MicroRNA roles in regeneration: Multiple lessons from zebrafish.

MicroRNAs (miRNAs) are small noncoding RNAs with pivotal roles in the control of gene expression. By comparing the miRNA profiles of uninjured vs. regenerating tissues and structures, several studies have found that miRNAs are potentially involved in the regenerative process. By inducing miRNA overexpression or inhibition, elegant experiments have directed regenerative responses validating relevant miRNA-to-target interactions. The zebrafish (Danio rerio) has been the epicenter of regenerative research because of its exceptional capability to self-repair damaged tissues and body structures. In this review, we discuss recent discoveries that have improved our understanding of the impact of gene regulation mediated by miRNAs in the context of the regeneration of fins, heart, retina, and nervous tissue in zebrafish. We compiled what is known about the miRNA control of regeneration in these tissues and investigated the links among up-regulated and down-regulated miRNAs, their putative or validated targets, and the regenerative process. Finally, we briefly discuss the forthcoming prospects, highlighting directions and the potential for further development of this field.

Variant expression signatures of microRNAs and protein related to growth in a crossbreed between two strains of Nile tilapia (Oreochromis niloticus).

Nile tilapia (Oreochromis niloticus) is a species of worldwide importance for aquaculture. A crossbred lineage was developed through introgressive backcross breeding techniques and combines the high growth performance of the Chitralada (CHIT) lwith attractive reddish color of the Red Stirling (REDS) strains. Since the crossbreed has an unknown genetically improved background, the objective of this work was to characterize expression signatures that portray the advantageous phenotype of the crossbreeds. We characterized the microRNA transcriptome by high throughput sequencing (RNA-seq) and the proteome through mass spectrometry (ESI-Q-TOF-MS) and applied bioinformatics for the comparative analysis of such molecular data on the three strains. Crossbreed expressed a distinct set of miRNAs and proteins compared to the parents. They comprised several microRNAs regulate traits of economic interest. Proteomic profiles revealed differences between parental and crossbreed in expression of proteins associated with glycolisis. Distinctive miRNA and protein signatures contribute to the phenotype of crossbreed.

A comparative analysis of heart microRNAs in vertebrates brings novel insights into the evolution of genetic regulatory networks.

BACKGROUND: During vertebrate evolution, the heart has undergone remarkable changes that lead to morphophysiological differences in the fully formed heart of these species, such as chamber septation, heart rate frequency, blood pressure, and cardiac output volume. Despite these differences, the heart developmental process is guided by a core gene set conserved across vertebrates. Nonetheless, the regulatory mechanisms controlling the expression of genes involved in heart development and maintenance are largely uncharted. MicroRNAs (miRNAs) have been described as important regulatory elements in several biological processes, including heart biology. These small RNA molecules are broadly conserved in sequence and genomic context in metazoans. Mutations may occur in miRNAs and/or genes that contribute to the establishment of distinct repertoires of miRNA-target interactions, thereby favoring the differential control of gene expression and, consequently, the origin of novel phenotypes. In fact, several studies showed that miRNAs are integrated into genetic regulatory networks (GRNs) governing specific developmental programs and diseases. However, studies integrating miRNAs in vertebrate heart GRNs under an evolutionary perspective are still scarce. RESULTS: We comprehensively examined and compared the heart miRNome of 20 species representatives of the five major vertebrate groups. We found 54 miRNA families with conserved expression and a variable number of miRNA families with group-specific expression in fishes, amphibians, reptiles, birds, and mammals. We also detected that conserved miRNAs present higher expression levels and a higher number of targets, whereas the group-specific miRNAs present lower expression levels and few targets. CONCLUSIONS: Both the conserved and group-specific miRNAs can be considered modulators orchestrating the core and peripheral genes of heart GRNs of vertebrates, which can be related to the morphophysiological differences and similarities existing in the heart of distinct vertebrate groups. We propose a hypothesis to explain evolutionary differences in the putative functional roles of miRNAs in the heart GRNs analyzed. Furthermore, we present new insights into the molecular mechanisms that could be helping modulate the diversity of morphophysiology in the heart organ of vertebrate species.

