Single-cell transcriptomes and runx2b-/- mutants reveal the genetic signatures of intermuscular bone formation in zebrafish.

Intermuscular bones (IBs) are mineralized spicules, present in the myosepta of many, but not all, teleost species. IBs are often small and sharp, and they consequently limit how the fish can be processed; the IBs may cause injury or trauma if lodged in consumers' throats or mouths, and therefore affect the appeal of the fish to many consumers. The development of IBs in teleosts is still not fully understood and the molecular basis of IB development remains to be established. Here, the characteristics of IB tissue are evaluated based on single-cell transcriptomics in wild-type zebrafish. The analysis defined 18 distinct cell types. Differentiation trajectories showed that IBs are derived from tendons and that a core tendon-osteoblast cell lineage is related to IB formation. In particular, the functions of 10 candidate genes were evaluated via CRISPR-Cas9 mutants. Among those, runx2b-/- mutants completely lost IBs, while swimming performance, growth and bone mineral density were not significantly different from runx2b+/+ zebrafish. Comparative single-cell RNA sequencing (scRNA-seq) analysis in runx2b-/- and runx2b+/+ zebrafish revealed the role of osteoblasts in IB formation. In addition, differentially expressed genes were enriched in the transforming growth factor ß/bone morphogenetic protein (TGF-ß/BMP) pathway after runx2b deletion. This study provides evidence for the crucial role of runx2b regulation in IB formation. Genetic breeding can target runx2b regulation and generate strains of commercial fish species without IBs, which can improve the safe consumption and economic value of many farmed fish species.

Functional Differentiation of BMP7 Genes in Zebrafish: bmp7a for Dorsal-Ventral Pattern and bmp7b for Melanin Synthesis and Eye Development.

Bone morphogenetic protein 7 (BMP7) belongs to the transforming growth factor ß (TGF-ß) family, which not only induces cartilage and bone formation, but also regulates eye development and melanoma tumorigenesis in mammals. In teleosts, BMP7 differentiates into two subtypes, bmp7a and bmp7b, which have clearly differentiated structures. To fully understand the functional differentiation of bmp7a and bmp7b in fish species, we successfully constructed bmp7a and bmp7b gene deletion mutants in zebrafish using CRISPR/Cas9-mediated gene editing technology. Our results showed that bmp7a mutation caused abnormal development of the embryo's dorsal-ventral pattern that led to death; bmp7b mutation induced growth inhibition and increased melanin production in the skin and eye of mutants. Histological analysis revealed that melanin in the retina of the eyes in bmp7b mutants increased, and behavioral observation showed that the vision and sensitivity to food of the mutants were reduced. Transcriptome analysis of the skin and eye tissues showed that the expression changes of wnt7ba and gna14 in bmp7b mutants might promote the increase of melanin. Additionally, the eye transcriptome analysis indicated that changes in the structure of the eyes in bmp7b mutants led to defects in phototransduction, and seven DEGs (rgs9a, rgs9b, rcvrn2, guca1d, grk1b, opn1mw4, and gc2) were identified as key candidate genes that affected the photonic response of the eyes. The study revealed the functional differentiation of bmp7a and bmp7b in teleosts and the first report about the inhibitory effect of bmp7b on melanogenesis may provide useful information for the future research on human melanoma-related diseases.

[Morin Improves Experimental Autoimmune Thyroiditis in Rats via NLRP3/Caspase-1 Pathway].

