High-throughput Genetically Modified Animal Experiments Achieved by Next-generation Mammalian Genetics.

Animal models are essential tools for modern scientists to conduct biological experiments and investigate their hypotheses in vivo. However, for the past decade, raising the throughput of such animal experiments has been a great challenge. Conventionally, in vivo high-throughput assay was achieved through large-scale mutagen-driven forward genetic screening, which took years to find causal genes. In contrast, reverse genetics accelerated the causal gene identification process, but its throughput was also limited by 2 barriers, that is, the genome modification step and the time-consuming crossing step. Defined as genetics without crossing, next-generation genetics is able to produce gene-modified animals that can be analyzed at the founder generation (F0). This method is or can be accomplished through recent technological advances in gene editing and virus-based efficient gene modifications. Notably, next-generation genetics has accelerated the process of cross-species studies, and it will be a useful technique during animal experiments as it can provide genetic perturbation at an individual level without crossing. In this review, we begin by introducing the history of animal-based high-throughput analysis, with a specific focus on chronobiology. We then describe ways that gene modification efficiency during animal experiments was enhanced and why crossing remained a barrier to reaching higher efficiency. Moreover, we mention the Triple CRISPR as a critical technique for achieving next-generation genetics. Finally, we discuss the potential applications and limitations of next-generation mammalian genetics.

The role of calcium and CaMKII in sleep.

Sleep is an evolutionarily conserved phenotype shared by most of the animals on the planet. Prolonged wakefulness will result in increased sleep need or sleep pressure. However, its mechanisms remain elusive. Recent findings indicate that Ca2+ signaling, known to control diverse physiological functions, also regulates sleep. This review intends to summarize research advances in Ca2+ and Ca2+/calmodulin-dependent protein kinase II (CaMKII) in sleep regulation. Significant changes in sleep phenotype have been observed through calcium-related channels, receptors, and pumps. Mathematical modeling for neuronal firing patterns during NREM sleep suggests that these molecules compose a Ca2+-dependent hyperpolarization mechanism. The intracellular Ca2+ may then trigger sleep induction and maintenance through the activation of CaMKII, one of the sleep-promoting kinases. CaMKII and its multisite phosphorylation status may provide a link between transient calcium dynamics typically observed in neurons and sleep-wake dynamics observed on the long-time scale.

Heavy Water Lengthens the Molecular Circadian Clock Period in the Suprachiasmatic Nucleus of Mice In Vitro.

Heavy water lengthens the periods of circadian rhythms in various plant and animal species. Many studies have reported that drinking heavy water lengthens the periods of circadian activity rhythms of rodents by slowing the clock mechanism in the suprachiasmatic nucleus (SCN), the mammalian circadian center. The SCN clock is stable and robust against disturbance, due to its intercellular network. It is unclear whether this robustness provides resistance to the effects of heavy water. Here, we report that heavy water lengthened the rhythm period of clock gene expression of the SCN and peripheral tissues in vitro using a PERIOD2::LUCIFERASE bioluminescence reporter. Our results show that the period-elongation rate of the SCN is similar to those of other tissues. Therefore, the intercellular network of the SCN is not resistant to the period-elongation effect of heavy water.

Effects of constant light exposure on allergic and irritant contact dermatitis in mice reared under constant light conditions.

Environmental light levels can affect physiological functions, such as general activity, body temperature and metabolism. Irregular lifestyles, such as those involving exposure to light during the night, can exacerbate the clinical symptoms of several inflammatory skin diseases. However, the effects of constant light exposure on immune responses are not fully understood. This study aimed to elucidate the effects of constant light exposure on two major types of skin reactions, allergic contact dermatitis (ACD) and irritant contact dermatitis (ICD). BALB/c mice were kept under constant light conditions or a normal light and dark cycle, and their ACD and ICD responses were assessed after the topical application of 2,4,6-trinitro-1-chlorobenzene and croton oil, respectively, to the ear skin. Interestingly, in both ACD and ICD, the ear-swelling response and local leukocyte infiltration were aggravated by constant exposure to light, which has previously been shown to severely disturb the behavioural rhythms of mice. In ACD, these findings were accompanied by increases in the numbers of degranulated mast cells and eosinophils. These results suggest that constant light exposure intensifies allergic and non-allergic skin inflammation.

