Systematic Identification of Long Noncoding RNAs during Three Key Organogenesis Stages in Zebrafish.

Thousands of lncRNAs have been found in zebrafish embryogenesis and adult tissues, but their identification and organogenesis-related functions have not yet been elucidated. In this study, high-throughput sequencing was performed at three different organogenesis stages of zebrafish embryos that are important for zebrafish muscle development. The three stages were 10 hpf (hours post fertilization) (T1), 24 hpf (T2), and 36 hpf (T3). LncRNA gas5, associated with muscle development, was screened out as the next research target by high-throughput sequencing and qPCR validation. The spatiotemporal expression of lncRNA gas5 in zebrafish embryonic muscle development was studied through qPCR and in situ hybridization, and functional analysis was conducted using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9, CRISPR/Cas9). The results were as follows: (1) A total of 1486 differentially expressed lncRNAs were identified between T2 and T1, among which 843 lncRNAs were upregulated and 643 were downregulated. The comparison with T3 and T2 resulted in 844 differentially expressed lncRNAs, among which 482 lncRNAs were upregulated and 362 lncRNAs were downregulated. A total of 2137 differentially expressed lncRNAs were found between T3 and T1, among which 1148 lncRNAs were upregulated and 989 lncRNAs were downregulated, including lncRNA gas5, which was selected as the target gene. (2) The results of spatiotemporal expression analysis showed that lncRNA gas5 was expressed in almost all detected embryos of different developmental stages (0, 2, 6, 10, 16, 24, 36, 48, 72, 96 hpf) and detected tissues of adult zebrafish. (3) After lncRNA gas5 knockout using CRISPR/Cas9 technology, the expression levels of detected genes related to muscle development and adjacent to lncRNA gas5 were more highly affected in the knockout group compared with the control group, suggesting that lncRNA gas5 may play a role in embryonic muscle development in zebrafish. (4) The results of the expression of the skeletal myogenesis marker myod showed that the expression of myod in myotomes was abnormal, suggesting that skeletal myogenesis was affected after lncRNA gas5 knockout. The results of this study provide an experimental basis for further studies on the role of lncRNA gas5 in the embryonic skeletal muscle development of zebrafish.

The Expression and Function of lincRNA-154324 and the Adjoining Protein-Coding Gene vmp1 in the Caudal Fin Regeneration of Zebrafish.

Caudal fin regeneration is regulated by a variety of mechanisms, but the role of long non-coding RNA (lncRNA) has rarely been studied. The present study aimed to describe the landscape of lncRNAs during caudal fin regeneration using whole transcriptome sequencing, and then to conduct a functional study on the target lncRNAs using real-time fluorescent quantitative PCR (RT-qPCR), in situ hybridization, and the CRISPR/Cas9 method for lncRNA gene knockout. The results of the transcriptome sequencing showed that a total of 381 lncRNAs were differentially expressed, among which ENSDART00000154324 (lincRNA-154324) was found to be highly related to caudal fin regeneration, and thus it was chosen as the target lncRNA for the subsequent functional study. The results regarding the temporal and spatial expression of lincRNA-154324 and the gene knockout results from CRISPR/Cas9 indicated that lincRNA-154324 is involved in the caudal fin regeneration of zebrafish. Importantly, we serendipitously discovered that the cis correlation coefficient between lincRNA-154324 and its neighboring gene vacuole membrane protein 1 (vmp1) is extremely high, and they are essential for the process of caudal fin regeneration. Moreover, studies have found that vmp1 plays an important role in protein secretion, organelle formation, multicellular development, and autophagy. Collectively, our result may provide a framework for the identification and analysis of lncRNAs involved in the regeneration of the zebrafish caudal fin.

High-throughput transcriptome sequencing reveals the key stages of cardiovascular development in zebrafish embryos.

