Introduction
The circadian clock coordinates multiple aspects of physiology, including digestion (Pal and Maitra, 2018), cell proliferation (Tamai et al., 2012), stress responses (S��nchez-V��zquez et al., 2019), spawning, and behavior (Marchesan et al., 2005). While the molecular architecture of the circadian system is well characterized in adult vertebrates, its developmental onset and early functional organization remain poorly understood, particularly in fish. In teleosts, circadian rhythms have been described at both behavioral and molecular levels (Cuesta et al., 2014); however, studies addressing their ontogeny are still limited. As a result, the timing of initial rhythmicity and the processes underlying the establishment of a functional circadian system during early development remain largely unresolved.
The aim of this study was to determine the onset and progression of circadian rhythmicity in Eurasian perch by combining high-resolution temporal sampling of core clock gene expression with transcriptomic profiling to identify rhythmically expressed genes and associated molecular pathways across key developmental stages.
Results and discussion
Using high-resolution temporal sampling, we demonstrated that core circadian clock genes in Eurasian perch begin to oscillate at 3 days post-hatching (DPH) and exhibit robust rhythmicity by 28 DPH under a controlled photoperiod (14L:10D). These two developmental stages were therefore selected for transcriptomic profiling using 24-hour round-the-clock sampling at 4-hour intervals to capture diel gene expression dynamics and identify molecular pathways involved in the emergence and consolidation of circadian regulation. Using MetaCycle, we identified 1,466 rhythmically expressed genes at 3 DPH and 478 at 28 DPH, of which 206 were shared between both stages, indicating a combination of conserved and stage-specific rhythmic processes.
Temporal clustering with TCseq revealed seven distinct expression clusters at each developmental stage, reflecting diverse rhythmic patterns across the diel cycle. Functional annotation and gene ontology analyses further indicated that these clusters were associated with a range of biological processes, suggesting a progressive organization of circadian-regulated pathways during development. This approach uncovered both shared and stage-specific rhythmic transcripts, including several genes not previously described as circadian biomarkers in fish. Among these were, for example, ciart and cipc, which are known components of the negative regulatory arm of the circadian clock in other vertebrates but have not yet been characterized in fish.
To further investigate candidate genes associated with early circadian development, we implemented two complementary validation strategies. First, a cross-species approach targeted genes previously reported in mammals or other model organisms but not in fish, including ciart and cipc, to assess their evolutionary conservation and potential functional relevance in teleost circadian systems. Second, we selected a set of ten putative novel circadian rhythm-associated genes identified from our transcriptomic dataset, representing candidates with no prior links to circadian regulation. Collectively, these findings expand current knowledge of the genetic architecture underlying early circadian development and lay the groundwork for further mechanistic exploration.
Acknowledgment
This research was funded by the National Science Centre, Poland (OPUS project, number UMO-2021/43/B/NZ9/03056).
References
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