- Transcriptome profiling analysis of sex- based differentially expressed mRNAs and lncRNAs in the brains of mature zebrafish ( Danio rerio. - However, systematic studies of the dimorphic patterns of gene expression in the brain of male and female zebrafish are lacking.. - Results: In this study, the mRNA and lncRNA expression profiles were obtained from the brain tissue samples of the three male and three female zebrafish by high-throughput transcriptome sequencing. - expressed genes for RT-qPCR verification and the results certified that the expression pattern showed a similar trend between RNA-seq and RT-qPCR results. - Protein-protein interaction network analysis, Gene Ontology (GO) analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed to obtain the biological significance of differentially expressed mRNA in the brain dimorphism of zebrafish. - Finally, a Pearson correlation analysis was performed to construct the co-expression network of the mRNAs and lncRNAs.. - Conclusions: We found that 12 new lncRNAs not only have significant gender specificity in the brain of zebrafish, and this finding may provide a clue to further study of the functional difference between male and female zebrafish brain.. - The zebrafish exposed to the sex hormone showed a regional (forebrain, midbrain and hindbrain) and gender-related differences in gene expression [6].. - The expression of 61 genes in the four zebrafish showed significant gender differences by RNA-seq sequencing data analysis [7]. - Long non-coding RNAs (long ncRNAs, lncRNAs), which may appear anywhere in the genome, are defined as transcripts longer than 200 nucleotides that are not. - 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. - The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.. - 1 Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources (Shanghai Ocean University), Ministry of Education, Shanghai 201306, China Full list of author information is available at the end of the article. - however the function of the sex-based lncRNAs in the brain is still unknown.. - In this study, we obtained the RNA expression data of the brains of the adult male and female zebrafish by using transcriptome sequencing (RNA-seq). - Some of the differential expres- sion mRNAs and lncRNAs have been verified by RT- qPCR. - Additionally, we found the mRNAs significantly enriched in many pathways according to the GO and KEGG functional enrichment analyses. - Finally, the lncRNA-mRNA interaction network was constructed using the Pearson correlation coefficients, based on the FPKM values of the lncRNAs and mRNAs. - This study expanded the zebrafish brain sex-based lncRNA cata- logue and constructed a regulatory network of the zebra- fish brains at the transcriptional level, providing clues for more in-depth depiction of gender differences in zeb- rafish brain neurons.. - Then, these high- quality reads were mapped to the reference zebrafish gen- ome by 89.2%. - Differentially expressed genes. - We analyzed the transcriptome data of the six zebrafish brain tissue samples to obtain the sex-based gene expres- sion of zebrafish in brain. - Using the TopHat2 and Cufflinks packages, 14,315 annotated genes were obtained, account- ing for 93.4% of the total genes assembled in the danRer10 zebrafish genome. - When taking into account the direction of expression, ap- proximately of the differentially expressed genes showed male-based expression (Additional file 2).. - Through literature search, we found that the detailed inter- pretation for majority of the differentially expressed male- based genes were not available. - The expression levels of f13a1a.1, zgc:114181 and hbaa2 were up-regulated in the fe- male zebrafish by 4.3, 2.8 and 2.6-folds, respectively, while apoa2, leg1.1, and c3a.1 were down-regulated by and 8.54-folds, respectively. - To verify the interrelationship between the differentially expressed genes, we constructed a protein-protein inter- action network of these regulated genes with 44 nodes and 54 interactions (Fig. - To investigate the function of the differentially expressed sex-based genes, GO enrichment analysis and KEGG pathway annotation were performed. - GO analysis showed that these differentially expressed sex-based genes were generally associated with the extracellular re- gion, cellular response to estrogen stimulus and endo- peptidase inhibitor activity (fold-change >. - We found that the enrichment degree of the male-bias genes in gene ontology was significantly higher than that of the female-biased genes. - The KEGG