Han Wang

Hepatic inflammation is a defining feature of Metabolic Dysfunction-Associated Steatohepatitis (MASH), yet the specific contributions of individual immune cell populations and their reciprocal interactions remain incompletely understood. In this study, we combined flow cytometry with single-cell transcriptomic profiling to characterize the hepatic immune landscape in a novel model of spontaneous MASH caused by hepatocyte-specific deficiency of endoplasmic reticulum-associated degradation (ERAD). Hepatic ERAD deficiency led to the expansion of multiple immune cell populations in the liver, including CD8+ T cells, macrophages, monocytes, and dendritic cells, accompanied by extensive functional reprogramming of both innate and adaptive immune compartments. Myeloid cells exhibited enhanced phagocytic activity and increased antigen processing and presentation, whereas CD8+ T cells displayed elevated proliferation capacity, DNA repair activity and cytotoxicity. Notably, two functionally distinct triggering receptor expressed on myeloid cells 2 (TREM2)-expressing macrophage subsets emerged during the progression of ERAD deficiency-induced MASH. Depletion of CD8+ T cells increased monocyte infiltration and aggravated liver injury, suggesting that CD8+ T cells exert a previously unrecognized protective role by restraining monocyte recruitment. Collectively, these findings reveal highly coordinated interactions between innate and adaptive cells during MASH progression and identify CD8+ T cells as potential regulators of monocyte infiltration and hepatic injury.

The vertebrate testis has long been regarded as lacking strong cellular daily rhythmicity. Here, we use time-series single-cell RNA sequencing to construct a temporal atlas of the adult zebrafish testis. We identify previously unknown cell-type–specific markers and previously unidentified testicular subtypes and reveal that Sertoli cells, spermatogonia, and spermatids exhibit robust oscillations in key circadian clock genes. In contrast, Leydig cells and spermatocytes primarily exhibit rhythmic expression of key functional genes. We show that intercellular communication networks also exhibit daily rhythmicity. Pseudotime trajectory analysis demonstrates a notable nocturnal preference for germ cell progression. Disrupting circadian input via constant light reduced germ cell numbers and impaired fertility. Genetic ablation of clock1a arrests spermatogonial differentiation, whereas mutation of the rhythmic spermatocyte gene hmgb1a substantially reduces meiotic cells. Together, our findings redefine the testis as a highly rhythmic organ in which a precisely timed, nocturnally coordinated cellular symphony drives spermatogenesis.

Introduction: Myostatin (MSTN), a transforming growth factor‐beta (TGF‐β) superfamily member, is an evolutionarily conserved negative regulator of skeletal muscle mass. Loss of MSTN commonly promotes augmentation in skeletal muscle mass in all animal species examined. Recent studies have demonstrated that circadian clock proteins play a critical role in the regulation of muscle mass and function, in part by modulating the expression of key muscle‐related genes. While myostatin has an important role in sustaining skeletal muscle protein turnover, it is unknown if circadian clock proteins regulate myostatin in a circadian pattern. Methods: We analysed time‐course muscle samples from 16 animal species ranging from Caenorhabditis elegans to humans and examined the rhythmic expression pattern of Mstn . We also used various circadian clock deficient models such as muscle‐specific Bmal1 knockout, Per1/Per2 double knockout, genetic knockout of per0 and tim0 genes in fruit flies, clocka gene in zebrafish and environmental perturbation. Results: Both mRNA and protein of MSTN exhibit rhythmic expression patterns in a variety of animal species ranging from Caenorhabditis elegans ( C. elegans ) to humans. The rhythmicity of Mstn orthologs in muscle is evolutionarily conserved along with their sequence evolution in C. elegans , Drosophila melanogaster , Crustacea , fish and mammals including mice (mRNA: amplitude = 0.188, p < 0.0001; protein: amplitude = 0.255, p < 0.05), goats, pigs and humans. In murine skeletal muscle, rhythmic expression of Mstn is synchronized with the core circadian genes, Per2 . We then constructed a muscle‐specific Bmal1 knockout mouse model ( Bmal1 ^MKO ). Notably, Bmal1 ^MKO mice had increased body weight (29.30 ± 0.85 vs. 32.16 ± 0.79, p < 0.05) and lean mass (WT 23.33 ± 0.35 vs. 25.35 ± 0.45, p < 0.01), while the difference in lean mass at 12 weeks of age (~1.996 g) closely matches the difference in total body weight (~ 2.000 g). Muscle‐specific Bmal1 knockout reduced the mRNA and protein levels of mstn /MSTN by ~ 50%. In addition, disruption of the circadian clock by constant light or Per1/Per2 double knockout also abolishes the rhythmicity of Mstn . Similarly, genetic knockout of per0 and tim0 genes in fruit flies, clocka gene in zebrafish ( mstna : p < 0.01 vs. p = 0.6397) and environmental perturbation ( Aplodinotus grunniens , mstn1 : p < 0.0001 vs. p = 0.04; mstn2 : p < 0.05 vs. p = 0.06) all alter Mstn oscillation profoundly. Conclusions: These findings reveal an evolutionarily conserved rhythmic expression pattern of Mstn in skeletal muscles.

