1. Introduction

Circadian rhythms constitute a universal endogenous timekeeping system that enables organisms to align gene expression, cellular physiology, and organ function with predictable daily environmental cycles, thereby maintaining homeostasis and optimizing fitness [1, 2]. Disruption of this intrinsic rhythmicity—whether due to lifestyle factors such as shift work and jet lag, environmental perturbations, or genetic mutations—is strongly associated with a wide spectrum of human disorders, including sleep disturbances, metabolic syndrome, cardiovascular diseases, neuropsychiatric conditions, and cancer [3]. Therefore, deciphering the molecular basis, regulatory networks, and physiological outputs of circadian rhythms is not only a central question in basic biology but also holds profound implications for translational medicine and public health.

Among the model organisms used in circadian research, zebrafish occupy a crucial evolutionary and experimental niche, bridging the gap between invertebrate models such as Drosophila melanogaster and mammalian systems like the mouse [4]. Unlike Drosophila, which utilize distinct clock genes and regulatory logic, or mammals, where circadian control is centralized in the hypothalamic suprachiasmatic nucleus (SCN), zebrafish exhibit a high degree of conservation in core clock genes (Clock, Bmal, Per, Cry) and pathways with humans. Concurrently, they possess unique biological features that expand the scope of inquiry. These include external fertilization, transparent embryos permitting real-time visualization of rhythmic processes, rapid organogenesis within 24-48 hours post-fertilization, and a robust genetic toolkit for manipulation via CRISPR/Cas9, transgenesis, and morpholino knockdown [5–7].

A defining characteristic that sets zebrafish apart is its decentralized circadian regulatory architecture [8]. In contrast to mammals, where peripheral tissues exhibit dampened autonomy and rely heavily on the SCN for synchronization, virtually all zebrafish cells—from classical photoreceptive tissues such as the retina and pineal gland to peripheral organs such as the liver, heart, and intestine, and even cultured cell lines—harbor intrinsic circadian oscillators and possess autonomous photosensitivity [4, 9, 10]. This allows zebrafish to integrate light signals directly at both central and peripheral levels, offering a powerful model to dissect the independent functions and synchronization mechanisms of distributed clocks [11]. The core circadian oscillator is built around a conserved transcription-translation feedback loop (TTFL). In this loop, CLOCK/BMAL heterodimers activate the transcription of the per and cry genes, and the resulting PER/CRY proteins subsequently inhibit CLOCK/BMAL activity, establishing an ~24-hour cycle [12, 13]. Teleost-specific genome duplication has further endowed zebrafish with multiple paralogs of clock genes, particularly within the Cry family, which have undergone subfunctionalization to mediate specialized roles in rhythm generation, light entrainment, and tissue-specific regulation [14–16]. This molecular oscillator is complemented by a redundant, multi-tiered light-input system encompassing the retina, deep-brain photoreceptors, and peripheral tissue photoreceptors, ensuring robust environmental signal detection and precise phase calibration [17]. As a fundamental form of energy and sensory input, light not only enables image perception through the eyes but also mediates non-image-forming (NIF) functions. The zebrafish genome encodes 42 opsin genes, providing a prototype for understanding NIF functions of light [18].

This sophisticated circadian network governs a wide array of physiological processes in zebrafish, including sleep-wake cycles, learning and memory, cardiovascular function, energy metabolism, immune responses, reproduction, and gut-microbiota interactions, many of which are evolutionarily conserved with humans [19–25]. For example, disruption of zebrafish clock genes recapitulates human disease phenotypes such as sleep fragmentation, metabolic dysregulation, and cardiovascular defects, providing valuable insights into disease mechanisms [26]. Despite significant progress, critical knowledge gaps persist. The intracellular signaling cascades linking light perception by opsins, Cryptochromes, and other photoreceptors to the core clock machinery have not been fully elucidated; the neural circuits mediating communication between the central pacemaker (in the telencephalon) and peripheral tissues remain incompletely mapped; and the dynamics of circadian rhythms across the zebrafish lifespan, from embryogenesis to aging, are not fully characterized [27–29].

This review comprehensively synthesizes recent progress in zebrafish circadian rhythm research. We begin with the molecular mechanisms of the core oscillator, followed by a detailed analysis of multi-tissue light-input pathways. We then summarize the circadian regulation of key physiological systems and address existing limitations and challenges. Finally, we outline future research directions that harness emerging technologies to resolve unanswered questions and promote translational applications. Our goal is to provide a consolidated overview of the zebrafish model in circadian research and to inform novel approaches for exploring the evolution, function, and dysfunction of biological clocks.