BACKGROUND: The cardiovascular developmental process is a tightly regulated network involving multiple genes. The current understanding of the molecular mechanism behind cardiovascular development is insufficient and requires further research. RESULTS: Transcriptome sequencing of three developmental stages in zebrafish embryos was performed and revealed three key cardiovascular developmental stages. Then, the differentially expressed genes (DEGs) involved in cardiovascular development were screened out. The three developmental stages were 18 (T1), 24 (T2), and 42 h post fertilization (hpf) (T3), and the three stages were confirmed by detecting differences in expression between cardiomyocyte and endothelial marker genes (cmlc2, fli1) using in situ hybridization, which represents the characteristics of cardiovascular development. Thousands of DEGs were identified using transcriptome analysis. Of them, 2605 DEGs were in T1-vs-T2, including 2003 up-regulated and 602 down-regulated genes, 6446 DEGs were in T1-vs-T3, consisting of 4608 up-regulated and 1838 down-regulated genes, and 3275 DEGs were in T2-vs-T3, including 2420 up-regulated and 855 down-regulated genes. There were 644 common DEGs and 167 common five-fold higher differentially expressed genes (HDEGs) identified, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID). Significant differences was observed in the levels of gene expression among different developmental stages in multiple GO terms and KEGG pathways, such as cell migration to the midline involved in heart development, cardiovascular system development, circulatory system process for biological processes of GO terms; and cardiac muscle contraction, adrenergic signaling in cardiomyocytes for KEGG pathways. These results demonstrated that these three stages were important period for the development of the cardiovascular system. Lastly, we used quantitative real-time PCR (qPCR) to validate the reliability of RNA-sequencing by selecting 21 DEGs. CONCLUSIONS: These results demonstrated that these three stages represented the important periods for cardiovascular system development of zebrafish and some candidate genes was obtained and provided a solid foundation for additional functional studies of the DEGs.

Alterations in transcription and protein expressions of HCC-related genes in HepG2 cells caused by microcystin-LR.

Microcystin-LR (MC-LR) is the most common and toxic hepatotoxin and it could induce human hepatitis and hepatocellular carcinoma (HCC) via the route of drinking water. The aim of the present study was to determine the expressions of oncogenes c-fos, c-jun, c-myc, c-met, and N-ras and tumor suppressor gene PTEN in HepG2 cells following MC-LR-exposure to understand the possible mechanism of MC-LR-related human primary liver cancer. The results of qPCR and Western blotting showed that MC-LR-exposure at non- or sub-cytotoxic concentrations promoted the expressions of oncogenes c-fos, c-jun, c-myc, c-met, and N-ras while suppressed tumor-suppressor gene PTEN in HepG2 cells at both transcription and protein levels. This result suggests that HCC-related genes may be involved in human hepatitis and primary liver cancer caused by MC-LR. The work might be useful for evaluating the human health risk resulted from the long-term of MC-LR-exposure at low dose via drinking water route.

Biochemical responses to the toxicity of the biocide abamectin on the freshwater snail Physa acuta.

The toxic effects of abamectin (ABM), an anthelmintic drug, on the snail, Physa Acuta, and the biochemical responses to the exposure stress were evaluated. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), acetylcholinesterase (AChE), and nitric oxide synthase (NOS), and the contents of malondialdehyde (MDA) were determined in snail soft tissues (head, foot, visceral mass, and the mantle) for up to 96h of exposure to 3.4, 9.6, 19.2, or 27.4µgL(-1) of ABM. The results showed that SOD and GST activities were promoted by ABM-exposure at the earlier periods of treatment (12-48h) while these activites were inhibited at the end of test. The tendency of CAT activity was similar to that of SOD, but it increased at the end of test. MDA levels of the snail soft tissues increased in all treatment groups, including the recovery group, indicating that lipid peroxidation occurred in snail soft tissues. ABM-exposure inhibited AChE activity. However, NOS activities increased by ABM-exposure. In addition, activities of antioxidant enzymes and AChE from the snail soft tissues resumed the normal levels after 96h of recovery period, but MDA level did not attain the original level. This study provides information on the biochemical mechanism of ABM toxicity on the snail.

[Effect of water stress in partial root zone on response of photosynthesis of Aconitum barbatum].

OBJECTIVE: To study the effect of different water stress mode on photosynthesis characteristics of Aconitum barbaturnm, aimed at providing the theoretical basis for cultivating this kind of plant for the arid area. METHOD: The transplanted seedlings of A. barbatum were processed with fixed partial root zone drying(TI) ,alternate partial root zone drying(T2) and both partial root zone irrigation (T3) for three cycles,and relative parameters were measured. RESULT: The net photosynthetic rate (Pn) in the treatment of T2 gradually increased and was higher than that in the treatment of T3 about 6.53%, mainly effected by non-stomatal factors. The water use efficiency (WUE) was higher than that in the treatments of T3 about 25.21%, while the transpiration rate (Tr) and the stomatal conductance was lower than those in the treatment of T1 and T3 separately. The maximum net photosynthesis rate (Pmax),the apparent quantum yield (AQY) and the dark respiration rate (Rd) were higher than those in comparison condition slightly. CONCLUSION: The alternate partial root zone drying is worth promoting vigorously, and it shall be a rational way to cultivate the A. barbatum under woodland.