OBJECTIVE: To investigate the effects of morin-regulated NLRP3/Caspase-1 pathway on experimental autoimmune thyroiditis in rats. METHODS: The rats were randomly assigned to 6 groups: control group, experimental autoimmune thyroiditis group (EAT), low-, medium- and high-dose morin groups (post-modeling gavage of 50, 100 and 200 mg/kg morin hydrate per day for 6 weeks) and tripterygium wilfordii polyglycosides group (LGT group, post-modeling gavage of 6.25 mg/kg tripterygium wilfordii polyglycosidesper day for 6 weeks). Except for the control group, the rat model of experimental autoimmune thyroiditis was established by subcutaneous injection of 0.1 mL incomplete Freund's adjuvant containing porcine thyroglobulin. The levels of serum thyroglobulin (TgAb), thyroid peroxidase antibody (TPOAb), triiodothyronine (T3) and tetraiodothyronine (T4) in serum were detected by radioimmunoassay. The mRNA levels of interleukin-17 ( IL-17), interleukin-4 ( IL-4) and interferon γ ( INF- γ) were detected by reverse transcription-polymerase chain reaction. The levels of serum protein carbonyl content, 8-hydroxydeoxyguanosine (8-OHdG), and malondialdehyde (MDA) activity were checked with test kits. Expressions of NLRP3, apoptosis-related speck-like protein (ASC), and Caspase-1 were detected by Western blot. RESULTS: Compared with the EAT group, serum levels of TPOAb, TgAb, T3, and T4 in low-, medium- and high-dose Morin groups and LGT group were reduced ( P<0.01) and the mRNA levels of IL-17, INF-γ and IL-4 were increased ( P<0.01), the protein hydroxyl content, MDA activity, and 8-OHdG levels were reduced ( P<0.01). The levels of NLRP3, ASC and Caspase-1 were reduced ( P<0.01), the levels of 8-OHdG were significantly reduced ( P<0.01), and the levels of NLRP3, ASC and Caspase-1 were significantly reduced ( P<0.01). There were statistically significant differences between the data from the low-dose and the medium-dose Morin groups and the data of the LGT group ( P<0.05), while data from the high-dose Morin group showed no significant difference compared with the data of the LGT group. Data from low-, medium- and high-dose Morin groups showed no statistically significant differences ( P<0.05). CONCLUSION: The findings suggest that Morin improved experimental autoimmune thyroiditis in rats through regulating NLRP3/Caspase-1 pathway.

A comparative genomic database of skeletogenesis genes: from fish to mammals.

Skeletogenesis is a complex process that requires a rigorous control at multiple levels during osteogenesis, such as signaling pathways and transcription factors. The skeleton among vertebrates is a highly conserved organ system, but teleost fish and mammals have evolved unique traits or have lost particular skeletal elements in each lineage. In present study, we constructed a skeletogenesis database containing 4101, 3715, 2996, 3300, 3719 and 3737 genes in Danio rerio, Oryzias latipes, Gallus gallus, Xenopus tropicalis, Mus musculus and Homo sapiens genome, respectively. Then, we found over 55% of the genes are conserved in the six species. Notably, there are 181 specific-genes in the human genome without orthologues in the other five genomes, such as the ZNF family (ZNF100, ZNF101, ZNF14, CALML6, CCL4L2, ZIM2, HSPA6, etc); and 31 genes are identified explicitly in fish species, which are mainly involved in TGF-beta, Wnt, MAPK, Calcium signaling pathways, such as bmp16, bmpr2a, eif4e1c, wnt2ba, etc. Particularly, there are 20 zebrafish-specific genes (calm3a, si:dkey-25li10, drd1a, drd7, etc) and one medaka-specific gene (c-myc17) that may alter skeletogenesis formation in the corresponding species. The database provides the new systematic genomic insights into skeletal development from teleosts to mammals, which may help to explain some of the complexities of skeletal phenotypes among different vertebrates and provide a reference for the treatment of skeletal diseases as well as for applications in the aquaculture industry.

Development of Teleost Intermuscular Bones Undergoing Intramembranous Ossification Based on Histological-Transcriptomic-Proteomic Data.

Intermuscular bones (IBs) specially exist in lower teleost fish and the molecular mechanism for its development remains to be clarified. In this study, different staining methods and comparative proteomics were conducted to investigate the histological structure and proteome of IB development in Megalobrama amblycephala, including four key IB developmental stages (S1-IBs have not emerged in the tail part; S2-several small IBs started to ossify in the tail part; S3-IBs appeared rapidly; S4-all the IBs appeared with mature morphology). Alcian blue and alizarin red S stained results indicated that IBs were gradually formed from S2 to S4, undergoing intramembranous ossification without a cartilaginous phase. A total of 3368 proteins were identified by using the isobaric tags for relative and absolute quantitation (iTRAQ) approach. Functional annotation showed that proteins which were differentially expressed among stages were involved in calcium, MAPK, Wnt, TGF-ß, and osteoclast pathways which played a critical role in bone formation and differentiation. Three proteins (collagen9α1, stat1, tnc) associated with chondrocytes did not exhibit significant changes through S2 to S4; however, proteins (entpd5, casq1a, pvalb, anxa2a, anxa5) which associated with osteoblasts and bone formation and differentiation showed significantly a higher expression level from S1 to S2, as well as to S3 and S4. These further demonstrated that development of IBs did not go through a cartilaginous phase. The inhibitors of TGF-ß and Wnt pathways were tested on zebrafish (sp7/eGFP) and the results indicated that both inhibitors significantly delayed IB development. This study provides a comprehensive understanding of the IB ossification pattern, which will help further elucidate the molecular mechanisms for IB development in teleosts.

Characterization and spatiotemporal expression analysis of nine bone morphogenetic protein family genes during intermuscular bone development in blunt snout bream.