Mimicking the tumor microenvironment: Fibroblasts reduce miR-29b expression and increase the motility of ovarian cancer cells in a co-culture model.

Ovarian cancer (OC) is a highly prevalent gynecological malignancy worldwide. Throughout ovarian carcinogenesis, the crosstalk between cellular components of the microenvironment, including tumor cells and fibroblasts, is proposed to play critical roles in cancer progression. The dysregulation of microRNA expression is also a pronounced feature of the OC. The screening of microRNAs, mainly those involved in OC microenvironment, could have diagnostic and/or therapeutic potential for this malignancy. Thus, we assessed the influence of fibroblasts on microRNA expression and the motility of OC cells. To achieve this goal, SKOV-3 cancer cells were co-cultured with human normal fibroblasts derived from primary culture (FP-96). Cell viability, expression of tumor suppressor microRNAs and oncomiRs by RT-qPCR, cell migration by wound healing assay and analysis of MMP-2 activity by zymography were performed in SKOV-3 cells. Moreover, α-smooth muscle actin (α-SMA) expression was evaluated by Western blot in FP-96 fibroblasts. Notably, the co-culture downregulated the tumor suppressor miR-29b and increased migration of SKOV-3 cells. In addition, co-culture increased the activity of MMP-2, which is a miR-29 target, and accounted for extracellular matrix remodeling and augmented cellular motility. Concomitantly, the co-culture system induced α-SMA expression in FP-96 fibroblasts, the commonly expressed marker in cancer-associated fibroblasts (CAFs). Our findings suggest that the potential crosstalk between OC cells and fibroblasts in tumor microenvironment may play a key role in the progression of OC.

Inadequate Dietary Phosphorus Levels Cause Skeletal Anomalies and Alter Osteocalcin Gene Expression in Zebrafish.

Phosphorus (P) is an essential mineral for the development and maintenance of the vertebrate skeletal system. Modulation of P levels is believed to influence metabolism and the physiological responses of gene expression. In this study, we investigated the influence of dietary P on skeletal deformities and osteocalcin gene expression in zebrafish (Danio rerio), and sought to determine appropriate levels in a diet. We analyzed a total of 450 zebrafish within 31 days of hatching. Animals were distributed in a completely randomized experimental design that consisted of five replications. After an eight-week experiment, fish were diaphanized to evaluate cranial and spinal bone deformities. Increases in dietary phosphorus were inversely proportional to the occurrence of partial spine fusions, the absence of spine fusions, absence of parallelism between spines, intervertebral spacing, vertebral compression, scoliosis, lordosis, ankylosis, fin caudal insertion, and craniofacial deformities. Additionally, osteocalcin expression was inversely correlated to P levels, suggesting a physiological recovery response for bone mineralization deficiency. Our data showed that dietary P concentration was a critical factor in the occurrence of zebrafish skeletal abnormalities. We concluded that 1.55% P in the diet significantly reduces the appearance of skeletal deformities and favors adequate bone mineralization through the adjustment of osteocalcin expression.

Evolution and genomic organization of muscle microRNAs in fish genomes.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNA molecules with an important role upon post-transcriptional regulation. These molecules have been shown essential for several cellular processes in vertebrates, including muscle biology. Many miRNAs were described as exclusively or highly expressed in skeletal and/or cardiac muscle. However, knowledge on the genomic organization and evolution of muscle miRNAs has been unveiled in a reduced number of vertebrates and mostly only reflects their organization in mammals, whereas fish genomes remain largely uncharted. The main goal of this study was to elucidate particular features in the genomic organization and the putative evolutionary history of muscle miRNAs through a genome-wide comparative analysis of cartilaginous and bony fish genomes. RESULTS: As major outcomes we show that (1) miR-208 was unexpectedly absent in cartilaginous and ray-finned fish genomes whereas it still exist in other vertebrate groups; (2) miR-499 was intergenic in medaka and stickleback conversely to other vertebrates where this miRNA is intronic; (3) the zebrafish genome is the unique harboring two extra paralogous copies of miR-499 and their host gene (Myh7b); (4) a rare deletion event of the intergenic and bicistronic cluster miR-1-1/133a-2 took place only into Tetraodontiformes genomes (pufferfish and spotted green puffer); (5) the zebrafish genome experienced a duplication event of miR-206/-133b; and (6) miR-214 was specifically duplicated in species belonging to superorder Acanthopterygii. CONCLUSIONS: Despite of the aforementioned singularities in fish genomes, large syntenic blocks containing muscle-enriched miRNAs were found to persist, denoting colligated functionality between miRNAs and neighboring genes. Based on the genomic data here obtained, we envisioned a feasible scenario for explaining muscle miRNAs evolution in vertebrates.

