503unc,a small and muscle‐specific zebrafish promoter

The muscle‐specific UNC‐45b assists in the folding of sarcomeric myosin. Analysis of the zebrafish unc‐45b upstream region revealed that unc‐45b promoter fragments reliably drive GFP expression after germline transmission. The muscle‐specific 503‐bp minimal promoter 503unc was identified to drive gene expression in the zebrafish musculature. In transgenic Tg(?503unc:GFP) zebrafish, GFP fluorescence was detected in the adaxial cells, their slow fiber descendants, and the fast muscle. At later stages, robust GFP fluorescence is eminent in the cardiac, cranial, fin, and trunk muscle, thereby recapitulating the unc‐45b expression pattern. We propose that the 503unc promoter is a small and muscle‐specific promoter that drives robust gene expression throughout the zebrafish musculature, making it a valuable tool for the exploration of zebrafish muscle. genesis 51:443–447. © 2013 Wiley Periodicals, Inc.     

Expression and Functional Characterization of Smyd1a in Myofibril Organization of Skeletal Muscles

Background

Smyd1, the founding member of the Smyd family including Smyd-1, 2, 3, 4 and 5, is a SET and MYND domain containing protein that plays a key role in myofibril assembly in skeletal and cardiac muscles. Bioinformatic analysis revealed that zebrafish genome contains two highly related smyd1 genes, smyd1a and smyd1b. Although Smyd1b function is well characterized in skeletal and cardiac muscles, the function of Smyd1a is, however, unknown.

Methodology/Principal Findings

To investigate the function of Smyd1a in muscle development, we isolated smyd1a from zebrafish, and characterized its expression and function during muscle development via gene knockdown and transgenic expression approaches. The results showed that smyd1a was strongly expressed in skeletal muscles of zebrafish embryos. Functional analysis revealed that knockdown of smyd1a alone had no significant effect on myofibril assembly in zebrafish skeletal muscles. However, knockdown of smyd1a and smyd1b together resulted in a complete disruption of myofibril organization in skeletal muscles, a phenotype stronger than knockdown of smyd1a or smyd1b alone. Moreover, ectopic expression of zebrafish smyd1a or mouse Smyd1 transgene could rescue the myofibril defects from the smyd1b knockdown in zebrafish embryos.

Conclusion/Significance

Collectively, these data indicate that Smyd1a and Smyd1b share similar biological activity in myofibril assembly in zebrafish embryos. However, Smyd1b appears to play a major role in this process.     

Faithful expression of green fluorescent protein (GFP) in transgenic zebrafish embryos under control of zebrafish gene promoters

Although the zebrafish has become a popular model organism for vertebrate developmental and genetic analyses, its use in transgenic studies still suffers from the scarcity of homologous gene promoters. In the present study, three different zebrafish cDNA clones were isolated and sequenced completely, and their expression patterns were characterized by whole‐mount in situ hybridization as well as by Northern blot hybridization. The first clone encodes a type II cytokeratin (CK), which is specifically expressed in skin epithelia in early embryos and prominently expressed in the adult skin tissue. The second clone is muscle specific and encodes a muscle creatine kinase (MCK). The third clone, expressed ubiquitously in all tissues, is derived from an acidic ribosomal phosphoprotein P0 (arp) gene. In order to test the fidelity of zebrafish embryos in transgenic expression, the promoters of the three genes were isolated using a rapid linker‐mediated PCR approach and subsequently ligated to a modified green fluorescent protein (gfp) reporter gene. When the three hybrid GFP constructs were introduced into zebrafish embryos by microinjection, the three promoters were activated faithfully in developing zebrafish embryos. The 2.2‐kb ck promoter was sufficient to direct GFP expression in skin epithelia, although a weak expression in muscle was also observed in a few embryos. This pattern of transgenic expression is consistent with the expression pattern of the endogenous cytokeratin gene. The 1.5‐kb mck promoter/gfp was expressed exclusively in skeletal muscles and not elsewhere. By contrast, the 0.8‐kb ubiquitous promoter plus the first intron of the arp gene were capable of expressing GFP in a variety of tissues, including the skin, muscle, lens, neurons, notochord, and circulating blood cells. Our experiments, therefore, further demonstrated that zebrafish embryos can faithfully express exogenously introduced genes under the control of zebrafish promoters. Dev. Genet. 25:158–167, 1999. © 1999 Wiley‐Liss, Inc.     

