Long non-coding RNAs mediate fish gene expression in response to ocean acidification.

The majority of the transcribed genome does not have coding potential but these non-coding transcripts play crucial roles in transcriptional and post-transcriptional regulation of protein-coding genes. Regulation of gene expression is important in shaping an organism's response to environmental changes, ultimately impacting their survival and persistence as population or species face global change. However, the roles of long non-coding RNAs (lncRNAs), when confronted with environmental changes, remain largely unclear. To explore the potential role of lncRNAs in fish exposed to ocean acidification (OA), we analyzed publicly available brain RNA-seq data from a coral reef fish Acanthochromis polyacanthus. We annotated the lncRNAs in its genome and examined the expression changes of intergenic lncRNAs (lincRNAs) between A. polyacanthus samples from a natural CO2 seep and a nearby control site. We identified 4728 lncRNAs, including 3272 lincRNAs in this species. Remarkably, 93.03% of these lincRNAs were species-specific. Among the 125 highly expressed lincRNAs and 403 differentially expressed lincRNAs in response to elevated CO2, we observed that lincRNAs were either neighboring or potentially trans-regulating differentially expressed coding genes associated with pH regulation, neural signal transduction, and ion transport, which are known to be important in the response to OA in fish. In summary, lncRNAs may facilitate fish acclimation and mediate the responses of fish to OA by modulating the expression of crucial coding genes, which offers insight into the regulatory mechanisms underlying fish responses to environmental changes.

Single-cell atlas of human infrapatellar fat pad and synovium implicates APOE signaling in osteoarthritis pathology.

The infrapatellar fat pad (IPFP) and synovium play essential roles in maintaining knee joint homeostasis and in the progression of osteoarthritis (OA). The cellular and transcriptional mechanisms regulating the function of these specialized tissues under healthy and diseased conditions are largely unknown. Here, single-cell and single-nuclei RNA sequencing of human IPFP and synovial tissues were performed to elucidate the cellular composition and transcriptional profile. Computational trajectory analysis revealed that dipeptidyl peptidase 4+ mesenchymal cells function as a common progenitor for IPFP adipocytes and synovial lining layer fibroblasts, suggesting that IPFP and synovium represent an integrated tissue unit. OA induced a profibrotic and inflammatory phenotype in mesenchymal lineage cells with biglycan+ intermediate fibroblasts as a major contributor to OA fibrosis. Apolipoprotein E (APOE) signaling from intermediate fibroblasts and macrophages was identified as a critical regulatory factor. Ex vivo incubation of human cartilage with soluble APOE accelerated proteoglycan degeneration. Inhibition of APOE signaling by intra-articular injection of an anti-APOE neutralizing antibody attenuated the progression of collagenase-induced OA in mice, demonstrating a detrimental effect of APOE on cartilage. Our studies provide a framework for designing further therapeutic strategies for OA by describing the cellular and transcriptional landscape of human IPFP and synovium in healthy versus OA joints.

Extracellular Vesicles in Infrapatellar Fat Pad from Osteoarthritis Patients Impair Cartilage Metabolism and Induce Senescence.

Infrapatellar fat pad (IPFP) is closely associated with the development and progression of knee osteoarthritis (OA), but the underlying mechanism remains unclear. Here, it is find that IPFP from OA patients can secret small extracellular vesicles (sEVs) and deliver them into articular chondrocytes. Inhibition the release of endogenous osteoarthritic IPFP-sEVs by GW4869 significantly alleviated IPFP-sEVs-induced cartilage destruction. Functional assays in vitro demonstrated that IPFP-sEVs significantly promoted chondrocyte extracellular matrix (ECM) catabolism and induced cellular senescence. It is further demonstrated that IPFP-sEVs induced ECM degradation in human and mice cartilage explants and aggravated the progression of experimental OA in mice. Mechanistically, highly enriched let-7b-5p and let-7c-5p in IPFP-sEVs are essential to mediate detrimental effects by directly decreasing senescence negative regulator, lamin B receptor (LBR). Notably, intra-articular injection of antagomirs inhibiting let-7b-5p and let-7c-5p in mice increased LBR expression, suppressed chondrocyte senescence and ameliorated the progression of experimental OA model. This study uncovers the function and mechanism of the IPFP-sEVs in the progression of OA. Targeting IPFP-sEVs cargoes of let-7b-5p and let-7c-5p can provide a potential strategy for OA therapy.

Gene losses, parallel evolution and heightened expression confer adaptations to dedicated cleaning behaviour.

