Weighted gene co-expression network analysis identifies important modules and hub genes involved in the regulation of breast muscle yield in broilers.

Objective: Increasing breast meat production is one of the primary goals of the broiler industry. Over the past few decades, tremendous progress has been made in genetic selection and the identification of candidate genes for improving the breast muscle mass. However, the molecular network contributing to muscle production traits in chickens still needs to be further illuminated. Methods: A total of 150 1-day-old male 817 broilers were reared in a floor litter system. At the market age of 50 d, eighteen healthy 817 broilers were slaughtered and the left pectoralis major muscle sample from each bird was collected for RNA-seq sequencing. The birds were then plucked and eviscerated and the whole breast muscle was removed and weighed. Breast muscle yield was calculated as the ratio of the breast muscle weight to the eviscerated weight. To identify the co-expression networks and hub genes contributing to breast muscle yield in chickens, we performed weighted gene co-expression network analysis (WGCNA) based on the 18 transcriptome datasets of pectoralis major muscle from eighteen 817 broilers. Results: The WGCNA analysis classified all co-expressed genes in the pectoral muscle of 817 broilers into 44 modules. Among these modules, the turquoise and skyblue3 modules were found to be most significantly positively (r=0.78, p=1e-04) and negatively (r=-0.57, p=0.01) associated with breast meat yield, respectively. Further analysis identified several hub genes (e.g., DLX3, SH3RF2, TPM1, CAV3, MYF6, and CFL2) that involved in muscle structure and muscle development were identified as potential regulators of breast meat production. Conclusion: The present study has advanced our understanding of the molecular regulatory networks contributing to muscle growth and breast muscle production and will contribute to the molecular breeding of chickens in the future.

Metformin Attenuates Neutrophil Recruitment through the H3K18 Lactylation/Reactive Oxygen Species Pathway in Zebrafish.

Beyond its well-established role in diabetes management, metformin has gained attention as a promising therapeutic for inflammation-related diseases, largely due to its antioxidant capabilities. However, the mechanistic underpinnings of this effect remain elusive. Using in vivo zebrafish models of inflammation, we explored the impact of metformin on neutrophil recruitment and the underlying mechanisms involved. Our data indicate that metformin reduces histone (H3K18) lactylation, leading to the decreased production of reactive oxygen species (ROS) and a muted neutrophil response to both caudal fin injury and otic vesicle inflammation. To investigate the precise mechanisms through which metformin modulates neutrophil migration via ROS and H3K18 lactylation, we meticulously established the correlation between metformin-induced suppression of H3K18 lactylation and ROS levels. Through supplementary experiments involving the restoration of lactate and ROS, our findings demonstrated that elevated levels of both lactate and ROS significantly promoted the inflammatory response in zebrafish. Collectively, our study illuminates previously unexplored avenues of metformin's antioxidant and anti-inflammatory actions through the downregulation of H3K18 lactylation and ROS production, highlighting the crucial role of epigenetic regulation in inflammation and pointing to metformin's potential in treating inflammation-associated conditions.

Effects of 0.4 T, 3.0 T and 9.4 T static magnetic fields on development, behaviour and immune response in zebrafish (Danio rerio).

Magnetic Resonance Imaging (MRI) is widely applied in medical diagnosis due to its excellent non-invasiveness. With the increasing intensity of static magnetic field (SMF), the safety assessment of MRI has been ongoing. In this study, zebrafish larvae were exposed to SMFs of 0.4, 3.0, and 9.4 T for 2 h (h), and we found that there was no significant difference in the number of spontaneous tail swings, heart rate, and body length of zebrafish larvae in the treatment groups. The expression of development-related genes shha, pygo1, mylz3 and runx2b in the three SMF groups was almost not significantly different from the control group. Behavior tests unveiled a notable reduction in both the average speed and duration of high-speed movements in zebrafish larvae across all three SMF groups. In addition, the 0.4 and 3.0 T SMFs increased the migration of neutrophils in caudal fin injury, and the expression of pro-inflammatory cytokines was also increased. To explore the mechanism of SMFs on zebrafish immune function, this study utilized aanat2-/- mutant fish to demonstrate the effect of melatonin (MT) involvement in SMFs on zebrafish immune function. This study provides experimental data for understanding the effects of SMFs on organisms, and also provides a new insight for exploring the relationship between magnetic fields and immune function.

