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The hsp90 1 and smyd1b genes were knocked down in zebrafish embryos by gene specific MO injection. murf1a gene expression was analyzed by RT-PCR in control (Con-MO), hsp90 1 (Hsp-MO) and smyd1b (Smy-MO) morphant embryos. 5A The effect of hsp90 1 and smyd1b knockdown on murf1a gene expression in zebrafish (Danio rerio) embryos. Corrigendum to "Generation of MuRF-GFP transgenic zebrafish models for investigating murf gene expression and protein localization in Smyd1b and Hsp90 1 knockdown embryos" [Comparative biochemistry and physiology. [Extracted from the article]

Muscle-specific RING-finger proteins (MuRFs) are E3 ubiquitin ligases that play important roles in protein quality control in skeletal and cardiac muscles. Here we characterized murf gene expression and protein localization in zebrafish embryos. We found that the zebrafish genome contains six murf genes, including murf1a , murf1b , murf2a , murf2b , murf3 and a murf2 -like gene that are specifically expressed in skeletal and cardiac muscles of zebrafish embryos. To analyze the subcellular localization, we generated transgenic zebrafish models expressing MurF1a-GFP or MuRF2a-GFP fusion proteins. MuRF1a-GFP and MuRF2a-GFP showed distinct patterns of subcellular localization. MuRF1a-GFP displayed a striated pattern of localization in myofibers, whereas MuRF2a-GFP mainly exhibited a random pattern of punctate distribution. The MuRF1a-GFP signal appeared as small dots aligned along the M-lines of the sarcomeres in skeletal myofibers. To determine whether knockdown of smyd1b or hsp90α1 that increased myosin protein degradation could alter murf gene expression or MuRF protein localization, we knocked down smyd1b or hsp90α1 in wild type, Tg(ef1a:MurF1a-GFP) and Tg(ef1a:MuRF2a-GFP) transgenic zebrafish embryos. Knockdown of smyd1b or hsp90α1 had no effect on murf gene expression. However, the sarcomeric distribution of MuRF1a-GFP was abolished in the knockdown embryos. This was accompanied by an increased random punctate distribution of MuRF1a-GFP in muscle cells of zebrafish embryos. Collectively, these studies demonstrate that MuRFs are specifically expressed in developing muscles of zebrafish embryos. The M-line localization MuRF1a is altered by sarcomere disruption in smyd1b or hsp90α1 knockdown embryos. Unlabelled Image • Zebrafish genomes contain six murf genes that show muscle-specific expression during myogenesis. • Transgenic zebrafish lines expressing MuRF1a-GFP and MuRF2a-GFP were generated. • MuRF1a-GFP and MuRF2a-GFP show a distinct pattern of localization in myofibers of zebrafish embryos. • The sarcomeric localization of MuRF1a-GFP is disrupted in smyd1b and hsp90 α 1 knockdown embryos. [ABSTRACT FROM AUTHOR]

Japanese flounder ( Paralichthys olivaceus ) undergoes metamorphosis by changing its body from the larval to the juvenile form, and this process involves muscle development. Smyd1, a histone methyltransferase, plays a role in the skeletal muscle. In the present study, the Smyd1a and Smyd1b expression patterns and their 5′ UTR and exon 1 DNA methylation levels were analyzed during metamorphosis of the Japanese flounder. Sample were analyzed 21 days post-hatching (dph) (with no migration of right eye; M1stage), 28 dph (during migration of right eye; M2 stage), and 35 dph (after migration of right eye; M3 stage). The results show that Smyd1a expression was highest in the M2 stage and then decreased, whereas Smyd1b expression continued to rise during the three stages. Methylation levels of CpG sites at positions −2318 and −2217 of the Smyd1a P region (−2462 to −2181 region of the 5′ UTR), and the CpG sites at positions −351, −330, −284, −190, and − 92 of the Smyd1b promoter, with both regions containing putative transcription factor binding sites, showed significant differences in the three stages (p < 0.05). Interestingly, the methylation levels of these CpG sites were negatively correlated with mRNA expression. We inferred that binding of the predicted transcription factors might be affected by methylation of the CpG sites and thus modulate gene expression. Taken together, our results suggest that DNA methylation in the Smyd1a and Smyd1b genes participates in the regulation of metamorphosis, and epigenetics may provide clues for further studies of the mechanisms of metamorphosis in the Japanese flounder. [ABSTRACT FROM AUTHOR]

Many genes related to muscle growth modulate myoblast proliferation and differentiation and promote muscle hypertrophy. MyoD is a myogenic determinant that contributes to myoblast determination, and insulin-like growth factor 1 (IGF-I) interacts with MyoD to regulate muscle hypertrophy and muscle mass. In this study, we aimed to assess DNA methylation and mRNA expression patterns of MyoD and IGF-I during different developmental stages of Japanese flounder, and to examine the relationship between MyoD and IGF-I gene. DNA and RNA were extracted from muscles, and DNA methylation of MyoD and IGF-I promoter and exons was detected by bisulfite sequencing. The relative expression of MyoD and IGF-I was measured by quantitative polymerase chain reaction. IGF-I was measured by radioimmunoassay. Interestingly, the lowest expression of MyoD and IGF-I emerged at larva stage, and the mRNA expression was negatively associated with methylation. We hypothesized that many skeletal muscle were required to complete metamorphosis; thus, the expression levels of MyoD and IGF-I genes increased from larva stage and then decreased. The relative expression levels of MyoD and IGF-I exhibited similar patterns, suggesting that MyoD and IGF-I regulated muscle growth through combined effects. Changes in the concentrations of IGF-I hormone were similar to those of IGF-I gene expression. Our results the mechanism through which MyoD and IGF-I regulate muscle development and demonstrated that MyoD interacted with IGF-I to regulate muscle growth during different developmental stages. [ABSTRACT FROM AUTHOR]

Objectives To identity the genetic causes of hearing loss in a Han Chinese family with enlarged vestibular aqueduct syndrome. Methods Multiplex PCR technology combined with Ion Torrent™ next-generation sequencing technology was used to search for pathogenic mutations. A group of 1500 ethnically-matched normal hearing subjects screened for mutations in deafness-related genes using the same method in previously studied were included as a control. Results The proband and his little sister suffered from typical features of sensorineural hearing loss with enlarged vestibular aqueduct (EVA). Both subjects harbored two compound heterozygous mutations in the SLC26A4 gene. A novel mutation named c.2110 G > C (p.Glu704Gln) in exon 19 and another previously reported mutation c.1673 A > T (p.Asn558Ile) were identified. These mutations were carried in the heterozygous state by the parents and therefore co-segregated with the genetic disease. The c.2110 G > C (p.Glu704Gln) mutation was absent in 1500 healthy newborns. Protein alignment indicated high evolutionary conservation of the p.E704 residue, and this mutation was predicted by online tools to be damaging and deleterious. Conclusion This study demonstrates that the novel mutation c.2110 G > C (p.Glu704Gln) in compound heterozygosity with c.1673 A > T (p.Asn558Ile) in the SLC26A4 gene corresponds to the EVA in this family. Our study will provide a foundation for elucidating the SLC26A4- related mechanisms of hearing loss. [ABSTRACT FROM AUTHOR]