Lack of Developmental Redundancy between Unc45 Proteins in Zebrafish Muscle Development

Since the majority of protein-coding genes in vertebrates have intra-genomic homologues, it has been difficult to eliminate the potential of functional redundancy from analyses of mutant phenotypes, whether produced by genetic lesion or transient knockdown. Further complicating these analyses, not all gene products have activities that can be assayed in vitro, where the efficiency of the various family members can be compared against constant substrates. Two vertebrate UNC-45 homologues, unc45a and unc45b, affect distinct stages of muscle differentiation when knocked down in cell culture and are functionally redundant in vitro. UNC-45 proteins are members of the UCS (UNC-45/CRO1/She4p) protein family that has been shown to regulate myosin-dependent functions from fungi to vertebrates through direct interaction with the myosin motor domain. To test whether the same functional relationship exists between these unc45 paralogs in vivo, we examined the developmental phenotypes of doubly homozygous unc45b^−/−; unc45a^−/− mutant zebrafish embryos. We focused specifically on the combined effects on morphology and gene expression resulting from the zygotic lack of both paralogs. We found that unc45b^−/− and unc45b^−/−; unc45a^−/− embryos were phenotypically indistinguishable with both mutants displaying identical cardiac, skeletal muscle, and jaw defects. We also found no evidence to support a role for zygotic Unc45a function in myoblast differentiation. In contrast to previous in vitro work, this rules out a model of functional redundancy between Unc45a and Unc45b in vivo. Instead, our phylogenetic and phenotypic analyses provide evidence for the role of functional divergence in the evolution of the UCS protein family.     

Enhanced hyperplasia in muscles of transgenic zebrafish expressing <Emphasis Type="Italic">Follistatin1</Emphasis>

Myostatin is a member of the transforming growth factor-β (TGF-β) super-family and functions as a negative regulator of muscle growth. Binding of the specific receptor, Activin receptor IIB (Act RIIB), with myostatin or other related TGF-β members, could be inhibited by the activin-binding protein follistatin (Fst) in mammals. Overexpressing Fst in mouse skeletal muscle leads to muscle hypertrophy and hyperplasia. To determine if Fst has similar roles in fish, we generated transgenic zebrafish expressing high levels of zebrafish Fst1 using the promoter of the zebrafish skeletal muscle-specific gene, myosin, light polypeptide 2, skeletal muscle (Mylz2). Independent transgenic zebrafish lines exhibited elevated expression levels of myogenic regulatory genes MyoD and Pax7 in muscle cells. Adult Fst1 overexpressing transgenic zebrafish exhibited a slight body weight increase. The high level of Fst1 expression dramatically increased myofiber numbers in skeletal muscle, without significantly changing the fiber size. Our findings suggest that Fst1 overexpression can promote zebrafish muscle growth by enhancing myofiber hyperplasia.     

<Emphasis Type="Italic">smyd1</Emphasis> and <Emphasis Type="Italic">smyd2</Emphasis> are expressed in muscle tissue in <Emphasis Type="Italic">Xenopus laevis</Emphasis>

Epigenetic modifications of histone play important roles for regulation of cell activity, such as cell division, cell death, and cell differentiation. A SET domain consisting of about 130 amino acids has lysine methyltransferase activity in the presence of the cosubstrate S-adenosyl-methionine. More than 60 SET domain-containing proteins have been predicted in various organisms. One of them, the SMYD family genes which contain a SET domain and a zinc-finger MYND domain are reported to regulate cell cycle and muscle formation. Here we examined the expression and function of smyd1 and 2 in Xenopus. smyd1 and 2 were expressed in various muscle tissues. While smyd1 expression was observed mainly in cardiac muscle and skeletal muscle, smyd2 expression was done abundantly in skeletal muscle and face region. Moreover, by loss-of-function experiments using antisense morpholino oligonucleotides, it was suggested that smyd1 and 2 related to muscle cells differentiation.     