Circadian clock1a coordinates neutrophil recruitment via nfe212a/duox-reactive oxygen species pathway in zebrafish.

Neutrophil recruitment to inflammatory sites appears to be an evolutionarily conserved strategy to fight against exogenous insults. However, the rhythmic characteristics and underlying mechanisms of neutrophil migration on a 24-h timescale are largely unknown. Using the advantage of in vivo imaging of zebrafish, this study explored how the circadian gene clock1a dynamically regulates the rhythmic recruitment of neutrophils to inflammatory challenges. We generated a clock1a mutant and found that neutrophil migration is significantly increased in caudal fin injury and lipopolysaccharide (LPS) injection. Transcriptome sequencing, chromatin immunoprecipitation (ChIP), and dual-luciferase reporting experiments suggest that the clock1a gene regulates neutrophil migration by coordinating the rhythmic expression of nfe212a and duox genes to control the reactive oxygen species (ROS) level. This study ultimately provides a visual model to expand the understanding of the rhythmic mechanisms of neutrophil recruitment on a circadian timescale in a diurnal organism from the perspective of ROS.

DUSP2 deletion with CRISPR/Cas9 promotes Mauthner cell axonal regeneration at the early stage of zebrafish.

Axon regeneration of central neurons is a complex process that is tightly regulated by multiple extrinsic and intrinsic factors. The expression levels of distinct genes are changed after central neural system (CNS) injury and affect axon regeneration. A previous study identified dusp2 as an upregulated gene in zebrafish with spinal cord injury. Here, we found that dual specificity phosphatase 2 (DUSP2) is a negative regulator of axon regeneration of the Mauthner cell (M-cell). DUSP2 is a phosphatase that mediates the dephosphorylation of JNK. In this study, we knocked out dusp2 by CRISPR/Cas9 and found that M-cell axons of dusp2-/- zebrafish had a better regeneration at the early stage after birth (within 8 days after birth), while those of dusp2+/- zebrafish did not. Overexpression of DUSP2 in Tg (Tol 056) zebrafish by single-cell electroporation retarded the regeneration of M-cell axons. Western blotting results showed that DUSP2 knockout slightly increased the levels of phosphorylated JNK. These findings suggest that knocking out DUSP2 promoted the regeneration of zebrafish M-cell axons, possibly through enhancing JNK phosphorylation.

Fluoxetine modifies circadian rhythm by reducing melatonin content in zebrafish.

Fluoxetine (FLX), a selective serotonin reuptake inhibitor (SSRI), increases the serotonin levels in the brain to treat depression. Antidepressants have been demonstrated to modulate circadian rhythm, but the underlying mechanisms by which antidepressants regulate circadian rhythm require more research. This study aimed to investigate the role of FLX on circadian rhythm by analyzing the movement behavior and internal circadian oscillations in zebrafish. The results showed that the expression of clock genes clock1a and bmal1b was significantly down-regulated, and the amplitude reduction and phase shift were observed after FLX treatment. Furthermore, FLX exposure inhibited the expression of aanat2, which led to a decrease in nocturnal melatonin secretion. aanat2-/- larvae showed disrupted circadian rhythm. These findings may help reveal the effect of FLX exposure on the circadian rhythm and locomotor activity. It may provide theoretical data for the clinical application of FLX.

The influences and regulatory mechanisms of magnetic fields on circadian rhythms.