Transgenic rats expressing dominant negative BMAL1 showed circadian clock amplitude reduction and rapid recovery from jet lag.

The circadian rhythms are endogenous rhythms of about 24 h, and are driven by the circadian clock. The clock centre locates in the suprachiasmatic nucleus. Light signals from the retina shift the circadian rhythm in the suprachiasmatic nucleus, but there is a robust part of the suprachiasmatic nucleus that causes jet lag after an abrupt shift of the environmental lighting condition. To examine the effect of attenuated circadian rhythm on the duration of jet lag, we established a transgenic rat expressing BMAL1 dominant negative form under control by mouse Prnp-based transcriptional regulation cassette [BMAL1 DN (+)]. The transgenic rats became active earlier than controls, just after light offset. Compared to control rats, BMAL1 DN (+) rats showed smaller circadian rhythm amplitudes in both behavioural and Per2 promoter driven luciferase activity rhythms. A light pulse during the night resulted in a larger phase shift of behavioural rhythm. Furthermore, at an abrupt shift of the light-dark cycle, BMAL1 DN (+) rat showed faster entrainment to the new light-dark cycle compared to controls. The circadian rhythm has been regarded as a limit cycle phenomenon, and our results support the hypothesis that modification of the amplitude of the circadian limit cycle leads to alteration in the length of the phase shift.

Towards organism-level systems biology by next-generation genetics and whole-organ cell profiling.

The system-level identification and analysis of molecular and cellular networks in mammals can be accelerated by "next-generation" genetics, which is defined as genetics that can achieve desired genetic makeup in a single generation without any animal crossing. We recently established a highly efficient procedure for producing knock-out (KO) mice using the "Triple-CRISPR" method, which targets a single gene by triple gRNAs in the CRISPR/Cas9 system. This procedure achieved an almost perfect KO efficiency (96-100%). We also established a highly efficient procedure, the "ES-mouse" method, for producing knock-in (KI) mice within a single generation. In this method, ES cells were treated with three inhibitors to keep their potency and then injected into 8-cell-stage embryos. These procedures dramatically shortened the time required to produce KO or KI mice from years down to about 3 months. The produced KO and KI mice can also be systematically profiled at a single-cell resolution by the "whole-organ cell profiling," which was realized by tissue-clearing methods, such as CUBIC, and an advanced light-sheet microscopy. The review describes the establishment and application of these technologies above in analyzing the three states (NREM sleep, REM sleep, and awake) of mammalian brains. It also discusses the role of calcium and muscarinic receptors in these states as well as the current challenges and future opportunities in the next-generation mammalian genetics and whole-organ cell profiling for organism-level systems biology.

Basement membrane recovery process in rat soleus muscle after exercise-induced muscle injury.

Purpose: Collagen IV is a component of the basement membrane (BM) that provides mechanical support for muscle fibers. In addition, transcription factor 4 (TCF4) is highly expressed in muscle connective tissue fibroblasts and regulates muscle regeneration. However, the expression of collagen IV and TCF4 (+) cells in response to exercise-induced muscle injury is not well known. Here, we investigated the expression and localization of collagen IV and TCF4 (+) cells during the recovery process after muscle injury induced by different exercise loads.Materials and Methods: Muscle injury was observed in the soleus muscle of young Wistar rats after 12 or 18 sets-downhill running (DR) on a treadmill. After running, the rats were permitted to recover for a period of 0.5 days, 2 days, or 7 days.Results: Ectopic localization of collagen IV in injured muscle fibers was observed after DR, and the number increased at 0.5 days after 18 sets DR and at 2 days after 12 or 18 sets DR as compared to the number observed at baseline. BM disruption was observed after DR. TCF4 (+) cells appeared in the inside and around injured muscle fibers at 0.5 day of recovery. After 18 sets DR, TCF4 (+) cells were more abundant for a longer period than that observed after 12 sets DR.Conclusions: DR induces BM disruption accompanied by muscle fiber damage. It is possible that BM destruction may be accompanied by muscle damage and that TCF4 (+) cells contribute to muscle fiber and BM recovery.

cAMP response element induces Per1 in vivo.