BACKGROUND: Cleaning symbioses are captivating interspecific interactions in which a cleaner fish removes ectoparasites from its client, contributing to the health and diversity of natural fish communities and aquaculture systems. However, the genetic signatures underlying this specialized behaviour remain poorly explored. To shed light on this, we generated a high-quality chromosome-scale genome of the bluestreak cleaner wrasse Labroides dimidiatus, a dedicated cleaner with cleaning as primary feeding mechanism throughout its life. RESULTS: Compared with facultative and non-cleaner wrasses, L. dimidiatus was found with notable contractions in olfactory receptors implying their limited importance in dedicated cleaning. Instead, given its distinct tactile pre-conflict strategies, L. dimidiatus may rely more heavily on touch sensory perception, with heightened gene expression in the brain in anticipation of cleaning. Additionally, a reduction in NLR family CARD domain-containing protein 3 might enhance innate immunity of L. dimidiatus, probably assisting to reduce the impacts from parasite infections. In addition, convergent substitutions for a taste receptor and bone development genes across cleaners (L. dimidiatus and facultative cleaners) may provide them with evolved food discrimination abilities and jaw morphology that differentiate them from non-cleaners. Moreover, L. dimidiatus may exhibit specialized neural signal transductions for cleaning, as evidenced by positive selection in genes related to the glutamatergic synapse pathway. Interestingly, numerous glutamate receptors also demonstrated significantly higher expression in L. dimidiatus not engaged in cleaning, as compared to those involved in cleaning. Besides, apparent contractions in L. dimidiatus for protocadherins, which are responsible for neuronal development, may further promote specialized neural signal transductions in this species. CONCLUSIONS: This study reveals that L. dimidiatus harbours substantial losses in specific gene families, convergent evolutions across cleaners and a large-scale high gene expression in preparation for cleaning, allowing for adaptation to the dedicated cleaning behaviour.

Enlarged fins of Tibetan catfish provide new evidence of adaptation to high plateau.

The uplift of the Tibetan Plateau significantly altered the geomorphology and climate of the Euroasia by creating large mountains and rivers. Fishes are more likely to be affected relative to other organisms, as they are largely restricted to river systems. Faced with the rapidly flowing water in the Tibetan Plateau, a group of catfish has evolved greatly enlarged pectoral fins with more numbers of fin-rays to form an adhesive apparatus. However, the genetic basis of these adaptations in Tibetan catfishes remains elusive. In this study, we performed comparative genomic analyses based on the chromosome-level genome of Glyptosternum maculatum in family Sisoridae and detected some proteins with conspicuously high evolutionary rates in particular in genes involved in skeleton development, energy metabolism, and hypoxia response. We found that the hoxd12a gene evolved faster and a loss-of-function assay of hoxd12a supports a potential role for this gene in shaping the enlarged fins of these Tibetan catfishes. Other genes with amino acid replacements and signatures of positive selection included proteins involved in low temperature (TRMU) and hypoxia (VHL) responses. Functional assays reveal that the G. maculatumTRMU allele generates more mitochondrial ATP than the ancestral allele found in low-altitude fishes. Functional assays of VHL alleles suggest that the G. maculatum allele has lower transactivation activity than the low-altitude forms. These findings provide a window into the genomic underpinnings of physiological adaptations that permit G. maculatum to survive in the harsh environment of the Tibetan Himalayas that mirror those that are convergently found in other vertebrates such as humans.

Circular RNA circNFKB1 promotes osteoarthritis progression through interacting with ENO1 and sustaining NF-κB signaling.

Inflammatory cytokines-induced activation of the nuclear factor κB (NF-κB) pathway plays a critical role in the pathogenesis of osteoarthritis (OA). Circular RNA (circRNA) has been identified as important epigenetic factor in numerous diseases. However, the biological roles of inflammation-related circRNAs in regulating OA pathogenesis remain elusive. Here, we revealed circRNA expression profiles in human primary chondrocytes with interleukin-1ß (IL-1ß) stimulation by circRNA sequencing. We identified a highly upregulated circRNA, termed as circNFKB1 in inflamed chondrocytes and osteoarthritic cartilage. As a circRNA derived from exon 2-5 of NFKB1, circNFKB1 is located in both cytoplasm and nucleus of chondrocytes. Furthermore, knockdown of circNFKB1 inhibited extracellular matrix (ECM) catabolism and rescued IL-1ß impaired ECM anabolism whereas ectopic expression of circNFKB1 significantly promoted chondrocytes degradation in vitro. Moreover, intraarticular injection of adenovirus-circNFKB1 in mouse joints triggered spontaneous cartilage loss and OA development. Mechanistically, circNFKB1 interacted with α-enolase (ENO1), regulated the expression of its parental gene NFKB1 and sustained the activation of NF-κB signaling pathway in chondrocytes. Therefore, this study highlights a novel ENO1-interacting circNFKB1 in OA pathogenesis, and provides valuable insights into understanding the regulatory mechanism of NF-κB signaling in chondrocytes and a promising therapeutic target for the treatment of OA.