MicroRNA Transcriptomes Reveal Prevalence of Rare and Species-Specific Arm Switching Events During Zebrafish Ontogenesis.

In metazoans, microRNAs (miRNAs) are essential regulators of gene expression, affecting critical cellular processes from differentiation and proliferation, to homeostasis. During miRNA biogenesis, the miRNA strand that loads onto the RNA-induced Silencing Complex (RISC) can vary, leading to changes in gene targeting and modulation of biological pathways. To investigate the impact of these "arm switching" events on gene regulation, we analyzed a diverse range of tissues and developmental stages in zebrafish by comparing 5p and 3p arms accumulation dynamics between embryonic developmental stages, adult tissues, and sexes. We also compared variable arm usage patterns observed in zebrafish to other vertebrates including arm switching data from fish, birds, and mammals. Our comprehensive analysis revealed that variable arm usage events predominantly take place during embryonic development. It is also noteworthy that isomiR occurrence correlates to changes in arm selection evidencing an important role of microRNA distinct isoforms in reinforcing and modifying gene regulation by promoting dynamics switches on miRNA 5p and 3p arms accumulation. Our results shed new light on the emergence and coordination of gene expression regulation and pave the way for future investigations in this field.

Selection of references for quantitative real-time PCR analysis of microRNAs in Nile tilapia (Oreochromis niloticus) under osmotic stress.

MicroRNAs play crucial regulatory roles in various aspects of development and physiology, including environmental adaptation and stress responses in teleosts. RT-qPCR is the most commonly used method for studying microRNA expression, with the accuracy and reliability of results depending on the use of an appropriate reference gene for normalization. This study aimed to evaluate seven miRNAs (U6, Let-7a, miR-23a, miR-25-3, miR-103, miR-99-5, and miR-455) expression stability in different tissues of Nile tilapia subjected to osmotic stress. Fish were divided into two groups: a control and an experimental group, raised in 0 and 12 ppt salinity water respectively. After 21 days, brain, gills, liver, and posterior intestine were collected for analysis. Different mathematical algorithms (geNorm, NormFinder, BestKeeper, and the comparative ΔCt method) were employed to identify the most suitable reference miRNAs. The results indicate that the miR-455/miR-23a combination is a robust reference for normalizing miRNA expression levels in studies of osmotic stress responses in Nile tilapia. The stability of miRNA expression can vary depending on specific stress conditions and biological processes, underscoring the necessity of selecting appropriate normalizing miRNAs for each experimental context. This study identifies reliable reference genes for future RT-qPCR analyses of miRNA expression, thereby enhancing our understanding of molecular responses in fish to environmental challenges. These insights are fundamental to the development of new technologies for the improved management and sustainability of aquaculture practices.

Assessing reproductive effects and epigenetic responses in Austrolebias charrua exposed to Roundup Transorb®: Insights from miRNA profiling and molecular interaction analysis.