Light is an important cue for resetting the circadian clock. In mammals, light signals are thought to be transmitted to the cAMP response element (CRE) via a binding protein (CREB) to induce the expression of Per1 and Per2 genes in the mammalian circadian pacemaker, the suprachiasmatic nuclei (SCN). Several in vitro studies have suggested candidate CRE sites that contribute to the Per1 and Per2 induction by light, resulting in a phase shift of the circadian rhythm. However, it remains unclear whether the CREs are responsible for the light-induced Per1/2 induction. To address this question, we generated CRE-deleted mice in the Per1 and Per2 promoter regions. Deletion of a cAMP-responsive CRE in the Per1 promoter blunted light-induced Per1 expression in the SCN at night, while deletion of an ATF4 (CREB-2)-associated CRE in the Per2 promoter had no effect on its expression. These results suggested that the CRE in the Per1 promoter works for light induction but not CRE in the Per2 promoter. Behavioral rhythms observed under some light conditions were not affected by the CRE-deletion in Per1 promoter, suggesting that the attenuated Per1 induction did not affect the entrainment in some light conditions.

Circadian rhythms of sorting nexin 25 in the mouse suprachiasmatic nucleus.

Entrainment of mammalian circadian rhythms requires receptor-mediated signaling in the hypothalamic suprachiasmatic nucleus (SCN), the site of the master circadian pacemaker. Receptor-mediated signaling is regulated by endocytosis, indicating that endocytosis-related proteins contribute to SCN pacemaking. Sorting nexin 25 (SNX25) belongs to the sorting nexin superfamily, whose members are responsible for membrane attachment to organelles of the endocytic system. In this study, we showed that Snx25 mRNA and SNX25 protein are highly expressed and exhibit remarkable circadian rhythms in the SCN of adult mice. Expression was maximal at about zeitgeber time (ZT) 16 in the subjective night and minimal at ZT8 in the subjective day. Prominent SNX25 immunoreactivity was found in the arginine vasopressin-positive neurons of the SCN. These findings suggest that SNX25 is a new actor in endocytic signaling, perhaps contributing to the circadian pacemaking system.

Chronic circadian misalignment accelerates immune senescence and abbreviates lifespan in mice.

Modern society characterized by a 24/7 lifestyle leads to misalignment between environmental cycles and endogenous circadian rhythms. Persisting circadian misalignment leads to deleterious effects on health and healthspan. However, the underlying mechanism remains not fully understood. Here, we subjected adult, wild-type mice to distinct chronic jet-lag paradigms, which showed that long-term circadian misalignment induced significant early mortality. Non-biased RNA sequencing analysis using liver and kidney showed marked activation of gene regulatory pathways associated with the immune system and immune disease in both organs. In accordance, we observed enhanced steatohepatitis with infiltration of inflammatory cells. The investigation of senescence-associated immune cell subsets from the spleens and mesenteric lymph nodes revealed an increase in PD-1+CD44high CD4 T cells as well as CD95+GL7+ germinal center B cells, indicating that the long-term circadian misalignment exacerbates immune senescence and consequent chronic inflammation. Our results underscore immune homeostasis as a pivotal interventional target against clock-related disorders.

Slow shift of dead zone after an abrupt shift of the light-dark cycle.

The suprachiasmatic nucleus (SCN) is the center of the mammalian circadian system. Environmental photic signals shifts the phase of the circadian rhythm in the SCN except during the dead zone, when the photic signal is gated somewhere on the way from the retina to the neurons in the SCN. Here we examined the phase of the dead zone after an abrupt delay of the LD cycles for several days by observing the mc-Fos induction in the SCN by light pulses. After an abrupt shift of the LD cycles, the dead zone showed a slow phase shift, about two hours per day, which was well corresponded with the slow phase shift of the rest-activity cycles. In our previous study we demonstrated that, after an abrupt shift of the LD cycles, the SCN showed transient endogenous desynchronization between shell and core regions that showed a slow and a rapid shift of the circadian rhythms, respectively. Therefore, the present findings on the phase shift of the dead zone after the LD cycles shift suggest that the phase of the dead zone is under the control of the timing signals from the shell region of the SCN.