The desire to increase life expectancy, coupled with the decline in biological functions that occurs as we age, represents one of the most significant challenges facing our society. Age-related declines in biological functions contribute to frailty and morbidity, demanding innovative strategies to promote healthy aging. The circadian clock, which controls daily physiological processes, is intricately linked to aging and overall health. Circadian disruptions can lead to metabolic dysfunction, impaired immune responses, increased DNA damage, and elevated disease susceptibility. On the other hand, maintaining robust circadian rhythms through interventions such as regular sleep–wake patterns, time-restricted feeding, and physical activity may extend health span and longevity. The circadian clock affects various molecular pathways associated with aging, including the insulin/IGF, mTOR, and sirtuin signaling pathways. Enhancing circadian rhythms presents a promising avenue for mitigating age-related disorders and promoting healthy aging. This review highlights the potential of circadian clock-based interventions as a transformative strategy to improve the quality of life and extend the healthspan of aging individuals.

To satisfy the needs of sleeping underwater, marine mammals, including cetaceans, sirenians, and pinnipeds, have evolved an unusual form of sleep, known as unihemispheric slow-wave sleep (USWS), in which one brain hemisphere is asleep while the other is awake. All aquatic cetaceans have only evolved USWS without rapid eye movement (REM) sleep, whereas aquatic sirenians and amphibious pinnipeds display both bihemispheric slow-wave sleep (BSWS) and USWS, as well as REM sleep. However, the molecular genetic changes underlying USWS remain unknown. The present study investigated the evolution of eight canonical circadian genes and found that positive selection occurred mainly within cetacean lineages. Furthermore, convergent evolution was observed in lineages with USWS at three circadian clock genes. Remarkably, in vitro assays showed that cetacean-specific mutations increased the nuclear localization of zebrafish clocka, and enhanced the transcriptional activation activity of Clocka and Bmal1a. In vivo, transcriptome analysis showed that the overexpression of the cetacean-specific mutant clocka (clocka-mut) caused the upregulation of the wakefulness-promoting glutamatergic genes and the differential expression of multiple genes associated with sleep regulation. In contrast, the GABAergic and cholinergic pathways, which play important roles in promoting sleep, were downregulated in the bmal1a-mut-overexpressing zebrafish. Concordantly, sleep time of zebrafish overexpressing clocka-mut and bmal1a-mut were significantly less than the zebrafish overexpressing the wild-type genes, respectively. These findings support our hypothesis that canonical circadian clock genes may have evolved adaptively to enhance circadian regulation ability relating to sleep in cetaceans and, in turn, contribute to the formation of USWS.

The circadian clock genes are known important for kidney development, maturation and physiological functions. However, whether and how they play a role in renal regeneration remain elusive. Here, by using the single cell RNA-sequencing (scRNA-seq) technology, we investigated the dynamic gene expression profiles and cell states after acute kidney injury (AKI) by gentamicin treatment in zebrafish. The core clock genes such as per1/2 and nr1d1, which encode transcriptional repressors of the circadian system, are strongly induced in the proximal tubule epithelial cells (PTECs). By generating mutant zebrafish lines, we show that per1a and nr1d1 are required for proper renal regeneration, by facilitating the expression of renal progenitor cell (RPC) genes. In per1a and nr1d1 mutants, the expression of RPC genes and the number of RPCs were decreased, resulting in a marked delay in nephron regeneration. lima1a, which encodes a cytoskeleton binding protein that functions to negatively regulate epithelial to mesenchymal transition (EMT), is identified as the direct target of the clock proteins. Down-regulation of lima1a is associated with enhanced EMT, increased expression of cell migration- and RPC markers, and accelerated nephron regeneration. We propose that per1a and nr1d1 are important for the formation of nephrongenic RPCs by repressing lima1a. Our findings using zebrafish provide important insights into the roles of the clock genes in kidney repair.