This study examines the effects of Roundup Transorb® (RDT) exposure on reproductive functions and ovarian miRNA expression in Austrolebias charrua. Exposure to RDT (at 0.065 or 5 mg. L-1 for 96 h) significantly disrupts fertility, evidenced by changes in fertilization rates and egg diameter. Profiling of ovarian miRNAs identified a total 205 miRNAs in A. charrua. Among these, three miRNAs were upregulated (miR-10b-5p, miR-132-3p, miR-100-5p), while ten miRNAs were downregulated (miR-499-5p, miR-375, miR-205-5p, miR-206-3p, miR-203a-3p, miR-133b-3p, miR-203b-5p, miR-184, miR-133a-3p, miR-2188-5p) compared to non-exposed fish. This study reveals that differentially expressed miRNAs are linked to molecular pathways such as steroid hormone biosynthesis, lipid and carbohydrate metabolism, bioenergetics, and antioxidant defense. It also analyzes molecular interactions between miRNAs and target genes during RDT exposure in annual killifish, providing insights into biomarkers in ecotoxicology. Moreover, it provides scope for developing environmental health assessment models based on epigenomic endpoints, supporting the protection of biodiversity and ecosystem services through the quantification of stress responses in living organisms exposed to pesticides.

Reconstruction of regulatory network predicts transcription factors driving the dynamics of zebrafish heart regeneration.

The limited regenerative capacity in mammals has serious implications for cardiac tissue damage. Meanwhile, zebrafish has a high regenerative capacity, but the regulation of the heart healing process has yet to be elucidated. The dynamic nature of cardiac regeneration requires consideration of the inherent temporal dimension of this process. Here, we conducted a systematic review to find genes that define the regenerative cell state of the zebrafish heart. We then performed an in silico temporal gene regulatory network analysis using transcriptomic data from the zebrafish heart regenerative process obtained from databases. In this analysis, the genes found in the systematic review were used to represent the final cell state of the transition process from a non-regenerative cell state to a regenerative state. We found 135 transcription factors driving the cellular state transition process during zebrafish cardiac regeneration, including Hand2, Nkx2.5, Tbx20, Fosl1, Fosb, Junb, Vdr, Wt1, and Tcf21 previously reported for playing a key role in tissue regeneration. Furthermore, we demonstrate that most regulators are activated in the first days post-injury, indicating that the transition from a non-regenerative to a regenerative state occurs promptly.

Fibronectin Modulates the Expression of miRNAs in Prostate Cancer Cell Lines.

Prostate cancer (PCa) is a significant cause of cancer-related deaths among men and companion animals, such as dogs. However, despite its high mortality and incidence rates, the molecular mechanisms underlying this disease remain to be fully elucidated. Among the many factors involved in prostate carcinogenesis, the extracellular matrix (ECM) plays a crucial role. This ECM in the prostate is composed mainly of collagen fibers, reticular fibers, elastic fibers, proteoglycans and glycoproteins, such as fibronectin. Fibronectin is a glycoprotein whose dysregulation has been implicated in the development of multiple types of cancer, and it has been associated with cell migration, invasion, and metastasis. Furthermore, our research group has previously shown that fibronectin induces transcriptional changes by modulating the expression of protein coding genes in LNCaP cells. However, potential changes at the post-transcriptional level are still not well understood. This study investigated the impact of exposure to fibronectin on the expression of a key class of regulatory RNAs, the microRNAs (miRNAs), in prostate cancer cell lines LNCaP and PC-3. Five mammalian miRNAs (miR-21, miR-29b, miR-125b, miR-221, and miR-222) were differentially expressed after fibronectin exposure in prostate cell lines. The expression profile of hundreds of mRNAs predicted to be targeted by these miRNAs was analyzed using publicly available RNA-Sequencing data (GSE64025, GSE68645, GSE29155). Also, protein-protein interaction networks and enrichment analysis were performed to gain insights into miRNA biological functions. Altogether, these functional analyzes revealed that fibronectin exposure impacts the expression of miRNAs potentially involved in PCa causing changes in critical signaling pathways such as PI3K-AKT, and response to cell division, death, proliferation, and migration. The relationship here demonstrated between fibronectin exposure and altered miRNA expression improves the comprehension of PCa in both men and other animals, such as dogs, which naturally develop prostate cancer.

The 5S rDNA family evolves through concerted and birth-and-death evolution in fish genomes: an example from freshwater stingrays.