2. Core Architecture and Multisystem Regulation of the Zebrafish Circadian Clock

2.1 A Highly Conserved Molecular Oscillator

The core circadian oscillator in zebrafish is constructed around a TTFL that is highly homologous to the mammalian system. CLOCK and BMAL proteins dimerize and bind E-box motifs (CACGTG) in the promoters of per and cry genes to drive their transcription [15]. Newly synthesized PER and CRY proteins form complexes in the cytoplasm, undergo phosphorylation by kinases such as CK1δ/ε, and translocate to the nucleus, where they directly inhibit CLOCK/BMAL activity, repressing their own transcription [30]. The rhythmic degradation of PER/CRY proteins, primarily via the ubiquitin-proteasome system with a peak at late night, releases this inhibition, allowing the next cycle of activation to begin [31].

This primary loop is reinforced by secondary stabilizing loops. REV-ERBα represses, while RORα activates, Bmal transcription through competitive binding to ROR elements (ROREs) in its promoter [32]. Notably, RORaa has been shown to directly regulate per2 expression via an RORE, adding a layer of cross-regulation [33, 34]. Additionally, D-box elements in the promoters of many clock-controlled genes are bound by PAR bZIP transcription factors (e.g., Tef, Hlf, Dbp). The expression of these factors is dually controlled by the core clock and acute light exposure, providing a direct conduit for photic input to shape circadian outputs [35].

A distinctive genomic feature of zebrafish is the presence of multiple paralogs for nearly all core clock genes, a result of teleost-specific genome duplication. The zebrafish genome contains two clock genes (clock1a, clock1b), two bmal genes (bmal1, bmal2), three per genes, and at least six cry genes. Functional analyses have revealed extensive subfunctionalization. For example, cry1a, cry1b, cry3a, and cry3b retain transcriptional repressor activity and participate in light-dependent phase resetting, with Cry1a acting as a bona fide photoreceptor undergoing light-induced conformational changes [36] (Figure 1). In contrast, Cry2 and Cry4 lack the motifs necessary for CLOCK/BMAL interaction and transcriptional repression [16]; Cry4 is enriched in UV cones and may function in non-circadian light detection, such as magnetoreception [37]. This expanded and diversified genetic repertoire enhances regulatory flexibility and provides a natural system for probing structure-function relationships within the circadian machinery.

Figure 1.

2.2 Redundant and Efficient Photoreceptive Input Systems

Light, a fundamental form of energy and sensory input, has profoundly shaped life on Earth [18]. Zebrafish possess a remarkable capacity for light detection, supported by redundant light-input pathways at both the cellular and molecular levels, ensuring reliable environmental signal capture by the circadian system (Figure 2).

Figure 2.

Retinal Pathway

The retina of zebrafish serves not only as a sensory organ but also as an intrinsic circadian system that maintains its own rhythmic cycles. Unlike mammals, which depend on the retinal-SCN pathway for centralized clock regulation, the zebrafish retina integrates light signals directly with its biological rhythms via inherent molecular clocks [10]. This integration enables the retina to regulate both visual function and behavioral rhythms independently [38]. The retinal circadian clock system responds to external light cues and, equally importantly, sustains intrinsic rhythms even in the absence of light, through multiple feedback loops [39–42]. These feedback mechanisms are driven by core clock genes, including clock and bmal1, which regulate the expression of genes crucial for maintaining the daily rhythm of visual function [39]. Specifically, the CLOCK/BMAL1 complex regulates the expression of long-wavelength cone opsin mRNA, exhibiting a circadian rhythm with peak levels in the afternoon and a trough in the early morning [39]. Notably, this rhythmic expression persists even in constant darkness, indicating the retina’s autonomous circadian nature [39]. In addition, light exposure directly induces the expression of light-responsive clock genes such as per2 and cry1a, which subsequently influence retinal and systemic circadian rhythms, facilitating synchronization with environmental light-dark cycles [40–42]. Studies have shown that mutations in per2 result in phase delays in zebrafish locomotor rhythms, lengthened cycles, and a reduction in both visual sensitivity and contrast sensitivity [42].