Disruption of circadian clockwork in in vivo reprogramming-induced mouse kidney tumors.

The circadian clock, which regulates cellular physiology, such as energy metabolism, resides in each cell level throughout the body. Recently, it has been elucidated that the cellular circadian clock is closely linked with cellular differentiation. Moreover, the misregulation of cellular differentiation in mouse embryonic stem cells (ESCs) induced abnormally differentiated cells with impaired circadian clock oscillation, concomitant with the post-transcriptional suppression of CLOCK proteins. Here, we show that the circadian molecular oscillation is disrupted in dysdifferentiation-mediated mouse kidney tumors induced by partial in vivo reprogramming, resembling Wilms tumors. The expression of CLOCK protein was dramatically reduced in the tumor cells despite the Clock mRNA expression. We also showed that a similar loss of CLOCK was observed in human Wilms tumors, suggesting that the circadian molecular clockwork may be disrupted in dysdifferentiation-mediated embryonal tumors such as Wilms tumors, similar to the in vivo reprogramming-induced mouse kidney tumors. These results support our previous reports and may provide a novel viewpoint for understanding the pathophysiological nature of cancers through the correlation between cellular differentiation and circadian clock.

Clock-dependent and system-driven oscillators interact in the suprachiasmatic nuclei to pace mammalian circadian rhythms.

Circadian clocks drive biological rhythms with a period of approximately 24 hours and keep in time with the outside world through daily resetting by environmental cues. While this external entrainment has been extensively investigated in the suprachiasmatic nuclei (SCN), the role of internal systemic rhythms, including daily fluctuations in core temperature or circulating hormones remains debated. Here, we show that lactating mice, which exhibit dampened systemic rhythms, possess normal molecular clockwork but impaired rhythms in both heat shock response gene expression and electrophysiological output in their SCN. This suggests that body rhythms regulate SCN activity downstream of the clock. Mathematical modeling predicts that systemic feedback upon the SCN functions as an internal oscillator that accounts for in vivo and ex vivo observations. Thus we are able to propose a new bottom-up hierarchical organization of circadian timekeeping in mammals, based on the interaction in the SCN between clock-dependent and system-driven oscillators.

Robust circadian clock oscillation and osmotic rhythms in inner medulla reflecting cortico-medullary osmotic gradient rhythm in rodent kidney.

Circadian clocks in mammals function in most organs and tissues throughout the body. Various renal functions such as the glomerular filtration and excretion of electrolytes exhibit circadian rhythms. Although it has been reported that the expression of the clock genes composing molecular oscillators show apparent daily rhythms in rodent kidneys, functional variations of regional clocks are not yet fully understood. In this study, using macroscopic bioluminescence imaging method of the PER2::Luciferase knock-in mouse kidney, we reveal that strong and robust circadian clock oscillation is observed in the medulla. In addition, the osmotic pressure in the inner medulla shows apparent daily fluctuation, but not in the cortex. Quantitative-PCR analysis of the genes contributing to the generation of high osmotic pressure or the water re-absorption in the inner medulla, such as vasopressin receptors (V1aR, V2R), urea transporter (UT-A2) and water channel (Aqp2) show diurnal variations as well as clock genes. Deficiency of an essential clock gene Bmal1 impairs day-night variations of osmotic pressure gradient in the inner medulla, suggesting that circadian clocks in the medulla part of the kidney may regulate the circadian rhythm of cortico-medullary osmotic pressure gradient, and may contribute physiological day-night rhythm of urination.

Involvement of posttranscriptional regulation of Clock in the emergence of circadian clock oscillation during mouse development.

