Significance We successfully identified the duper allele as a null mutation of Cryptochrome 1 in Syrian hamsters. Here, we have shown the use of fast homozygosity mapping as an effective approach to identify causal mutations in mammals, despite lacking chromosomal genome information. In the course of this work, we improved the draft Syrian hamster genome and generated datasets necessary to exploit Syrian hamsters as a modern genetic research model. The unique physiological features of Syrian hamsters make them a desirable model to investigate human diseases, including circadian disorders, cancer, heart function, metabolism, and infectious diseases (e.g., severe acute respiratory syndrome coronavirus 2).
The Cryptochrome 1 ( Cry1 )–deficient duper mutant hamster has a short free-running period in constant darkness (τ DD ) and shows large phase shifts in response to brief light pulses. We tested whether this measure of the lability of the circadian phase is a general characteristic of Cry1 -null animals and whether it indicates resistance to jet lag. Upon advance of the light:dark (LD) cycle, both duper hamsters and Cry1 −/− mice re-entrained locomotor rhythms three times as fast as wild types. However, accelerated re-entrainment was dissociated from the amplified phase-response curve (PRC): unlike duper hamsters, Cry1 −/− mice show no amplification of the phase response to 15’ light pulses. Neither the amplified acute shifts nor the increased rate of re-entrainment in duper mutants is due to acceleration of the circadian clock: when mutants drank heavy water to lengthen the period, these aspects of the phenotype persisted. In light of the health consequences of circadian misalignment, we examined effects of duper and phase shifts on a hamster model of heart disease previously shown to be aggravated by repeated phase shifts. The mutation shortened the lifespan of cardiomyopathic hamsters relative to wild types, but this effect was eliminated when mutants experienced 8-h phase shifts every second week, to which they rapidly re-entrained. Our results reveal previously unsuspected roles of Cry1 in phase shifting and longevity in the face of heart disease. The duper mutant offers new opportunities to understand the basis of circadian disruption and jet lag.
Mitosis is an essential process in which the duplicate genome is segregated equally into two daughter cells. CTCF has been reported to be present in mitosis but its importance for mitotic fidelity remains to be determined. To evaluate the importance of CTCF in mitosis, we tracked mitotic behaviors in wild type and two different CTCF CRISPR-based genetic knockdowns. We find that knockdown of CTCF results in prolonged mitoses and failed anaphase segregation via time lapse imaging of SiR-DNA. CTCF knockdown did not alter cell cycling or the mitotic checkpoint, which was activated upon nocodazole treatment. Immunofluorescence imaging of the mitotic spindle in CTCF knockdowns revealed disorganization via tri/tetrapolar spindles and chromosomes behind the spindle pole. Imaging of interphase nuclei showed that nuclear size increased drastically, consistent with failure to divide the duplicated genome in anaphase. Population measurements of nuclear shape in CTCF knockdowns do not display decreased circularity or increased nuclear blebbing relative to wild type. However, failed mitoses do display abnormal nuclear morphologies relative to successful mitoses, suggesting population images do not capture individual behaviors. Thus, CTCF is important for both proper metaphase organization and anaphase segregation which impacts the size and shape of the interphase nucleus.
Cell birth and survival in the adult hippocampus are regulated by a circadian clock. Rotating shift work and jet lag disrupt circadian rhythms and aggravate disease. Internal misalignment, a state in which abnormal phase relationships prevail between and within organs, is proposed to account for adverse effects of circadian disruption. This hypothesis has been difficult to test because phase shifts of the entraining cycle inevitably lead to transient desynchrony. Thus, it remains possible that phase shifts, regardless of internal desynchrony, account for adverse effects of circadian disruption and alter neurogenesis and cell fate.In order to address this question, we examined cell birth and differentiation in the duper Syrian hamster (Mesocricetus auratus), aCry1-null mutant in which re-entrainment of locomotor rhythms is greatly accelerated. Adult females were subjected to alternating 8h advances and delays at eight 16-day intervals. BrdU, a cell birth marker, was given midway through the experiment. Repeated phase shifts decreased the number of newborn non-neuronal cells in wt, but not duper hamsters. The duper mutation increased the number of BrdU-ir cells that stained for NeuN, which marks neuronal differentiation. Immunocytochemical staining for proliferating cell nuclear antigen (PCNA) indicated no overall effect of genotype or repeated shifts on cell division rates at the time of sacrifice. Cell differentiation, assessed by doublecortin (DCX), was higher in duper hamsters but was not significantly altered by repeated phase shifts. Our results support the internal misalignment hypothesis, and indicate thatCry1regulates cell differentiation. Phase shifts may determine neuronal stem cell survival and time course of differentiation after cell birth.Significance StatementThe birth of neurons in adult brain impacts learning and memory. Circadian disruption, such as occurs in jet lag, adversely affects neurogenesis. It is unclear whether shifts of the light:dark cycle are inherently deleterious, or whether misalignment of the phase of circadian oscillators is responsible. Repeated shifts decreased the number of non-neuronal cells born in adult dentate gyrus of wild type hamsters, but increased the percentage that developed neuronal phenotype. Duper mutants, which are deficient in the core clock geneCryptochrome 1and re-entrain 4 times as fast as wild types, experienced increased neurogenesis but showed no effect of phase shifts. These results implicateCry1in regulation of neurogenesis and indicate that circadian misalignment is critical in jet lag.
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