Rapid evolution fuels transcriptional plasticity to ocean acidification.

Ocean acidification (OA) is postulated to affect the physiology, behavior, and life-history of marine species, but potential for acclimation or adaptation to elevated pCO2 in wild populations remains largely untested. We measured brain transcriptomes of six coral reef fish species at a natural volcanic CO2  seep and an adjacent control reef in Papua New Guinea. We show that elevated pCO2 induced common molecular responses related to circadian rhythm and immune system but different magnitudes of molecular response across the six species. Notably, elevated transcriptional plasticity was associated with core circadian genes affecting the regulation of intracellular pH and neural activity in Acanthochromis polyacanthus. Gene expression patterns were reversible in this species as evidenced upon reduction of CO2 following a natural storm-event. Compared with other species, Ac. polyacanthus has a more rapid evolutionary rate and more positively selected genes in key functions under the influence of elevated CO2 , thus fueling increased transcriptional plasticity. Our study reveals the basis to variable gene expression changes across species, with some species possessing evolved molecular toolkits to cope with future OA.

Integrated Transcriptomics and Metabolomics Analyses Provide Insights Into the Response of Chongyi Wild Mandarin to Candidatus Liberibacter Asiaticus Infection.

Candidatus Liberibacter asiaticus (CLas) is the causative agent of Huanglongbing (HLB), which has caused great economic losses to the citrus industry. The molecular mechanism of the host response to CLas in wild citrus germplasm has been reported less. Eighteen weeks after inoculation via grafting, all the CLas-inoculated Chongyi wild mandarin (Citrus reticulata) were positive and showed severe anatomical aberrations, suggesting its susceptibility to HLB. Transcriptomics and metabolomics analyses of leaves, barks, and roots from mock-inoculated (control) and CLas-inoculated seedlings were performed. Comparative transcriptomics identified 3,628, 3,770, and 1,716 differentially expressed genes (DEGs) between CLas-infected and healthy tissues in the leaves, barks, and roots, respectively. The CLas-infected tissues had higher transcripts per kilobase per million values and more genes that reached their maximal expression, suggesting that HLB might cause an overall increase in transcript accumulation. However, HLB-triggered transcriptional alteration showed tissue specificity. In the CLas-infected leaves, many DEGs encoding immune receptors were downregulated. In the CLas-infected barks, nearly all the DEGs involved in signaling and plant-pathogen interaction were upregulated. In the CLas-infected roots, DEGs encoding enzymes or transporters involved in carotenoid biosynthesis and nitrogen metabolism were downregulated. Metabolomics identified 71, 62, and 50 differentially accumulated metabolites (DAMs) in the CLas-infected leaves, barks and roots, respectively. By associating DEGs with DAMs, nitrogen metabolism was the only pathway shared by the three infected tissues and was depressed in the CLas-infected roots. In addition, 26 genes were determined as putative markers of CLas infection, and a hypothesized model for the HLB susceptibility mechanism in Chongyi was proposed. Our study may shed light on investigating the molecular mechanism of the host response to CLas infection in wild citrus germplasm.

Analysis of Multiplicity of Hypoxia-Inducible Factors in the Evolution of Triplophysa Fish (Osteichthyes: Nemacheilinae) Reveals Hypoxic Environments Adaptation to Tibetan Plateau.

HIF (Hypoxia-inducible factor) gene family members function as master regulators of cellular and systemic oxygen homeostasis during changes in oxygen availability. Qinghai-Tibet Plateau is a natural laboratory for for long-term hypoxia and cold adaptation. In this context, T. scleroptera that is restricted to >3500 m high-altitude freshwater rivers was selected as the model to compare with a representative species from the plain, P. dabryanus. We cloned different HIF-α and carried out a phylogenetic analysis from invertebrates to vertebrates for identifying HIF-α genes and analyzing their evolutionary history. Intriguingly, the HIF-α has undergone gene duplications might be due to whole-genome duplication (WGD) events during evolution. PAML analysis indicated that HIF-1αA was subjected to positive selection acted on specific sites in Triplophysa lineages. To investigate the relationship between hypoxia adaptation and the regulation of HIF-α stability by pVHL in plateau and plain fish, a series of experiments were carried out. Comparison the luciferase transcriptional activity and protein levels of HIF-αs and the differing interactions of HIF-αs with pVHL, show clear differences between plateau and plain fish. T. scleroptera pVHL could enhance HIF-α transcriptional activity under hypoxia, and functional validation through pVHL protein mutagenesis showed that these mutations increased the stability of HIF-α and its hetero dimerization affinity to ARNT. Our research shows that missense mutations of pVHL induced evolutionary molecular adaptation in Triplophysa fishes living in high altitude hypoxic environments.