Circadian clock oscillation emerges in mouse embryo in the later developmental stages. Although circadian clock development is closely correlated with cellular differentiation, the mechanisms of its emergence during mammalian development are not well understood. Here, we demonstrate an essential role of the posttranscriptional regulation of Clock subsequent to the cellular differentiation for the emergence of circadian clock oscillation in mouse fetal hearts and mouse embryonic stem cells (ESCs). In mouse fetal hearts, no apparent oscillation of cell-autonomous molecular clock was detectable around E10, whereas oscillation was clearly visible in E18 hearts. Temporal RNA-sequencing analysis using mouse fetal hearts reveals many fewer rhythmic genes in E10-12 hearts (63, no core circadian genes) than in E17-19 hearts (483 genes), suggesting the lack of functional circadian transcriptional/translational feedback loops (TTFLs) of core circadian genes in E10 mouse fetal hearts. In both ESCs and E10 embryos, CLOCK protein was absent despite the expression of Clock mRNA, which we showed was due to Dicer/Dgcr8-dependent translational suppression of CLOCK. The CLOCK protein is required for the discernible molecular oscillation in differentiated cells, and the posttranscriptional regulation of Clock plays a role in setting the timing for the emergence of the circadian clock oscillation during mammalian development.

Constant light exposure impairs immune tolerance development in mice.

BACKGROUND: An intrinsic daily physiological rhythm called circadian rhythm has been indicated to affect the immune system and its related diseases. Immune tolerance development is closely associated with the onset of immunological disorders. However, the effect of circadian rhythm in the mechanisms of immune tolerance development has not yet been fully understood. OBJECTIVE: The purpose of this study was to investigate the effects of circadian rhythm disruption on the development of immune tolerance by the perturbation of light environment, using a mouse model of neonatally induced cutaneous tolerance. METHODS: Mice were kept under constant light (LL) or light-dark (LD) conditions, and hapten was applied at 2days after birth. Six weeks later, hapten was reapplied to abdominal skin, followed by hapten application to ear skin 5days later. RESULTS: The ear-swelling responses and cell infiltration into inflamed skin significantly increased in LL mice compared with those in LD mice. Interestingly, the percentage and the number of Foxp3+-regulatory T cells notably decreased in inflamed skin and draining lymph nodes of LL mice compared with that in LD mice. Loss-of-function mutation of a key circadian gene, Bmal1, also exacerbated the ear-swelling responses and cell infiltration into inflamed skin in mice. CONCLUSION: These results suggest that circadian rhythm may be implicated in immune tolerance development in allergic inflammation.

A PTH-responsive circadian clock operates in ex vivo mouse femur fracture healing site.

The circadian clock contains clock genes including Bmal1 and Period2, and it maintains an interval rhythm of approximately 24 hours (the circadian rhythm) in various organs including growth plate and articular cartilage. As endochondral ossification is involved not only in growth plate but also in fracture healing, we investigated the circadian clock functions in fracture sites undergoing healing. Our fracture models using external fixation involved femurs of Period2::Luciferase knock-in mice which enables the monitoring of endogenous circadian clock state via bioluminescence. Organ culture was performed by collecting femurs, and fracture sites were observed using bioluminescence imaging systems. Clear bioluminescence rhythms of 24-hour intervals were revealed in fracture healing sites. When parathyroid hormone (PTH) was administered to fractured femurs in organ culture, peak time of Period2::Luciferase activity in fracture sites and growth plates changed, indicating that PTH-responsive circadian clock functions in the mouse femur fracture healing site. While PTH is widely used in treating osteoporosis, many studies have reported that it contributes to improvement of fracture healing. Future studies of the role of this local clock in wound healing may reveal a novel function of the circadian timing mechanism in skeletal cells.

Effect of Multiple Clock Gene Ablations on the Circadian Period Length and Temperature Compensation in Mammalian Cells.