Intermuscular bones (IBs) only exist in the myosepta of lower teleosts and its molecular mechanism remains to be clarified. Bone morphogenetic proteins (BMPs) have been demonstrated to be involved in various physiological processes, including bone and cartilage formation. In this study, we firstly obtained and characterized nine bmp genes for Megalobrama amblycephala, which belongs to Cyprinidae and have a certain amount of IBs. Sequence alignment and phylogenetic analysis both documented that the mature proteins of M. amblycephala bmp genes were highly conserved with other corresponding homologs, respectively, indicating that the function of each bmp gene has been conserved throughout evolution. As a step to characterize potential involvement of bmp genes in IB formation and development, spatiotemporal expressions of nine bmp genes (bmp2a, bmp2b, bmp3, bmp4, bmp5, bmp7b, bmp8a, bmp14 and bmp16) were investigated during the key development stages of IBs. During the ossification process from stage I (the IBs haven't emerged) to stage IV (all of the IBs ossified in the tail with the mature morphology), the expression profiles revealed that bmp16 was the most abundant transcript while bmp4 had the lowest abundance. The mRNA levels of bmp3, bmp4, bmp5 and bmp8a increased significantly at stage II, suggesting their roles in stimulating IB formation. The expression of bmp7b reached the highest level at stage III (the rapid period of IB development), suggesting potential involvement of bmp7b in promoting osteoblast differentiation. With the exception of bmp7b and bmp16, most bmp genes appeared a significant increase at IB maturation phase (stage IV), which means that they may play important roles in maintenance of IB morphogenesis. Spatial tissue distribution of bmp genes showed that most bmp genes were observed at the highest level in developing IBs at one year old fish. Spatiotemporal expression patterns suggest the potential key roles of these bmp genes in IBs formation and maintenance in fish, being as possible promoters or inhibitors.

Construction of a high-density linkage map and fine mapping of QTLs for growth and gonad related traits in blunt snout bream.

High-density genetic maps based on SNPs are essential for fine mapping loci controlling specific traits for fish species. Using restriction-site associated DNA tag sequencing (RAD-Seq) technology, we identified 42,784 SNPs evenly distributed across the Megalobrama amblycephala genome. Based on 2 parents and 187 intra-specific hybridization progenies, a total of 14,648 high-confidence SNPs were assigned to 24 consensus linkage groups (LGs) of maternal and paternal map. The total length of the integrated map was 3,258.38 cM with an average distance of 0.57 cM among 5676 effective loci, thereby representing the first high-density genetic map reported for M. amblycephala. A total of eight positive quantitative trait loci (QTLs) were detected in QTL analysis. Of that, five QTL explained ≥35% of phenotypic variation for growth traits and three QTL explained ≥16% phenotypic variation for gonad related traits. A total of 176 mapped markers had significant hits in the zebrafish genome and almost all of the 24 putative-chromosomes of M. amblycephala were in relatively conserved synteny with chromosomes of zebrafish. Almost all M. amblycephala and zebrafish chromosomes had a 1:1 correspondence except for putative-chromosome 4, which mapped to two chromosomes of zebrafish caused by the difference in chromosome numbers between two species.

Comparative proteomics analysis of teleost intermuscular bones and ribs provides insight into their development.

BACKGROUND: Intermuscular bones (IBs) and ribs both are a part of skeletal system in teleosts, but with different developing process. The chemical composition of fish IBs and ribs as well as the underlying mechanism about their development have not been investigated. In the present study, histological structures showed that one bone cavity containing osteoclasts were existed in ribs, but not in IBs of Megalobrama amblycephala. We constructed the first proteomics map for fish bones including IBs and ribs, and identified the differentially expressed proteins between IBs and ribs through iTRAQ LC-MS/MS proteomic analysis. RESULTS: The proteins extracted from IBs and ribs at 1- to 2-year old M. amblycephala were quantified 2,342 proteins, with 1,451 proteins annotated with GO annotation in biological processes, molecular function and cellular component. A number of bone related proteins as well as pathways were identified in the study. A total of 93 and 154 differently expressed proteins were identified in comparison groups of 1-IB-vs-1-Rib and 2-IB-vs-2-Rib, which indicated the obvious differences of chemical composition between these two bone tissues. The two proteins (vitronectin b precursor and matrix metalloproteinase-2) related to osteoclasts differentiation were significantly up-regulated in ribs compared with IBs (P < 0.05), which was in accordance with the results from histological structures. In comparison groups of 1-IB-vs-2-IB and 1-Rib-vs-2-Rib, 33 and 51 differently expressed proteins were identified and the function annotation results showed that these proteins were involved in regulating bone development and differentiation. Subsequently, 11 and 13 candidate proteins in comparison group of 1-IB-vs-1-Rib and 1-IB-vs-2-IB related to bone development were validated by MRM assays. CONCLUSIONS: Our present study suggested the different key proteins involved in the composition of fish ribs and IBs as well as their growth development. These findings could provide important clues towards further understanding of fish skeletal system and the roles of proteins playing in regulating diverse biological processes in fish.