BACKGROUND: Ribosomal 5S genes are well known for the critical role they play in ribosome folding and functionality. These genes are thought to evolve in a concerted fashion, with high rates of homogenization of gene copies. However, the majority of previous analyses regarding the evolutionary process of rDNA repeats were conducted in invertebrates and plants. Studies have also been conducted on vertebrates, but these analyses were usually restricted to the 18S, 5.8S and 28S rRNA genes. The recent identification of divergent 5S rRNA gene paralogs in the genomes of elasmobranches and teleost fishes indicate that the eukaryotic 5S rRNA gene family has a more complex genomic organization than previously thought. The availability of new sequence data from lower vertebrates such as teleosts and elasmobranches enables an enhanced evolutionary characterization of 5S rDNA among vertebrates. RESULTS: We identified two variant classes of 5S rDNA sequences in the genomes of Potamotrygonidae stingrays, similar to the genomes of other vertebrates. One class of 5S rRNA genes was shared only by elasmobranches. A broad comparative survey among 100 vertebrate species suggests that the 5S rRNA gene variants in fishes originated from rounds of genome duplication. These variants were then maintained or eliminated by birth-and-death mechanisms, under intense purifying selection. Clustered multiple copies of 5S rDNA variants could have arisen due to unequal crossing over mechanisms. Simultaneously, the distinct genome clusters were independently homogenized, resulting in the maintenance of clusters of highly similar repeats through concerted evolution. CONCLUSIONS: We believe that 5S rDNA molecular evolution in fish genomes is driven by a mixed mechanism that integrates birth-and-death and concerted evolution.

Genome assembly and annotation of the tambaqui (Colossoma macropomum): an emblematic fish of the Amazon River Basin.

Colossoma macropomum, known as "tambaqui", is the largest Characiformes fish in the Amazon River Basin and a leading species in Brazilian aquaculture and fisheries. Good quality meat and excellent adaptability to culture systems are some of its remarkable farming features. To support studies into the genetics and genomics of the tambaqui, we have produced the first high-quality genome for the species. We combined Illumina and PacBio sequencing technologies to generate a reference genome, assembled with 39× coverage of long reads and polished to a consensus quality value (QV) of 36 with 130× coverage of short reads. The genome was assembled into 1269 scaffolds (a total of 1,221,847,006 bases), with a scaffold N50 size of 40 Mb, where 93% of all assembled bases were placed in the largest 54 scaffolds corresponding to the diploid karyotype of the tambaqui. Furthermore, the NCBI Annotation Pipeline annotated genes, pseudogenes, and non-coding transcripts using the RefSeq database as evidence, guaranteeing a high-quality annotation. A Genome Data Viewer for the tambaqui was produced, which will benefit groups interested in exploring the unique genomic features of the species. The availability of a highly accurate genome assembly for tambaqui provides the foundation for the discovery of novel ecological and evolutionary insights, and is a helpful resource for aquaculture.

GH Overexpression Alters Spermatic Cells MicroRNAome Profile in Transgenic Zebrafish.

Overexpression of growth hormone (GH) in gh-transgenic zebrafish of a highly studied lineage F0104 has earlier been reported to cause increased muscle growth. In addition to this, GH affects a broad range of cellular processes in transgenic fish, such as morphology, physiology, and behavior. Reports show changes such as decreased sperm quality and reduced reproductive performance in transgenic males. It is hypothesized that microRNAs are directly involved in the regulation of fertility potential during spermatogenesis. The primary aim of our study was to verify whether gh overexpression disturbs the sperm miRNA profile and influences the sperm quality in transgenic zebrafish. We report a significant increase in body weight of gh-transgenic males along with associated reduced sperm motility and other kinetic parameters in comparison to the non-transgenic group. MicroRNA transcriptome sequencing of gh-transgenic zebrafish sperms revealed expressions of 186 miRNAs, among which six miRNA were up-regulated (miR-146b, miR-200a-5p, miR-146a, miR-726, miR-184, and miR-738) and sixteen were down-regulated (miR-19d-3p, miR-126a-5p, miR-126b-5p, miR-22a-5p, miR-16c-5p, miR-20a-5p, miR-126b-3p, miR-107a-3p, miR-93, miR-2189, miR-202-5p, miR-221-3p, miR-125a, miR-125b-5p, miR-126a-3p, and miR-30c-5p) in comparison to non-transgenic zebrafish. Some of the dysregulated miRNAs were previously reported to be related to abnormalities in sperm quality and reduced reproduction ability in other species. In this study, an average of 134 differentially expressed miRNAs-targeted genes were predicted using the in silico approach. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis demonstrated that the genes of affected pathways were primarily related to spermatogenesis, sperm motility, and cell apoptosis. Our results suggested that excess GH caused a detrimental effect on sperm microRNAome, consequently reducing the sperm quality and reproductive potential of zebrafish males.