analysis showed that the differentially expressed mRNAs remarkably enriched in PPAR signal- ing pathway, glycolysis/gluconeogenesis, starch and su- crose metabolism, tryptophan metabolism, and cysteine and methionine metabolism pathways (Fig. - Previous studies found that PPAR signaling pathway plays import- ant roles in mammalian reproductive system during the processes of the ovarian cycle, luteal formation, embryo implantation, placentation and male reproduction [20].. - To investigate the biological function of the sex- related lncRNAs in zebrafish brain, the lncRNAs were. - 1 Characterization and verification of differentially expressed genes. - (b) Hierarchical clustering analysis of mRNAs that are differentially expressed between the female and male zebrafish samples. - (c) Significantly differentially expressed genes by RT-qPCR. - 2 GO and KEGG analyses of differentially expressed genes. - a GO enrichment analysis of the differentially expressed genes. - (b) Enriched pathway of the differentially expressed genes. - Using coding prediction and ORF (open reading frame) identification software, we found that 3709 potential lncRNAs were expressed in the six zebrafish samples. - The scatter plot showed a high degree of positive correlation (P = 0.913) between lncRNA expression in the female and male zebrafish samples (Fig. - The volcano plots showed the differential expression levels of the lncRNA in the female and male samples (Fig. - To examine the collaboration between the lncRNAs and mRNAs in the gender-based zebrafish brain tissues, a co- expression analysis of the differentially expressed lncRNAs and the corresponding differentially expressed mRNAs was performed based on their FPKM values. - The lncRNA-mRNA coupling suggested that the regulation of cldn7a by multiple lncRNAs was likely to occur in the brain. - mRNAs in the co-expression network were enriched on. - The information of the 12 lncRNAs in the lncRNA–. - For each of these lncRNAs, we found that the mRNA co-expressed with them was not within 300 kb from the same chromo- some, indicating that they did not have cis regulatory functions and were not directly involved in the regula- tion of gene transcription or post-transcriptional levels. - The ZFLNC database was used to perform a conservative analysis of the lncRNAs in the co- expression network. - We found that XLOC_038516 and human pseudogene HSPA8P5 were considered as orthologs. - Further analysis showed that HSPA8P5 was differentially expressed in multiple human brain neurological diseases (Fig. - In this study, we investigated the differential gene expres- sion between 8-month-old male and female zebrafish brain tissues by using RNA-Seq sequencing technique, with a re- sult of differentially expressed seven female-based genes and 101 male-based genes (fold-change >. - These differentially expressed genes account for less than 1% of all genes identified from the brains of the male. - 3 Characterization and verification of the differentially expressed lncRNAs. - a the scatter plot of the lncRNA expression in the female and male zebrafish samples. - (b) The volcano plots analysis of the lncRNAs that are differentially expressed between the female and male zebrafish samples.. - Inf indicates that the FPKM value of the gene in female zebrafish is 0. - -inf indicates that the FPKM value of the gene in male zebrafish is 0. - and female zebrafish. - found that 7478 expressed genes does not show a clear separation of the individual transcriptomes according to gender, sug- gesting that gender is not the main determinant of the vari- ation between individual brain gene expression profiles [5].. - Available evidence showed that many genes in the list of 108 sex- based genes we obtained in this study in- volves in neural circuit or brain development, e.g., egr2b (early growth response 2b, also known as KROX20) [24], mych (myelocytomatosis oncogene homolog) [25], and hrh3 (histamine receptor H3, [26. - In addition, we found that some genes play a similar role in the brain of zebrafish and humans. - a Up- or down-regulated expression compared to expression levels in the female zebrafish FC fold change. - When performing GO enrichment analyses, an inter- esting result was obtained in the differentially expressed genes. - The result showed that five genes (NUPR1, ESR1, SERPINC1, ZGC: 66313, and FKBP11) enriched in cellu- lar response to estrogen stimulus, but these five genes were expressed in the brain of the male zebrafish. - Furthermore, previous work had also reported that the sexually