Retinal circadian rhythms also impact visual processing through the modulation of neurotransmitter systems, notably dopamine and melatonin [43, 44]. During daylight, dopamine release in the retina promotes cone cell function, enhancing visual sensitivity and color discrimination [43]. Furthermore, dopamine regulates the transmission of visual signals by altering electrical coupling between photoreceptors and bipolar and horizontal cells [43]. At night, increased melatonin secretion shifts retinal function towards rod-dominated, low-light vision [44]. In this manner, the retina adjusts its photoreceptor activity in response to the specific visual demands of day and night. Mutations in per1b cause damage to dopaminergic cells in the retina, resulting in dopamine deficiency and decreased contrast sensitivity [45]. These findings emphasize the critical role of retinal clocks and dopamine in maintaining both visual perception and behavioral rhythms [45]. In addition to regulating visual function, retinal clock genes play a role in the development of the visual system [46]. For example, the transcription factor NeuroD is regulated by clock genes after photoreceptor differentiation, contributing to the rhythmic expression of phototransduction genes [46]. Moreover, circadian rhythms regulate synaptogenesis along the retinal-tectal pathway, further underscoring the involvement of retinal clocks in the development of visual neural circuits [47].

The effects of retinal rhythms extend beyond local visual functions, influencing systemic behavioral rhythms as well [38]. Even under constant darkness, zebrafish exhibit clear circadian rhythms in visual sensitivity, with faster recovery of visual responses in the morning, better suited for rapid visual tasks, and slower recovery at night [48]. The retinal clock adjusts the dominance of cone and rod photoreceptors and modulates visual sensitivity, ensuring optimal performance under varying light conditions [43]. In larvae, light regulates the rhythmic phagocytosis of retinal rod and cone outer segment (OS) tips by the retinal pigment epithelium (RPE), thereby preventing the accumulation of harmful compounds in photoreceptors [49, 50]. Furthermore, zebrafish larvae undergo disassembly of cone synaptic ribbons at night, exhibiting “night blindness” as an energy-saving mechanism [48, 51]. Retinal rhythms also synchronize with the central nervous system through neural projections, thus influencing behavioral responses in zebrafish [17]. Pathways from the retina to the optic tectum and hypothalamus regulate circadian changes in behaviors such as visual escape responses, optokinetic responses (OKR), and visual motor responses (VMR) [17]. Although larvae lacking retinal ganglion cells can still maintain basic locomotor rhythms, the retina plays an essential role in adjusting the phase of behavioral rhythms, particularly during subjective night, when behavioral phase advancement occurs [41, 52].

Deep Brain Photoreceptors

The network composed of multiple neural pacemakers and light-responsive regions in the zebrafish brain expresses clock genes in regions including the ventral thalamic nuclei, periventricular gray zone (PGZ), dorsal nucleus of the ventral telencephalic area (Vd), hypothalamus, torus longitudinalis (TL), preglomerular nuclei, and valvula cerebelli. Among these regions, only the ventral thalamic nuclei, PGZ, and Vd receive retinal input in zebrafish, while the hypothalamus, TL, preglomerular nuclei, and valvula cerebelli are directly photosensitive [53, 54]. In vitro, clock genes in multiple brain regions of zebrafish, including the telencephalon, diencephalon, mesencephalon, optic tectum, pituitary, and rhombencephalon, can all be photo-induced [54]. Therefore, it appears that retinofugal inputs are not necessary for clock gene expression. The zebrafish brain may contain various types of photosensitive cells, and photosensitive neurons are also widely distributed in non-image-forming brain regions, such as the thalamus, hypothalamus, and preoptic area. Each type of cell may contain the same or different photopigments, including opsins, cryptochrome 4, and flavin-containing oxidases [8]. Among the family of non-visual opsins, TMT-opsin is expressed broadly in neural and non-neural tissues, paralleling clock gene expression [53]. VAL-opsin subtypes in the thalamus are distinctly regulated: valopa rhythm is endogenous, whereas valopb is light-modulated [55]. Furthermore, in larvae lacking eyes and pineal glands, *opn4m-1* mediates dark phototaxis, a behavior that is absent in otpa mutants but rescued by *opn4m-1* overexpression in the otp domain [56]. Melanopsin double mutants exhibit reduced daytime locomotor activity, requiring sustained melatonin inhibition to restore normal activity levels [57].