MicroRNAs post-transcriptionally regulate gene expression and contribute to numerous life processes, including circadian rhythms. However, whether miRNAs contribute to zebrafish circadian regulation has not yet been investigated. Here, we showed that mature miR-219-5p, and its three pre-miRNAs, mir-219-1, mir-219-2, and mir-219-3, are rhythmically expressed primarily in Tectum opticum (TeO), Corpus cerebelli (CCe), and Crista cerellaris (CC) of the zebrafish brain. While mir-219-1 and mir-219-2 are regulated by the circadian clock through the E-like box, mir-219-3 is regulated by light via the D-box. Deleting mir-219-1, mir-219-2, or mir-219-3 individually or knocking down miR-219-5p all results in a shortened period of locomotor rhythms and up-regulation of bmal1b. RIP assays with Ago2 and miRNA pull-down assays show that miR-219-5p binds to bmal1b in the RISC. Cell transfection and in Vivo assays show that miR219-5p inhibits bmal1b through binding to its 3’UTR. Further, transcriptome analysis of miR-219-5p knockdown zebrafish adult brain reveals possible roles of miR-219-5p in phototransduction and neuroactive ligand-receptor interaction. Together, our findings demonstrate that mir-219-1, mir-219-2, and mir-219-3 are controlled directly by the circadian clock; and in turn, miR-219-5p contributes to circadian regulation by targeting bmal1b, highlighting a miR-219-5p-bmal1b negative feedback loop in the zebrafish circadian circuit.

The circadian clock generates and maintains ∼24-hour oscillations in almost all organs. The testis, however, remains mysterious, without a clear understanding of its circadian functions. Our time-series transcriptome analysis reveals more than 1000 rhythmically expressed genes in the zebrafish and mouse testes, respectively. Canonical circadian clock genes are rhythmically expressed in Sertoli cells and regulate retinoic acid (RA) production, which is also evidenced by their co-expression with RA synthesis genes in single Sertoli cells. Genetic and pharmacological manipulations and temporal desynchronization revealed that the circadian clock-regulated RA signaling synchronizes spermatogonial differentiation via zbtb16a and promotes fertilization via izumo1 in zebrafish. Our findings indicate that the testicular circadian clock contributes to reproduction in a cell-specific manner through RA signaling, highlighting circadian roles in male fertility.

The zebrafish (Danio rerio) has emerged as a model organism for investigating lncRNAs-driven fundamental biological processes, such as circadian rhythms, physiology, metabolism, and various diseases. While state-of-the-art sequencing technologies have identified an increasing number of lncRNAs in zebrafish, their annotations are far from complete. In this study, we collect 28,925 lncRNAs from both the published studies and our own RNA-seq analyses and establish a novel webserver-based database called SUDAZFLNC (https://sudarna.website/). The database, containing 28,925 lncRNAs, 25,432 mRNAs, and 368 miRNAs, provides several crucial features and annotations for the zebrafish RNAs, such as sequence identifiers (IDs), sequence length, hexamer score, coding probabilities, GO and KEGG annotations, and micropeptides. SUDAZFLNC also includes time-course expression profiles of 3288 lncRNAs, 25,432 mRNAs, and 342 miRNAs generated from our RNA-seq experiments, and 149, 4407, and 43 rhythmically expressed lncRNAs, mRNAs, and miRNAs, respectively. Based on the peak expression patterns, we classified these RNAs into morning RNAs, evening RNAs, and night RNAs. Users of the database can access the RNA sequences and their expression profiles by searching the corresponding IDs from the Graphical User Interface (GUI) of the database. The database supports several features to investigate RNA sequences and expression profiles, including BLAST, search of sequence and data, ID conversion, and RNA-RNA interaction prediction. This is the largest curated database of zebrafish RNAs and their expression profiles to date.