Stat5b Regulates Sexually Dimorphic Gene Expression in Zebrafish Liver.

Sexual size dimorphism is an interesting phenomenon occurred in many fish species. Wildtype zebrafish exhibits a significant sexual dimorphism in body size at the adult stage. Previous studies indicated that sexual size dimorphism was eliminated in stat5b-mutated zebrafish. Herein, the comparative transcriptome analysis was conducted to observe the genes and pathways involved in sexual size dimorphism. The number of male-biased and female-biased genes was much less in the liver of stat5b mutant zebrafish than in wildtype. Gene ontology (GO) enrichment and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis indicated that multiple pathways related to metabolism were affected upon loss of stat5b function. qRT-PCR results also validated that sexually dimorphic expression of a set of genes was lost when stat5b was mutated. Furthermore, the weighted correlation network analysis (WGCNA) detected many candidate genes related to the growth traits and stat5b function, such as greb1, lepr, and igf2b. Our data suggest that stat5b should regulate the sexually dimorphic gene expression in zebrafish liver and add in understanding of the molecular mechanisms underlying sexual size dimorphism in fish species.

Author Correction: Population genetics analysis of the Nujiang catfish Creteuchiloglanis macropterus through a genome-wide single nucleotide polymorphisms resource generated by RAD-seq.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Essential roles of stat5.1/stat5b in controlling fish somatic growth.

Signal transducer and activator of transcription 5b (STAT5b) has been identified as a key downstream mediator of growth hormone (GH) signaling in somatic growth of mammalian. However, the corresponding homologue gene of Stat5b is unknown in fish species. In this study, we generated loss-of-function mutants in stat5.1 and stat5.2, two stat5 homologues existing in zebrafish. In stat5.1-deficient zebrafish, a significant reduction of body length and body weight was detected in the embryos/larvae and adults compared with the wild-type control fish, and sexual size dimorphism in adult zebrafish was also eliminated. However, the stat5.2-deficient zebrafish displayed a normal developmental phenotype during all lifespan. Chromatin immunoprecipitation combined with deep sequencing (ChIP-seq) method was adopted to further investigate the potential transcriptional targets of Stat5 protein and cast much light upon the biological function of Stat5. We identified more than 800 genes as transcriptional targets of Stat5 during zebrafish embryogenesis. KEGG analysis indicated that the Stat5 target gene network is predominantly linked to the metabolic pathways, neuroactive ligand-receptor interaction and JAK-STAT signaling pathways. Further validation studies suggested that Stat5.1 protein could directly regulate the expression of gh1, and stat5.1-mutated zebrafish showed a reduction of gh1 mRNA level. In the present study, stat5.1 was revealed as the corresponding homologue gene of Stat5b in fish species. Additionally, we found a novel molecular interaction between Stat5.1/Stat5b and GH, and unraveled a positive feedback loop Stat5.1-GH-Stat5.1 which is necessary for somatic growth and body development in zebrafish.

Evidence of high-altitude adaptation in the glyptosternoid fish, Creteuchiloglanis macropterus from the Nujiang River obtained through transcriptome analysis.

BACKGROUND: Organisms living at high altitudes face low oxygen and temperature conditions; thus, the genetic mechanisms underlying the adaptations in these organisms merit investigation. The glyptosternoid fish, Creteuchiloglanis macropterus mainly inhabits regions with gradual increases in altitudes along the Nujiang River and might serve as an appropriate evolutionary model for detecting adaptation processes in environments with altitude changes. RESULTS: We constructed eleven RNA-sequencing (RNA-seq) libraries of C. macropterus collected from five locations at different altitudes to identify the genetic signatures of high-altitude adaptation. The comparative genomic analysis indicated that C. macropterus has an accelerated evolutionary rate compared with that of fishes in the lowland, and fishes at higher altitudes might evolve faster. Functional enrichment analysis of the fast-evolving and positively selected genes, differentially expressed genes and highly expressed genes, showed that these genes were involved in many functions related to energy metabolism and hypoxia. CONCLUSIONS: Our study provides evidence of high-altitude adaptation in C. macropterus, and the detected adaptive genes might be a resource for future investigations of adaptations to high-altitude environments in other fishes.

