose uptake of iBAT, peaking at approximately9hours into the light phase of the12hour light,12hour dark day. The observation of a24-hour rhythm in glucoseuptake in iBAT makes this tissue a candidate site of interaction between metabolicand circadian systems. Moreover, FDG uptake was scanned at different times-of-daywithin an individual mouse, and also compared to different times-of-day betweenindividuals, showing both biological and technical reproducibility of the24-hourpattern in FDG uptake.