A variety of devices used in daily life and biomedical field will generate magnetic fields with different parameters, raising concern about their influences on people's physiological functions. Multiple experimental works have been devoted to the influences of magnetic fields on circadian rhythms, yet the findings were not always consistent due to the differences in magnetic field parameters and experimental organisms. Also, clear regulatory mechanisms have not been found. By systematizing the major achievements in research on magnetic and circadian rhythms based on magnetic flux density and analyzing the potential mechanisms of the magnetic fields affecting circadian rhythms, this review sheds light on the effects of magnetic fields on circadian rhythms and the potential applications in biomedicine.

Muscarinic acetylcholine receptors regulate inflammatory responses through arginases 1/2 in zebrafish.

Muscarinic acetylcholine receptors (mAChRs) are widely expressed in various effector cells and have been proved to play vital roles in smooth muscle contraction and digestive secretion. However, there are relatively few literatures revealing the roles of mAChRs in inflammatory processes, and its underlying regulatory mechanisms have not been elucidated. Taking the advantages of live imaging of zebrafish, we found that inhibition of mAChRs resulted in increased neutrophils recruitment and proinflammatory cytokines expression, whereas activation of mAChRs led to opposite outcome. Subsequently, we found that mAChRs regulated the expression of arginases (args), and pharmacological intervention of args level could reverse the effects of mAChRs on neutrophils migration and cytokines expression, suggesting that args are important downstream proteins of mAChRs that mediate the regulation of inflammatory response. In this study, we identified args as novel downstream proteins of mAChRs in inflammatory responses, providing additional evidence for system immune regulation of cholinergic receptors.

Hypoxia regulates cytokines expression and neutrophils migration by ERK signaling in zebrafish.

Normal dissolved oxygen in water is essential for maintaining the physiological functions of fish, but environmental pollution, such as eutrophication can lead to a decrease in oxygen content in water. How this reduction of dissolved oxygen in water affects the immune functions of fish and the potential regulatory mechanisms have not been thoroughly elucidated. In this study, we made full use of the aquatic model animal zebrafish to explore this question. In a model of LPS-induced inflammation, we found that hypoxia induced by infusing nitrogen into water increased the expression of pro-inflammatory cytokines, such as il-1ß, il-6, and il-8. In vivo imaging also showed that hypoxia significantly increased neutrophil migration to the site of caudal fin injury in the transgenic line. Subsequently, we found that the phosphorylation level of ERK protein was significantly activated upon hypoxia and proved the roles of ERK signaling in the expression of pro-inflammatory cytokines and neutrophil migration in zebrafish. This study indicated that reduced water oxygen significantly increases the inflammatory response of the zebrafish.

N-acetylcholine receptors regulate cytokines expression and neutrophils recruitment via MAPK/ERK signaling in zebrafish.

N-acetylcholine receptors (AChRs) are mainly distributed in the postsynaptic membrane and have been widely studied for their control of muscle contraction by regulating neural action potentials. However, the influences of AChRs on immune responses and potential mechanisms remain unclear. Here, we used the advantages of live imaging of zebrafish to explore the regulation process of AChRs on inflammatory responses. Pharmacologically activating of the receptor, we found that the expression of pro-inflammatory cytokines il-1ß, il-6, tnf-α and il-8 was significantly up-regulated and neutrophil migration to injury sites was also significantly increased. However, these phenomena were reversed under antagonism of the receptor activity. Results showed that interfering with nAChRs functions did not significantly affect zebrafish motion behavior. Results also showed that activation and antagonism of nAChRs function could regulate the phosphorylation of ERK protein respectively. We further demonstrated that ERK participated in the regulation of AChRs in cytokines expression and neutrophils migration in zebrafish. This study preliminarily revealed the roles of AChRs in inflammatory processes and their potential mechanism, providing additional evidence of peripheral immune regulation by cholinergic receptors.

Cordycepin inhibits inflammatory responses through suppression of ERK activation in zebrafish.