Population genetics analysis of the Nujiang catfish Creteuchiloglanis macropterus through a genome-wide single nucleotide polymorphisms resource generated by RAD-seq.

Advances in genome scanning using high-throughput sequencing technologies has led to a revolution in studies of non-model organisms. The glyptosternoid fish Creteuchiloglanis macropterus, is widely distributed in the main stem and tributaries of the Nujiang River basin. Here, we analyzed IIB restriction-site-associated DNA (2b-RAD) sequences and mitochondrial DNA sequences, to assess the genomic signature of adaptation by detecting and estimating the degree of genetic differentiation among ten Creteuchiloglanis macropterus populations from the Nujiang River. The analyses revealed significant population differentiation among the up-tributaries, main stem, mid-tributary and low-tributary. Annotation of contigs containing outlier SNPs revealed that the candidate genes showed significant enrichment in several important biological process terms between up-tributaries and low-tributary, and exhibited prominent enrichment in the term macromolecular metabolic process between all tributaries and the main stem. Population dynamics analyses indicated that the Late Pleistocene glaciations strongly influenced the demographic history of C. macropterus. Our results provide strong evidence for the utility of RAD-seq in population genetics studies, and our generated SNP resource should provide a valuable tool for population genomics studies of C. macropterus in the future.

Comprehensive Transcriptome Analysis of Six Catfish Species from an Altitude Gradient Reveals Adaptive Evolution in Tibetan Fishes.

Glyptosternoid fishes (Siluriformes), one of the three broad fish lineages (the two other are schizothoracines and Triplophysa), have a limited distribution in the rivers in the Tibetan Plateau and peripheral regions. To investigate the genetic mechanisms underlying adaptation to the Tibetan Plateau in several fish species from gradient altitudes, a total of 20,659,183-37,166,756 sequence reads from six species of catfish were generated by Illumina sequencing, resulting in six assemblies. Analysis of the 1,656 orthologs among the six assembled catfish unigene sets provided consistent evidence for genome-wide accelerated evolution in the three glyptosternoid lineages living at high altitudes. A large number of genes refer to functional categories related to hypoxia and energy metabolism exhibited rapid evolution in the glyptosternoid lineages relative to yellowhead catfish living in plains areas. Genes showing signatures of rapid evolution and positive selection in the glyptosternoid lineages were also enriched in functions associated with energy metabolism and hypoxia. Our analyses provide novel insights into highland adaptation in fishes and can serve as a foundation for future studies aiming to identify candidate genes underlying the genetic basis of adaptation in Tibetan fishes.

Biogeographic history and high-elevation adaptations inferred from the mitochondrial genome of Glyptosternoid fishes (Sisoridae, Siluriformes) from the southeastern Tibetan Plateau.

BACKGROUND: The distribution of the Chinese Glyptosternoid catfish is limited to the rivers of the Tibetan Plateau and peripheral regions, especially the drainage areas of southeastern Tibet. Therefore, Glyptosternoid fishes are ideal for reconstructing the geological history of the southeastern Tibet drainage patterns and mitochondrial genetic adaptions to high elevations. RESULTS: Our phylogenetic results support the monophyly of the Sisoridae and the Glyptosternoid fishes. The reconstructed ancestral geographical distribution suggests that the ancestral Glyptosternoids was widely distributed throughout the Brahmaputra drainage in the eastern Himalayas and Tibetan area during the Late Miocene (c. 5.5 Ma). We found that the Glyptosternoid fishes lineage had a higher ratio of nonsynonymous to synonymous substitutions than those found in non-Glyptosternoids. In addition, ωpss was estimated to be 10.73, which is significantly higher than 1 (p-value 0.0002), in COX1, which indicates positive selection in the common ancestral branch of Glyptosternoid fishes in China. We also found other signatures of positive selection in the branch of specialized species. These results imply mitochondrial genetic adaptation to high elevations in the Glyptosternoids. CONCLUSIONS: We reconstructed a possible scenario for the southeastern Tibetan drainage patterns based on the adaptive geographical distribution of the Chinese Glyptosternoids in this drainage. The Glyptosternoids may have experienced accelerated evolutionary rates in mitochondrial genes that were driven by positive selection to better adapt to the high-elevation environment of the Tibetan Plateau.