miRTil: An Extensive Repository for Nile Tilapia microRNA Next Generation Sequencing Data.

Nile tilapia is the third most cultivated fish worldwide and a novel model species for evolutionary studies. Aiming to improve productivity and contribute to the selection of traits of economic impact, biotechnological approaches have been intensively applied to species enhancement. In this sense, recent studies have focused on the multiple roles played by microRNAs (miRNAs) in the post-transcriptional regulation of protein-coding genes involved in the emergence of phenotypes with relevance for aquaculture. However, there is still a growing demand for a reference resource dedicated to integrating Nile Tilapia miRNA information, obtained from both experimental and in silico approaches, and facilitating the analysis and interpretation of RNA sequencing data. Here, we present an open repository dedicated to Nile Tilapia miRNAs: the "miRTil database". The database stores data on 734 mature miRNAs identified in 11 distinct tissues and five key developmental stages. The database provides detailed information about miRNA structure, genomic context, predicted targets, expression profiles, and relative 5p/3p arm usage. Additionally, miRTil also includes a comprehensive pre-computed miRNA-target interaction network containing 4936 targets and 19,580 interactions.

Transfection of exogenous DNA complexed to cationic dendrimer induces alterations of bovine sperm microRNAome.

MicroRNAs have been hypothesized to be involved in the regulation of male fertility potential. The primary aim of our study was to demonstrate the effects of transfection with dendrimer nanostructure on the parameters of bovine sperm quality and to investigate whether the microRNA profile could be disturbed after cationic dendrimer-mediated exogenous DNA transfection of bovine spermatozoa. The binding of exogenous DNA was significantly increased when dendrimer-based transfection was implemented. However, cationic dendrimer transfection induced detrimental changes in the kinetics and sperm quality parameters, such as membrane integrity, acrosome reaction, and mitochondrial membrane potential, when compared to the control group. Sperm microRNA sequencing revealed 218 known and 106 novel microRNAs in the sperm samples, among which nine were dysregulated after transfection (one was upregulated and eight were downregulated), in comparison to the non-transfected sperm. All the dysregulated microRNAs were related to sperm quality and embryonic development. These results suggest that the transfection process using the dendrimer nanostructure has an impact on the quality and microRNA profile of bovine sperm.

Molecular organization of 5S rDNA in sharks of the genus Rhizoprionodon: insights into the evolutionary dynamics of 5S rDNA in vertebrate genomes.

In this study, we attempted a molecular characterization of the 5S rDNA in two closely related species of carcharhiniform sharks, Rhizoprionodon lalandii and Rhizoprionodon porosus, as well as a further comparative analysis of available data on lampreys, several fish groups and other vertebrates. Our data show that Rhizoprionodon sharks carry two 5S rDNA classes in their genomes: a short repeat class (termed class I) composed of approximately 185 bp repeats, and a large repeat class (termed class II) arrayed in approximately 465 bp units. These classes were differentiated by several base substitutions in the 5S coding region and by completely distinct non-transcribed spacers (NTS). In class II, both species showed a similar composition for both the gene coding region and the NTS region. In contrast, class I varied extensively both within and between the two shark species. A comparative analysis of 5S rRNA gene sequences of elasmobranchs and other vertebrates showed that class I is closely related to the bony fishes, whereas the class II gene formed a separate cartilaginous clade. The presence of two variant classes of 5S rDNA in sharks likely maintains the tendency for dual ribosomal classes observed in other fish species. The present data regarding the 5S rDNA organization provide insights into the dynamics and evolution of this multigene family in the fish genome, and they may also be useful in clarifying aspects of vertebrate genome evolution.