dimorphic gene expression in the zebrafish does not correspond to specific pathways, from which we can ascertain that commonalities in their regulatory mechanisms have the sex determining path- ways in mammals [8].. - In the lncRNAs and mRNAs co-expression networks, some of the differen- tially expressed genes were previously reported to be in- volved in neural circuit such as egr2b and hrh3, indicating that lncRNA XLOC_038516 and XLOC_. - Further ana- lysis revealed that HSPA8P5, one of the orthologs of XLOC_038516, was differentially expressed in multiple human brain neurological diseases. - Further in vivo studies will help to fully understand the role of these lncRNAs in the brain of zebrafish.. - In this study, the mRNAs and lncRNAs with the sex- based differential expression were screened by transcrip- tome sequencing (RNA-seq) in the zebrafish brains.. - Based on the various biological analyses, we found that 12 new lncRNAs have significant gender specificity in the brain of zebrafish by analyzing the biological func- tions of the co-expressing mRNA. - provide a clue to further study of the functional differ- ence between male and female zebrafish brain.. - Acquisition of the Zebrafish Specimens. - The wild-type AB zebrafish were purchased from the Wuhan Institute of Hydrobiology, Chinese Academy of Sciences, China, and the adult zebrafish of the same size (3 male and 3 female) were selected as experimental specimens.. - We followed the NIH guidelines for zebrafish euthanasia (https://oacu.oir.nih.gov/sites/default/files/uploads/arac- guidelines/zebrafish.pdf). - The fish were left in the solu- tion for 15 min following cessation of opercular move- ment. - Total RNA was extracted using Invitrogen Ambion RNA Extraction Kit according to the manufacturer’s protocol (ThermoFisher Scientific, MA, USA). - Library Preparation was created using VAHTS Stranded mRNA-seq Library Prep Kit according to the manufac- turer’s protocol (Vazyme, Nanjing, China). - The purified double-stranded cDNA was added an A and ligated to the sequencing linker,. - The concentration of the library was ac- curately quantified using KAPA Library Quantification Kits according to the manufacturer’s protocol (KAPA Biosystems, MA, USA). - By using Trimmomatic software [35], Low-quality reads were filtered according to the following criteria: quality scores are less than 20, and reads with average quality scores of each read less than 20. - babraham.ac.uk/projects/fastqc/) [36] was employed to assess the quality of the raw reads.. - to map the reads to the reference genome. - TopHat ini- tially removed a small portion of the reads based on the quality information of each reads and then mapped the qualified reads to the reference genome [37]. - Differential expressed genes were characterized according to the criterion of a fold change >. - Every node in the diagram represented a term that reflected the relationships between nodes, and the color of the nodes reflected the enrichment of the node classification.. - For the characterization of the zebrafish brain, RT-qPCR of mRNAs was performed using SYBR Green PCR Mas- ter Mix (Fermentas, Guangzhou, China) following the manufacturer’s instructions. - The STRING online software [45] was employed to con- struct the interaction network of the proteins encoded. - by the differentially expressed genes. - According to the FPKM values of the different expression of the lncRNAs and the mRNAs in the six samples, the Pear- son correlation coefficient between the lncRNAs and the mRNAs were calculated according to their FPKM values.. - Differentially expressed mRNAs in the brains of male and female zebrafish.. - Differentially expressed lnRNAs in the brains of male and female zebrafish.. - The funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.. - All experiments were performed in strict accordance with the requirements of the Animal Ethics Procedures and Guidelines of the People ’ s Republic of China.. - The zebrafish brain: a neuroanatomical comparison with the goldfish. - Sexually dimorphic gene expression in the brains of mature zebrafish. - Transcriptome analysis reveals a ribosome constituents disorder involved in the RPL5 downregulated zebrafish model of diamond- Blackfan anemia. - Representing sex in the brain, one module at a time.. - The zebrafish reference genome sequence and its relationship to the human genome. - Transcriptomic analyses reveal novel genes with sexually dimorphic expression in the zebrafish gonad and brain
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