In the absence of a discrete SCN, zebrafish utilize distributed deep-brain photoreceptive centers [58]. The pineal gland, a photosensitive neuroendocrine organ, generates robust circadian rhythms in melatonin secretion [59, 60]. Rhythmic melatonin is detectable by 2 days post-fertilization and originates from the differentiating pineal before retinal photoreceptors become functional [61]. Even in mutants lacking eyes and forebrains, the pineal maintains rhythmic expression of clock and melatonin synthesis genes, confirming its autonomy as a pacemaker [62, 63]. Larvae without pineal glands exhibit blunted locomotor responses to darkness, underscoring their behavioral role [64]. Collectively, these findings highlight a complex non-visual light perception system essential for phase entrainment and specific light-driven behaviors. However, the complete molecular cascades linking deep-brain photoreceptor activation to the core clock remain to be fully defined.

Peripheral Photoreceptors

Unlike the SCN-centric model in mammals, zebrafish exhibit a decentralized circadian system [5]. Their peripheral organs (e.g., heart, liver), tissues (e.g., skin, muscle, intestine), and embryonic cell lines can sustain endogenous, undamped circadian oscillations in vitro for multiple cycles without central input [25, 65–68]. Moreover, these tissues can respond directly to light-dark cycles to adjust their circadian phase. This autonomy stems from the widespread expression of photopigments and associated signaling components in peripheral tissues [69]. The zebrafish genome encodes 42 opsin genes (10 visual, 32 non-visual), providing a molecular basis for tissue-specific light detection [70] (Figure 3). For example, opn6b, opn7, opn8, and opn9 are highly expressed in the heart, whereas opn5 is specific to the skin. Among the family of non-visual opsins, the zebrafish-specific TMT-opsin subfamily shares homology with known photopigments; in vitro studies have confirmed its activation by blue light and initiation of G protein signaling, supporting its role as a photopigment [71]. Despite the widespread expression of opsins in these non-retinal tissues, the light-sensitive physiological functions of many of these opsins remain to be fully elucidated.

Figure 3.

In addition to opsins, cryptochromes and flavin-containing oxidases are also candidate photoreceptive proteins. In zebrafish, Cry proteins possess multiple isoforms. Cry1a functions as an ultraviolet/blue light photoreceptor that can directly communicate with the molecular oscillator [72]. cry2b shows high-amplitude rhythmic expression across all tissues, underpinning peripheral light perception [73]. Notably, light can induce hydrogen peroxide (H₂O₂) production, which activates the MAPK pathway [74]. This may involve light-responsive flavoenzymes that absorb near-UV/blue light and promote H₂O₂ generation [75]. Light-responsive D-box enhancers can serve as nuclear targets for reactive oxygen species (ROS), and the antioxidant enzyme catalase can modulate clock gene expression by regulating H₂O₂ levels [76]. Thus, ROS signaling may complement classical pathways in a sophisticated circadian regulatory network.

2.3 Circadian Orchestration of Multi-System Physiology

Neural Rhythmicity

Zebrafish display clear diurnal sleep-wake patterns, with consolidated rest at night and daytime activity [20] (Figure 4). Genetic ablation of clock or bmal1 causes severe sleep fragmentation and arrhythmic locomotion, modeling human circadian sleep disorders. At the cellular level, the clock regulates synaptic homeostasis in the optic tectum, where synapse number and strength increase during wakefulness and are pruned during sleep—a process dependent on intact sleep-wake cycles [47]. Learning and memory also fluctuate diurnally; enhanced avoidance learning at specific phases correlates with peak per1 expression in the brain. Disruption of rhythms impairs hippocampal-like O-GlcNAcylation rhythms and downregulates nuclear OGT, leading to cognitive deficits [22]. Anxiety-like behaviors show circadian variation, suggesting a model for mood disorders. For example, protein kinase Cα (prkcaa), which is linked to neuropsychiatric disorders, modulates the morning expression of immediate early genes; its loss attenuates their normal nocturnal repression [77].

Figure 4.

