Mutant animals Flashcards
Whitmore et al. (1998)
Striking discovery in the zebrafish circadian system.
o They found that in the heart and kidney, in addition to cell lines derived from zebrafish embryos, there contained both circadian oscillators and photoreceptive mechanisms sufficient for entrainment by LD cycles.
o This was demonstrated by monitoring rhythms of Clock mRNA accumulation in cultured organs and cells.
o In vivo, Clock mRNA levels were found to be rhythmic in heart, kidney, and spleen and in the brain, pineal, and eye.
o When heart and kidney are removed to organ culture, these rhythms persist for two or more cycles in constant conditions, indicating that these organs contain circadian oscillators.
o When heart and kidney are exposed to LD cycles in vitro, the damping of Clock mRNA rhythms is prevented, and reversal of the LD cycle in vitro re-entrains the rhythm to the opposite phase (Whitmore et al. 2000).
o This shows that these organs contain phototransduction mechanisms sufficient for entrainment, in addition to circadian oscillators.
o Similar results have been obtained with cell lines derived from zebrafish embryos, suggesting that many cell types in zebrafish contain photoentrainable circadian oscillators.
Kaneko et al. (2006),
in vivo luciferase assay with period3-luciferase (per3-luc) transgenic zebrafish, found tissue-dependent differences in free-running period, phase, response to light, and temperature compensation.
In a similar experiment, Moore and Whitmore (2014) found that clock genes also oscillate throughout the zebrafish brain.
Moutsakia et al. (2003)
Identified a novel opsin family, tmt-opsin, that has a genomic structure characteristic of vertebrate photopigments, an amino acid identity equivalent to the known photopigment opsins, and the essential residues required for photopigment function.
o Significantly, tmt-opsin is expressed in a wide variety of neural and non-neural tissues, including a zebrafish embryonic cell line that exhibits a light entrainable clock.
o Collectively the data suggest that tmt-opsin is a strong candidate for the photic regulation of zebrafish peripheral clocks. Despite this there is no functional data/proof yet.
Plautz et al. (1997)
Transgenic Drosophila that expressed either luciferase or green fluorescent protein driven from the promoter of the clock gene period to monitor the circadian clock in explanted head, thorax, and abdominal tissues.
o The tissues (including sensory bristles in the leg and wing) showed rhythmic bioluminescence, and the rhythms could be reset by light. The photoreceptive properties of the explanted tissues indicate that unidentified photoreceptors are likely to contribute to photic signal transduction to the clock.
o These results show that autonomous circadian oscillators are present throughout the body, and they suggest that individual cells in Drosophila are capable of supporting their own independent clocks.
Stephan and Zucker (1972)
showed that bilateral electrolytic lesions in the suprachiasmatic nuclei permanently eliminated nocturnal and circadian rhythms in drinking behavior and locomotor activity of albino rats. In contrast the destruction of the medial preoptic area had no effect on 24-hr drinking rhythms. This suggests that the generation of 24-hr behavioral rhythms and the entrainment of these rhythms to the light-dark cycle of environmental illumination may be coordinated by neurons in the suprachiasmatic region of the rat brain. The suprachiasmatic nucleus (SCN) refers to the two bilateral nuclei in ventral hypothalamus located above optic chiasm and either side of 3rd ventricle which each contains approx. 10,000 neurons (densely packed). They are directly light responsive via the retinohypothalamic tract .
Ralph and Menaker (1988)
discovered the first mammalian period mutant known as the tau hamster. They found a mutation that dramatically shortens the period of the circadian locomotor rhythm of golden hamsters which occurred at a single, autosomal locus (tau). The wild-type hamsters had rhythms with a free-running period averaging 24.1 hours whereas heterozygous mutants had a period average of 22 hours and homozygous mutants had a period close to 20 hours. Animals that carry the mutant alleles exhibit abnormal entrainment to 24-hour light:dark cycles or are unable to entrain.
Ralph et al. (1990)
tested the pacemaker role of the SCN in the mammalian circadian system. The SCN of the mutant tau hamster, with a period of 22 hours, was transplanted into a wild-type hamster with an ablated SCN and vice versa, with the SCN of the wild-type hamster was transplanted into the mutant tau hamster. Small neural grafts from the WT suprachiasmatic region restored circadian rhythms to arrhythmic animals and neural grafts from mutant SCN caused WT hamsters to become arrhythmic. Thus the restored rhythms always exhibited the period of the donor genotype regardless of the direction of the transplant or genotype of the host. They therefore concluded that the period of the circadian rhythm was determined by cells of the suprachiasmatic region.