As a natural extract, cordycepin has been shown to play important regulatory roles in many life activities. In the study, the effects of cordycepin on inflammatory responses and the underlying mechanisms was explored using a zebrafish model. In the model of LPS-induced inflammation, cordycepin was found to significantly inhibited the expression of pro-inflammatory cytokines such as tnf-α, il-1ß, il-6, and il-8. Using in vivo imaging model, cordycepin significantly inhibited fluorescent-labeled neutrophils migrating towards injury sites. Furthermore, results showed that the phosphorylation level of ERK protein dramatically decreased after cordycepin treatment. Meanwhile, the ERK inhibitor, PD0325901, significantly inhibited the expression of pro-inflammatory cytokines in LPS-induced inflammatory model and neutrophils migration in the caudal fin injury model. This study indicated the important roles of cordycepin in inhibiting LPS and injury-induced inflammation and preliminarily explained the role of ERK protein in this process.

Dual Oxidase Mutant Retards Mauthner-Cell Axon Regeneration at an Early Stage via Modulating Mitochondrial Dynamics in Zebrafish.

Dual oxidase (duox)-derived reactive oxygen species (ROS) have been correlated with neuronal polarity, cerebellar development, and neuroplasticity. However, there have not been many comprehensive studies of the effect of individual duox isoforms on central-axon regeneration in vivo. Here, we explored this question in zebrafish, an excellent model organism for central-axon regeneration studies. In our research, mutation of the duox gene with CRISPR/Cas9 significantly retarded the single-axon regeneration of the zebrafish Mauthner cell in vivo. Using deep transcriptome sequencing, we found that the expression levels of related functional enzymes in mitochondria were down-regulated in duox mutant fish. In vivo imaging showed that duox mutants had significantly disrupted mitochondrial transport and redox state in single Mauthner-cell axon. Our research data provide insights into how duox is involved in central-axon regeneration by changing mitochondrial transport.

In vivo imaging of evoked calcium responses indicates the intrinsic axonal regenerative capacity of zebrafish.

Calcium is an important messenger in the neuronal system, but its specific role in axonal regeneration has not been fully investigated. To clarify it, we constructed a noninvasive in vivo calcium-imaging model of zebrafish Mauthner cells and monitored subcellular calcium dynamics during axonal regeneration. Using the calcium indicator GCamp6f, we observed that the regenerative length correlated with the peak amplitude of the evoked calcium response before axotomy, which suggested that the evoked calcium response might serve as a useful indicator of evoked neuronal activity and axonal regenerative capacity. To investigate this possibility, we overexpressed an inward rectifying potassium channel protein, Kir2.1a, to decrease the Mauthner neuronal activity and found that the inhibition of the calcium response correlated with decreased axonal regeneration. In contrast, treatment of pentylenetetrazol and knockout of the sodium voltage-gated channel α subunit 1 gene increased the calcium response and thus enhanced axonal regeneration. Our results therefore increased the understanding of the correlation between the neural activity and the vertebrate axonal regeneration.-Chen, M., Huang, R.-C., Yang, L.-Q., Ren, D.-L., Hu, B. In vivo imaging of evoked calcium responses indicates the intrinsic axonal regenerative capacity of zebrafish.

Hypoxia Delays Oligodendrocyte Progenitor Cell Migration and Myelin Formation by Suppressing Bmp2b Signaling in Larval Zebrafish.

Hypoxia in newborns tends to result in developmental deficiencies in the white matter of the brain. As previous studies of the effects of hypoxia on neuronal development in rodents and human infants have been unable to use in vivo imaging, insight into the dynamic development of oligodendrocytes (OLs) in the central nervous system under hypoxia is limited. Here, we developed a visual model to study OL development using sublethal postnatal hypoxia in zebrafish larvae. We observed that hypoxia significantly suppressed OL progenitor cell migration toward the dorsum using in vivo imaging. Further, we found that hypoxia affected myelination, as indicated by thinner myelin sheaths and by a downregulation of myelin basic protein expression. Bmp2b protein expression was also significantly downregulated following hypoxia onset. Using gain of function and loss of function experiments, we demonstrated that the Bmp2b protein was associated with the regulation of OL development. Thus, our work provides a visual hypoxia model within which to observe OL development in vivo, and reveals the underlying mechanisms involved in these processes.