Cardiovascular Homeostasis

The molecular clock plays specific roles within each type of cardiovascular tissue [66, 78] (Figure 4). The occurrence of adverse cardiovascular events also exhibits circadian rhythmicity. Heart rate and cardiac output are under strict circadian control [78]. bmal2 mutants lose heart rate rhythms and develop bradycardia and reduced stroke volume, directly linking the core clock to cardiovascular function [51]. Vascular development and regeneration also exhibit daily rhythms. Clock genes in endothelial cells rhythmically regulate VEGF and other angiogenic factors. Circadian disruption severely impairs developmental angiogenesis and adult fin regeneration [79]. Notably, bmal1 knockdown inhibited vessel formation, whereas per2 knockdown accelerated vessel formation, revealing antagonistic effects. Mechanistically, BMAL1 binds E-boxes in the vegf promoter, and its deficiency compromises Notch inhibition-induced sprouting, highlighting crosstalk between circadian and developmental pathways [19]. Hypoxia-inducible factors (HIFs) and their targets, such as EPO and VEGF, are also clock-modulated. Circadian disruption in developing zebrafish impairs hypoxic responses, alters erythropoiesis, disrupts vascular patterning, and increases mortality [80–82].

Hepatic Rhythmicity

The liver exemplifies peripheral clock-metabolism crosstalk. Transcriptomic analyses have revealed circadian oscillations in hundreds of metabolic genes involved in lipogenesis, gluconeogenesis, and detoxification [22] (Figure 4). Mutations in core clock genes induce hepatic steatosis, dyslipidemia, glucose intolerance, and insulin resistance, modeling the metabolic syndrome observed in shift workers [82, 83]. Constant darkness reduces metabolic and immune gene expression and induces fatty liver [84]. Melatonin exerts beneficial metabolic effects by increasing satiety signals in the liver and gut and suppressing Igf-I expression [85]. roraa⁻/⁻ mutants show downregulation of fatty acid oxidation genes (cpt2) and transporters (fabp2), which are direct RORaa targets and part of the PPAR signaling axis, positioning RORaa as a key integrator of circadian and metabolic regulation [33]. PAR bZIP factors and AhR targets also exhibit light- and clock-dependent rhythms, aligning detoxification capacity with daily environmental exposure [23].

Immune Rhythmicity

Innate immune responses are under circadian regulation (Figure 5) [24]. The migratory efficiency of zebrafish neutrophils to injury sites peaks during the day and is modulated by both the immune cell-intrinsic clock and systemic glucocorticoid signaling. Inflammatory cytokine expression (e.g., *i1-1β*, tnfα) also oscillates, indicating preprogramming of the inflammatory pathway by the clock [86, 87]. The core molecular clock component Clock1a regulates the rhythmic recruitment behavior of neutrophils by modulating the antioxidant response through the *nfe212a/duox* pathway [88]. per1b and per2 mutations have distinct effects: Per2 is required for neutrophil bactericidal activity, driving ROS production and enhancing infection-induced hmgb1a expression to clear bacteria [89]. The Cry-binding domain of Per2 is essential for this regulation. Conversely, Cryla-deficient neutrophils show increased bactericidal activity and hmgb1a expression [90]. Conserved BMAL1 and NF-κB motifs in the hmgbla promoter constrain its induction to the light phase. Mutating the BMAL1 motif impairs the light-dependent priming of bactericidal activity, indicating that light optimizes neutrophil function via the circadian timer. In contrast, per1b mutation downregulates ERK and subsequent p65 phosphorylation, reducing NF-κB activation and proinflammatory gene expression [91].

Figure 5.

Reproductive Rhythmicity

Circadian rhythmicity is an inherent feature of the reproductive system. Circadian clock-controlled retinoic acid (RA) signaling plays a significant role in spermatogonial differentiation and fertilization [92] (Figure 4). In Sertoli cells, the circadian clock regulates RA synthesis and receptor expression by binding to the E-box elements of the aldh1a2 and rarga genes [92]. After RA diffuses to spermatogonia, it inhibits the transcriptional repressor Zbtb16a to promote spermatogonial differentiation. Meanwhile, RA signaling upregulates the expression of the sperm surface fusion factor Izumo1, enhancing sperm fertilization capacity without affecting sperm count [92]. Circadian disruption, either global or Sertoli cell-specific disruption of clock1a/bmal1, or temporal perturbation or desynchronization of clock1a expression, all result in arrested spermatogonial differentiation and reduced fertilization [92]. Clock genes also regulate ovarian function [93]. Loss-of-function mutations in Per1/Per2 lead to premature depletion of the ovarian reserve, resulting in declined reproductive capacity. In addition, circadian rhythm disruption disturbs reproductive hormone levels and ultimately induces ovarian tumorigenesis in zebrafish [94, 95]. Embryos inherit maternal clock gene products through oogenesis and initiate their own rhythms [96]. Furthermore, preconceptional circadian rhythm disruption impairs the ovarian function of female offspring, specifically manifested as compromised follicular development, reduced oocyte quality, and decreased embryonic developmental potential, an impairment closely associated with abnormal lipid metabolism and disruption of the ovarian immune microenvironment in the offspring [96, 97].