Most organisms have cell-autonomous circadian clocks to coordinate their activity and physiology according to 24-h environmental changes. Despite recent progress in circadian studies, it is not fully understood how the period length and the robustness of mammalian circadian rhythms are determined. In this study, we established a series of mouse embryonic stem cell (ESC) lines with single or multiplex clock gene ablations using the CRISPR/Cas9-based genome editing method. ESC-based in vitro circadian clock formation assay shows that the CRISPR-mediated clock gene disruption not only reproduces the intrinsic circadian molecular rhythms of previously reported mice tissues and cells lacking clock genes but also reveals that complexed mutations, such as CKIδ(m/m):CKIε(+/m):Cry2(m/m) mutants, exhibit an additively lengthened circadian period. By using these mutant cells, we also investigated the relation between period length alteration and temperature compensation. Although CKIδ-deficient cells slightly affected the temperature insensitivity of period length, we demonstrated that the temperature compensation property is largely maintained in all mutants. These results show that the ESC-based assay system could offer a more systematic and comprehensive approach to the genotype-chronotype analysis of the intracellular circadian clockwork in mammals.

Disruption of MeCP2 attenuates circadian rhythm in CRISPR/Cas9-based Rett syndrome model mouse.

Methyl-CpG-binding protein 2 (Mecp2) is an X-linked gene encoding a methylated DNA-binding nuclear protein which regulates transcriptional activity. The mutation of MECP2 in humans is associated with Rett syndrome (RTT), a neurodevelopmental disorder. Patients with RTT frequently show abnormal sleep patterns and sleep-associated problems, in addition to autistic symptoms, raising the possibility of circadian clock dysfunction in RTT. In this study, we investigated circadian clock function in Mecp2-deficient mice. We successfully generated both male and female Mecp2-deficient mice on the wild-type C57BL/6 background and PER2(Luciferase) (PER2(Luc)) knock-in background using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Generated Mecp2-deficient mice recapitulated reduced activity in mouse models of RTT, and their activity rhythms were diminished in constant dark conditions. Furthermore, real-time bioluminescence imaging showed that the amplitude of PER2(Luc)-driven circadian oscillation was significantly attenuated in Mecp2-deficient SCN neurons. On the other hand, in vitro circadian rhythm development assay using Mecp2-deficient mouse embryonic stem cells (ESCs) did not show amplitude changes of PER2(Luc) bioluminescence rhythms. Together, these results show that Mecp2 deficiency abrogates the circadian pacemaking ability of the SCN, which may be a therapeutic target to treat the sleep problems of patients with RTT.

Parathyroid hormone resets the cartilage circadian clock of the organ-cultured murine femur.

BACKGROUND AND PURPOSE: The circadian clock governs endogenous day-night variations. In bone, the metabolism and growth show diurnal rhythms. The circadian clock is based on a transcription-translation feedback loop composed of clock genes including Period2 (Per2), which encodes the protein period circadian protein homolog 2. Because plasma parathyroid hormone (PTH) levels show diurnal variation, we hypothesized that PTH could carry the time information to bone and cartilage. In this study, we analyzed the effect of PTH on the circadian clock of the femur. PATIENTS AND METHODS: Per2::Luciferase (Per2::Luc) knock-in mice were used and their femurs were organ-cultured. The bioluminescence was measured using photomultiplier tube-based real-time bioluminescence monitoring equipment or real-time bioluminescence microscopic imaging devices. PTH or its vehicle was administered and the phase shifts were calculated. Immunohistochemistry was performed to detect PTH type 1 receptor (PTH1R) expression. RESULTS: Real-time bioluminescence monitoring revealed that PTH reset the circadian rhythm of the Per2::Luc activity in the femurs in an administration time-dependent and dose-dependent manner. Microscopic bioluminescence imaging revealed that Per2::Luc activity in the growth plate and the articular cartilage showed that the circadian rhythms and their phase shifts were induced by PTH. PTH1R was expressed in the growth plate cartilage. INTERPRETATION: In clinical practice, teriparatide (PTH (1-34)) treatment is widely used for osteoporosis. We found that PTH administration regulated the femoral circadian clock oscillation, particularly in the cartilage. Regulation of the local circadian clock by PTH may lead to a more effective treatment for not only osteoporosis but also endochondral ossification in bone growth and fracture repair.