Understanding the Modus Operandi of MicroRNA Regulatory Clusters.

MicroRNAs (miRNAs) are non-coding RNAs that regulate a wide range of biological pathways by post-transcriptionally modulating gene expression levels. Given that even a single miRNA may simultaneously control several genes enrolled in multiple biological functions, one would expect that these tiny RNAs have the ability to properly sort among distinctive cellular processes to drive protein production. To test this hypothesis, we scrutinized previously published microarray datasets and clustered protein-coding gene expression profiles according to the intensity of fold-change levels caused by the exogenous transfection of 10 miRNAs (miR-1, miR-7, miR-9, miR-124, miR-128a, miR-132, miR-133a, miR-142, miR-148b, miR-181a) in a human cell line. Through an in silico functional enrichment analysis, we discovered non-randomic regulatory patterns, proper of each cluster identified. We demonstrated that miRNAs are capable of equivalently modulate the expression signatures of target genes in regulatory clusters according to the biological function they are assigned to. Moreover, target prediction analysis applied to ten vertebrate species, suggest that such miRNA regulatory modus operandi is evolutionarily conserved within vertebrates. Overall, we discovered a complex regulatory cluster-module strategy driven by miRNAs, which relies on the controlled intensity of the repression over distinct targets under specific biological contexts. Our discovery helps to clarify the mechanisms underlying the functional activity of miRNAs and makes it easier to take the fastest and most accurate path in the search for the functions of miRNAs in any distinct biological process of interest.

Corrigendum: Gene and Blood Analysis Reveal That Transfer from Brackish Water to Freshwater Is More Stressful to the Silverside Odontesthes humensis.

[This corrects the article DOI: 10.3389/fgene.2018.00028.].

Differential expression of myogenic regulatory factor MyoD in pacu skeletal muscle (Piaractus mesopotamicus Holmberg 1887: Serrasalminae, Characidae, Teleostei) during juvenile and adult growth phases.

Skeletal muscle is the edible part of the fish. It grows by hypertrophy and hyperplasia, events regulated by differential expression of myogenic regulatory factors (MRFs). The study of muscle growth mechanisms in fish is very important in fish farming development. Pacu (Piaractus mesopotamicus) is one of the most important food species farmed in Brazil and has been extensively used in Brazilian aquaculture programs. The aim of this study was to analyze hyperplasia and hypertrophy and the MRF MyoD expression pattern in skeletal muscle of pacu (P. mesopotamicus) during juvenile and adult growth stages. Juvenile (n=5) and adult (n=5) fish were anaesthetized, sacrificed, and weight (g) and total length (cm) determined. White dorsal region muscle samples were collected and immersed in liquid nitrogen. Transverse sections (10 microm thick) were stained with Haematoxilin-Eosin (HE) for morphological and morphometric analysis. Smallest fiber diameter from 100 muscle fibers per animal was calculated in each growth phase. These fibers were grouped into three classes (<20, 20-50, and >50 microm) to evaluate hypertrophy and hyperplasia in white skeletal muscle. MyoD gene expression was determined by semi-quantitative RT-PCR. PCR products were cloned and sequenced. Juvenile and adult pacu skeletal muscle had similar morphology. The large number of <20 microm diameter muscle fibers observed in juvenile fish confirms active hyperplasia. In adult fish, most fibers were over 50 microm diameter and denote more intense muscle fiber hypertrophy. The MyoD mRNA level in juveniles was higher than in adults. A consensus partial sequence for MyoD gene (338 base pairs) was obtained. The Pacu MyoD nucleotide sequence displayed high similarity among several vertebrates, including teleosts. The differential MyoD gene expression observed in pacu white muscle is possibly related to differences in growth patterns during the phases analyzed, with hyperplasia predominant in juveniles and hypertrophy in adult fish. These results should provide a foundation for understanding the molecular control of skeletal muscle growth in economically important Brazilian species, with a view to improving production quality.