Gut Homeostasis

The zebrafish intestine functions as a peripheral clock tissue with autonomous regulatory capacity. Its intrinsic pacemaker can be directly photoentrained for precise daily mitosis, regulating cell cycle genes such as cdc2, wee1, and p21, with modest effects on cyclins [25] (Figure 4). The proliferation and apoptosis of intestinal epithelial cells and the expression of nutrient absorption genes are systemically clock-regulated [98]. Notably, the composition and function of the commensal gut microbiota exhibit diurnal fluctuations synchronized with the host clock. Disruption of clock genes or constant light alters the microbial composition, increasing opportunistic bacteria and reducing probiotics, leading to dysbiosis [99]. This makes zebrafish an ideal model for studying the “circadian clock-gut-brain axis.” For example, constant light in larvae elevates cortisol, inhibits intestinal peristalsis, and creates a stress-induced constipation model characterized by inflammation, impaired neural activity, and dysregulated aquaporin/VIP expression [99]. Probiotic supplementation, particularly with Bifidobacterium longum, alleviates these phenotypes by reducing cortisol, modulating inflammation, and restoring gut motility and neural activity [99, 100], demonstrating functional crosstalk within the circadian-gut-microbiota network.

3. Future Perspectives

Compared with classical circadian models, zebrafish are distinguished by their unique combination of vertebrate-level physiological complexity, optical accessibility, and distributed photoreceptive capacity. The presence of decentralized photoreceptors further enhances the theoretical value of this model. In mammals, peripheral clocks are typically regarded as subordinate oscillators indirectly regulated by a central pacemaker. In zebrafish, however, peripheral tissues function as primary light-responsive units capable of autonomous entrainment. This distributed sensory architecture suggests that the relationship between central and peripheral clocks may not strictly follow a hierarchical structure, but instead be dynamically modulated across tissues depending on environmental conditions, developmental stage, or metabolic state. Such a framework redefines a central question in chronobiology. Whereas previous research has focused on how the central clock imposes order on passive peripheral oscillators, future investigations may shift toward understanding how multiple semi-autonomous oscillators achieve coordinated coherence. Does the brain integrate temporal signals from peripheral tissues, or do peripheral clocks collectively constrain central timing? By enabling direct experimental manipulation of both central and peripheral photoreceptive units within the same organism, zebrafish provide a powerful platform to address these questions. Future studies integrating tissue-specific optogenetic control, longitudinal whole-body imaging, and network modeling may reveal that vertebrate circadian systems operate as adaptive, multi-node temporal networks rather than rigid hierarchical structures. Such insights could reshape our understanding of how central and peripheral clocks coordinate under physiological conditions and how their uncoupling contributes to disease.

Exploring clock genes holds profound significance for disease treatment, as their value extends beyond circadian rhythm regulation to encompass complex organismal interactions and multi-dimensional functional roles. Increasing evidence supports the existence of bidirectional communication between biological clocks and physiological systems, as well as tight coupling among clocks across organs. Intestinal clocks influence the sleep-wake cycle by temporally maintaining the homeostasis of glutamatergic neurons in hypothalamic nuclei, and can also participate in regulating brain cognitive processes, whereas defects in gut biological clocks exert a significant negative impact on cognitive performance [101]. In parallel, the synergistic interaction between the gut microbiota and liver biological clocks has also attracted considerable attention [102]. To meet the host’s glucose demand, the gut microbiota can activate hepatic gluconeogenesis through extracellular vesicles, and the production of these extracellular vesicles exhibits circadian rhythmicity related to the host’s nutritional status, which further confirms the synergistic regulatory network of biological clocks among systems [103].