Circadian gene period1b regulates proinflammatory cytokine expression through NF-κB signalling in zebrafish.

The circadian clock plays a critical role in regulating the immune system. Our previous publication revealed that a mutation in the circadian gene period1b (per1b) in zebrafish significantly decreased proinflammatory gene expression, particularly under constant darkness (DD) conditions; however, the underlying mechanisms remain unclear. In this study, using per1b-null mutant zebrafish and a larval tail fin injury model, we observed that the loss of per1b resulted in the downregulation expression of proinflammatory cytokines, such as IL-6 and TNF-α, at protein level. Furthermore, the loss of per1b downregulated ERK phosphorylation and inhibited p65 phosphorylation, leading to reduced NF-κB activation, which could downregulate the expression of proinflammatory cytokines, such as IL-6 and TNF-α, in zebrafish. These results provided insight into the communication between the circadian clock and immune functions.

Circadian genes period1b and period2 differentially regulate inflammatory responses in zebrafish.

The circadian clock has been shown to regulate various immune processes in different animals. Our previous report demonstrated that the innate immune responses in zebrafish show significant rhythmicity that could be regulated by melatonin. Here, we used diurnal zebrafish to determine the role of circadian genes in the inflammatory responses. Our results indicate that circadian genes exhibit rhythmic oscillations in zebrafish leukocytes, and mutations of the clock genes period1b (per1b) and period2 (per2) considerably affect these oscillations. Using a wounded zebrafish inflammation model, we found that under constant dark conditions (DD), the expression of pro-inflammatory cytokines is significantly downregulated in per1b gene mutant zebrafish and significantly upregulated in the per2 gene mutant zebrafish. Furthermore, using real-time imaging technology, we found that the per1b gene markedly disturbs the rhythmic recruitment of neutrophils toward the injury, whereas the per2 gene does not show a significant effect. Taken together, our results reveal differential functions of the circadian genes per1b and per2 in the inflammatory responses, serving as evidence that circadian rhythms play a vital role in immune processes.

Zebrafish Lacking Circadian Gene per2 Exhibit Visual Function Deficiency.

The retina has an intrinsic circadian clock, but the importance of this clock for vision is unknown. Zebrafish offer many advantages for studying vertebrate vision and circadian rhythm. Here, we explored the role of zebrafish per2, a light-regulated gene, in visual behavior and the underlying mechanisms. We observed that per2 mutant zebrafish larvae showed decreased contrast sensitivity and visual acuity using optokinetic response (OKR) assays. Using a visual motor response (VMR) assay, we observed normal OFF responses but abnormal ON responses in mutant zebrafish larvae. Immunofluorescence showed that mutants had a normal morphology of cone photoreceptor cells and retinal organization. However, electron microscopy showed that per2 mutants displayed abnormal and decreased photoreceptor ribbon synapses with arciform density, which resulted in retinal ON pathway defect. We also examined the expression of three cone opsins by quantitative real-time PCR (qRT-PCR), and the expression of long-wave-sensitive opsin (opn1lw) and short-wave-sensitive opsin (opn1sw) was reduced in mutant zebrafish larvae. qRT-PCR analyses also showed a down-regulation of the clock genes cry1ba and bmal1b in the adult eye of per2 mutant zebrafish. This study identified a mechanism by which a clock gene affects visual function and defined important roles of per2 in retinal information processing.

Effects of circadian clock protein Per1b on zebrafish visual functions.