Herzog and Block (1997)
used multimicroelectrode plates to measure extracellular action potential activity simultaneously from multiple sites within the cultured mouse SCN. They found that rhythms in firing rate in the mouse SCN that were either in phase or ≈6 or 12 h out of phase. They concluded that neurons in the SCN are electrically active, rhythmic, express neuropeptides.
Webb et al. (2009)
measured the firing rate and per2 gene expression in single SCN neurons to see if these cells show 24 hour rhythms. By plating SCN neurons and eliminating synaptic inputs, they found that both the circadian cells as well as the arrhythmic cells express the neuropeptides arginine vasopressin (AVP) or vasoactive intestinal polypeptide (VIP). Furthermore, neurons were observed to lose or gain circadian rhythmicity in these dispersed cell cultures, both spontaneously and in response to forskolin stimulation (used to increase intracellular cAMP levels). They concluded that individual SCN neurons can generate circadian oscillations and that their results indicate that AVP, VIP, and other SCN neurons are intrinsic but unstable circadian oscillators that rely on network interactions to stabilize their otherwise noisy cycling.
Long et al. (2005)
by using paired whole-cell recordings, found that many neurons in the rat and mice SCN communicate via electrical synapses (gap junctions). These electrical synapses were absent in connexin36 (Cx36)-knockout mice and these mice also showed dampened circadian activity rhythms and a delayed onset of activity during transition to constant darkness. These results suggest that electrical synapses in the SCN help to synchronize its spiking activity, and that such synchrony is necessary for normal circadian behavior
Aton et al. (2005)
found that Vip-/- and Vipr2-/- mice showed two daily bouts of activity in a skeleton photoperiod and multiple circadian periods in constant darkness. Loss of VIP or VPAC2 also abolished circadian firing rhythms in approximately half of all SCN neurons and disrupted synchrony between rhythmic neurons. They found that daily application of a VPAC2 agonist restored rhythmicity and synchrony to VIP-/- SCN neurons, but not to Vipr2-/- neurons. We conclude that VIP coordinates daily rhythms in the SCN and behavior by synchronizing a small population of pacemaking neurons and maintaining rhythmicity in a larger subset of neurons.
Vitaterna et al. (1994)
were the first to identify the first mutant Clock mouse by using the highly potent mutagen N-ethyl-N-nitrosourea (ENU) to treat a progeny of mice. A semidominant mutation, Clock, that lengthens circadian period and abolishes persistence of rhythmicity was identified. Clock segregated as a single gene that mapped to the midportion of mouse chromosome 5 (or human chromosome 4). CLOCK = Circadian Locomotor Output Cycles Kaput.
Gekakis et al. (1998)
performed a yeast two-hybrid screen using a LEXA-CLOCK hybrid as bait to identify CLOCK protein interactions and found that BMAL1 (MOP3) binds to CLOCK. Using in situ hybridisation to examine the Clock and mper1 transcripts, it was suggested that CLOCK, BMAL1, and mPER1 are colocalised within circadian clock cells. Next, using yeast one-hybrid assays, they tested CLOCK-BMAL1 heterodimers for binding to the E-box element within the Drosophila per clock control region. They found that CLOCK and BMAL1 formed heterodimers that robustly bound to the E-box sequence known to mediate positive regulation of the Drosophila per gene. CLOCK-BMAL1 heterodimers appear to drive the positive component of per transcriptional oscillations, which are thought to underlie circadian rhythmicity.
Bunger et al. (2000)
showed that BMAL1 deficient mice results in immediate and complete loss of circadian rhythmicity in constant darkness. Additionally, locomotor activity in light–dark (LD) cycles is impaired and activity levels are reduced in Bmal-/- (Mop3−/−) mice. Using in situ hybridisation, they found extremely low expression of mPer1 and mPer2 in the SCN of BMAL1 deficient mice. These results provide genetic evidence that BMAL1 is the bona fide heterodimeric partner of mCLOCK.
Reppert et al. (1998)
cloned and characterised the 3 mammalian homologues of the drosophila per genes; per1, per2, per3. Each mammalian Per gene encodes a protein with a protein dimerisation PAS domain that is homologous to the PAS domain of insect PER.