Notably, in addition to their core function in circadian rhythm regulation, clock genes also possess significant “non-canonical functions” and can participate in numerous pathways outside traditional circadian control. The CLOCK gene can reshape neuronal connectivity networks, and its abnormal expression is one of the important contributing factors to neuropsychiatric disorders such as autism and schizophrenia [104]. BMAL1 exhibits even more diverse functions: its deletion can cause pluripotent stem cells to partially reverse to a totipotent-like state, a process independent of CLOCK involvement but achieved by synergistically inhibiting the activation of MERVL and 2C genes with TRIM28 [105]. In the field of vascular diseases, the regulatory role of BMAL1 has also been re-evaluated; relevant studies have revealed its pathogenic role in vascular calcification and suggested that targeting dysregulated circadian rhythm factors may serve as a novel therapeutic strategy for preventing diabetic vascular calcification [105]. The discovery of these non-canonical functions has opened new avenues for understanding the pathogenesis of complex diseases such as cognitive disorders and developing targeted therapeutic regimens. Furthermore, the research and development of small-molecule drugs targeting clock gene regulatory pathways have achieved phased progress. Some compounds capable of regulating the activity of the BMAL1/CLOCK complex have entered the preclinical research phase and are expected to be used in combination with chronochemotherapy, enabling dual precision therapy of “targeted + temporal” [106, 107].

Although chronotherapy mediated by clock genes has achieved numerous breakthroughs in clinical applications, it still faces many challenges in translating from basic research to clinical practice. First, the regulatory network of clock genes is extremely complex: there are significant differences in the expression rhythms and regulatory mechanisms of clock genes across different tissues and disease states, and the clock gene regulatory mechanisms of most diseases have not been fully elucidated, limiting the development of targeted chronotherapeutic regimens. In the future, with the development of multi-omics technologies such as genomics, transcriptomics, and metabolomics, as well as the popularization of wearable devices (which can monitor rhythmic indicators and physiological parameters in real-time), clock gene research and chronotherapy will move towards a more precise, personalized, and systematically integrated direction. As an excellent model for circadian rhythm biology research, zebrafish has unique advantages in mechanism elucidation, drug screening, and regimen validation. By leveraging the zebrafish model to deeply analyze the inter-system regulatory mechanisms and non-canonical functions of clock genes, and to identify key regulatory targets for different diseases and individuals, it can provide more precise theoretical support for the development of chronotherapeutic regimens. Meanwhile, using the zebrafish model to rapidly verify the effectiveness of clock gene-targeted drugs and the rationality of chronotherapeutic administration schedules can strongly support improving therapeutic efficacy, reducing adverse reactions, and enhancing patients’ quality of life, thus ushering in a new era of “temporal precision + systematic synergy” in disease treatment.

4. Conclusion

Zebrafish circadian research has established a comprehensive framework encompassing molecular mechanisms, multi-tiered photic inputs, and systemic physiological regulation. While conserving the canonical TTFL architecture of vertebrates, zebrafish uniquely combine genomic expansion (e.g., subfunctionalized cry paralogs), a tripartite redundant photoreceptive system, and autonomous peripheral photoreception—collectively illuminating the evolutionary adaptability and functional sophistication of biological clocks. This model has yielded profound insights into how circadian networks orchestrate neural plasticity, cardiovascular dynamics, metabolic flux, immune vigilance, reproductive function, and host-microbiome symbiosis, providing an indispensable in vivo platform to investigate “clock-organ-environment” interactions and model human diseases rooted in circadian disruption.

Despite these advances, fundamental questions remain—most critically, the intracellular signaling cascades coupling photoreception to the oscillator and the molecular identity of synchronizing cues between central and peripheral clocks. Addressing these gaps through interdisciplinary approaches will not only deepen our understanding of circadian biology but also catalyze the development of novel diagnostics, therapeutics, and preventive strategies for a wide spectrum of human disorders. Owing to its unparalleled combination of genetic tractability, optical accessibility, physiological relevance, and evolutionary position, the zebrafish is poised to remain a cornerstone model in advancing both foundational chronobiology and the emerging era of precision time medicine.

Acknowledgements

This work was supported by the Anhui Provincial Natural Science Foundation (2208085Y10), the National Natural Science Foundation of China (32573167), the Major Special Science and Technology Project of Anhui Province (202103b06020015), the Open Project of Provincial and Ministerial Scientific Research Platform, and Fuyang Normal University (FYKFKT24038).

Additional information

Funding

MOST | National Natural Science Foundation of China (NSFC) (2021YFD1301200)

• Da-long Ren