The circadian clock is an endogenous and entrainable time-keeping mechanism with a period of approximately 24 h, operated by transcription/translation feedback loops composed of circadian clock genes and their proteins. The visual system displays robust circadian changes. Relatively little, however, is known about the mechanisms underlying visual circadian rhythmicity. Zebrafish period1b (per1b), as a canonical circadian clock gene, is involved in circadian regulation. Here, we observed that zebrafish per1b mutants exhibit visual defects including reduced behavioral contrast sensitivity and significant retinal dopaminergic deficiency. Further, partially damaged dopaminergic interplexiform cells in wild-type larvae also led to reduced behavioral contrast sensitivity, while exogenous dopamine administration effectively restored the contrast sensitivity of per1b mutants. Taken together, these results suggest that retinal dopaminergic deficiency derived from loss of per1b results in visual defects in zebrafish. ABBREVIATIONS: per1b, period1b; per, period; per1, period1; per2, period2; per3, period3; ERG, electroretinogram; DA-IPCs, dopaminergic interplexiform cells; IRBP, interphotoreceptor retinoid binding protein; MS-222, methane-sulfonate; USTC, University of Science and Technology of China; OKR, optokinetic response; dpf, day postfertilization; 6-OHDA, 6-hydroxydopamine; TH, tyrosine hydroxylase; DA, dopaminergic; INL, inner nuclear; IPL, innerplexiform layers; hpf, hours postfertilization; cpd, cycle per degree; ADHD, attention deficit and hyperactivity disorder.

Endogenous melatonin promotes rhythmic recruitment of neutrophils toward an injury in zebrafish.

Neutrophil recruitment to injured tissue appears to be an evolutionarily conserved strategy for organisms to fight against exogenous insults. Recent studies have shown rhythmic migration of neutrophils and several factors, including melatonin, have been implicated in regulating this rhythmic migration. The mechanisms underlying how endogenous melatonin regulates rhythmic neutrophils migration, however, are unclear. Here we generated a zebrafish annat2 mutant that lacks endogenous melatonin and, subsequently, a Tg(lyz:EGFP);aanat2 -/- transgenic line that allows for monitoring neutrophils migration visually in live zebrafish. We observed that migrating neutrophils are significantly reduced in aanat2 -/- mutant zebrafish under a light/dark condition, and the disrupted migrating rhythmicity of neutrophils in aanat2 -/- zebrafish is independent of the circadian clock. Further, we also found that endogenous melatonin enhances neutrophils migration likely by inducing the expression of cytokines such as interleukin-8 and interleukin-1ß. Together, our findings provide evidence that endogenous melatonin promotes rhythmic migration of neutrophils through cytokines in zebrafish.

Exogenous melatonin inhibits neutrophil migration through suppression of ERK activation.

Neutrophil migration to inflammatory sites is the fundamental process of innate immunity among organisms against pathogen invasion. As a major sleep adjusting hormone, melatonin has also been proved to be involved in various inflammatory events. This study aimed to evaluate the impact of exogenous melatonin on neutrophil migration to the injury site in live zebrafish and further investigate whether ERK signaling is involved in this process. Using the tail fin transection model, the fluorescently labeled neutrophil was in vivo visualized in transgenic Tg(lyz:EGFP), Tg(lyz:DsRed) zebrafish. We found that exogenous melatonin administration dramatically inhibited the injury-induced neutrophil migration in a dose-dependent and time-dependent manner. The inhibited effect of melatonin on neutrophil migration could be attenuated by melatonin receptor 1, 2, and 3 antagonists. The ERK phosphorylation level was significantly decreased post injury when treated with melatonin. The blocking of ERK activation with inhibitor PD0325901 suppressed the number of migrated neutrophils in response to injury. However, the activation of ERK with the epidermal growth factor could impair the inhibited effect of melatonin on neutrophil migration. We also detected that PD0325901 significantly suppressed the in vivo neutrophils transmigrating over the vessel endothelial cell using the transgenic Tg(flk:EGFP);(lyz:DsRed) line labeled as both vessel and neutrophil. Taking all of these data together, the results indicated that exogenous melatonin had an anti-migratory effect on neutrophils by blocking the ERK phosphorylation signal, and it led to the subsequent adhesion molecule expression. Thus, the crossing of the vessel endothelial cells of neutrophils became difficult.