Dynamic mRNA and miRNA expression analysis in response to intermuscular bone development of blunt snout bream (Megalobrama amblycephala).

Intermuscular bone (IB), which occurs only in the myosepta of lower teleosts, is attracting more attention because they are difficult to remove and make the fish unpleasant to eat. By gaining a better understanding of the genetic regulation of IB development, an integrated analysis of miRNAs and mRNAs expression profiling was performed on Megalobrama amblycephala. Four key development stages were selected for transcriptome and small RNA sequencing. A number of significantly differentially expressed miRNAs/genes associated with bone formation and differentiation were identified and the functional characteristics of these miRNAs/genes were revealed by GO function and KEGG pathway analysis. These were involved in TGF-ß, ERK and osteoclast differentiation pathways known in the literature to affect bone formation and differentiation. MiRNA-mRNA interaction pairs were detected from comparison of expression between different stages. The function annotation results also showed that many miRNA-mRNA interaction pairs were likely to be involved in regulating bone development and differentiation. A negative regulation effect of two miRNAs was verified through dual luciferase reporter assay. As a unique public resource for gene expression and regulation during the IB development, this study is expected to provide forwards ideas and resources for further biological researches to understand the IBs' development.

Mitochondrial Genome Variation after Hybridization and Differences in the First and Second Generation Hybrids of Bream Fishes.

Hybridization plays an important role in fish breeding. Bream fishes contribute a lot to aquaculture in China due to their economically valuable characteristics and the present study included five bream species, Megalobrama amblycephala, Megalobrama skolkovii, Megalobrama pellegrini, Megalobrama terminalis and Parabramis pekinensis. As maternal inheritance of mitochondrial genome (mitogenome) involves species specific regulation, we aimed to investigate in which way the inheritance of mitogenome is affected by hybridization in these fish species. With complete mitogenomes of 7 hybrid groups of bream species being firstly reported in the present study, a comparative analysis of 17 mitogenomes was conducted, including representatives of these 5 bream species, 6 first generation hybrids and 6 second generation hybrids. The results showed that these 17 mitogenomes shared the same gene arrangement, and had similar gene size and base composition. According to the phylogenetic analyses, all mitogenomes of the hybrids were consistent with a maternal inheritance. However, a certain number of variable sites were detected in all F1 hybrid groups compared to their female parents, especially in the group of M. terminalis (♀) × M. amblycephala (♂) (MT×MA), with a total of 86 variable sites between MT×MA and its female parent. Among the mitogenomes genes, the protein-coding gene nd5 displayed the highest variability. The number of variation sites was found to be related to phylogenetic relationship of the parents: the closer they are, the lower amount of variation sites their hybrids have. The second generation hybrids showed less mitogenome variation than that of first generation hybrids. The non-synonymous and synonymous substitution rates (dN/dS) were calculated between all the hybrids with their own female parents and the results indicated that most PCGs were under negative selection.

Identification of MicroRNA for Intermuscular Bone Development in Blunt Snout Bream (Megalobrama amblycephala).

Intermuscular bone (IB), which occurs only in the myosepta of the lower teleosts, is attracting more attention of researchers due to its particular development and lack of genetic information. MicroRNAs (miRNAs) are emerging as important regulators for biological processes. In the present study, miRNAs from IBs and connective tissue (CT; encircled IBs) from six-month-old Megalobrama amblycephala were characterized and compared. The results revealed the sequences and expression levels of 218 known miRNA genes (belonging to 97 families). Of these miRNAs, 44 known microRNA sequences exhibited significant expression differences between the two libraries, with 24 and 20 differentially-expressed miRNAs exhibiting higher expression in the CT and IBs libraries, respectively. The expressions of 11 miRNAs were selected to validate in nine tissues. Among the high-ranked predicted gene targets, differentiation, cell cycle, metabolism, signal transduction and transcriptional regulation were implicated. The pathway analysis of differentially-expressed miRNAs indicated that they were abundantly involved in regulating the development and differentiation of IBs and CT. This study characterized the miRNA for IBs of teleosts for the first time, which provides an opportunity for further understanding of miRNA function in the regulation of IB development.