A tetracycline‐inducible and skeletal muscle‐specific Cre recombinase transgenic mouse

We have generated a transgenic mouse that expresses Cre recombinase only in skeletal muscle and only following tetracycline treatment. This spatiotemporal specificity is achieved using two transgenes. The first transgene uses the human skeletal actin (HSA) promoter to drive expression of the reverse tetracycline‐controlled transactivator (rtTA). The second transgene uses a tetracycline responsive promoter to drive the expression of Cre recombinase. We monitored transgene expression in these mice by crossing them with ROSA26 loxP‐LacZ reporter mice, which express β‐galactosidase when activated by Cre. We find that the expression of this transgene is only detectable within skeletal muscle and that Cre expression in the absence of tetracycline is negligible. Cre is readily induced in this model with tetracycline analogs at a range of embryonic and postnatal ages and in a pattern consistent with other HSA transgenic mice. This mouse improves upon existing transgenic mice in which skeletal muscle Cre is expressed throughout development by allowing Cre expression to begin at later developmental stages. This temporal control of transgene expression has several applications, including overcoming embryonic or perinatal lethality due to transgene expression. This mouse is especially suited for studies of steroid hormone action, as it uses tetracycline, rather than tamoxifen, to activate Cre expression. In summary, we find that this transgenic induction system is suitable for studies of gene function in the context of hormonal regulation of skeletal muscle or interactions between muscle and motoneurons in mice. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Colored medaka and zebrafish: Transgenics with ubiquitous and strong transgene expression driven by the medaka β‐actin promoter

Conditional cell labeling, cell tracing, and genetic manipulation approaches are becoming increasingly important in developmental and regenerative biology. Such approaches in zebrafish research are hampered by the lack of an ubiquitous transgene driver element that is active at all developmental stages. Here, we report the isolation and characterization of the medaka fish (Oryzias latipes) β‐actin (Olactb) promoter, which drives constitutive transgene expression during all developmental stages, and the analysis of adult organs except blood cell types. Taking advantage of the compact medaka promoter, we succeeded in generating a zebrafish transgenic (Tg) line with unprecedentedly strong and widespread transgene expression from embryonic to adult stages. Moreover, the Tg carries a pair of loxP sites, which enables the reporter fluorophore to switch from DsRed2 to enhanced green fluorescent protein (EGFP). We induced Cre/loxP recombination with Tg(hsp70l: mCherry‐t2a‐Cre^ERt2) in the double Tg embryo and generated a Tg line that constitutively expresses EGFP. We further demonstrate the powerful application of Olactb‐driven Tgs for cell lineage tracing using transplantation experiments with embryonic cells at the shield stage and adult cells of regenerating fin. Thus, the use of promoter elements from medaka is an alternative approach to generate Tgs with stronger and even novel expression patterns in zebrafish. The Olactb promoter and the Tg lines presented here represent an important advancement for the broader use of Cre/loxP‐based Tg applications in zebrafish.     

Histone deacetylase inhibition regulates miR-449a levels in skeletal muscle cells

microRNAs (miRNAs) are small non-coding RNAs that regulate cellular processes by fine-tuning the levels of their target mRNAs. However, the regulatory elements determining cellular miRNA levels are not well studied. Previously, we had described an altered miRNA signature in the skeletal muscle of db/db mice. Here, we sought to explore the role of epigenetic mechanisms in altering these miRNAs. We show that histone deacetylase (HDAC) protein levels and activity are upregulated in the skeletal muscle of diabetic mice. In C2C12 cells, HDAC inhibition using suberoylanilide hydroxamic acid (SAHA) altered the levels of 24 miRNAs: 15 were downregulated and 9 were upregulated. miR-449a, an intronic miRNA localized within the Cdc20b gene, while being downregulated in the skeletal muscle of diabetic mice, was the most highly upregulated during HDAC inhibition. The host gene, Cdc20b, was also significantly upregulated during HDAC inhibition. Bioinformatics analyses identified a common promoter for both Cdc20b and miR-449a that harbors significant histone acetylation marks, suggesting the possibility of regulation by histone acetylation-deacetylation. These observations suggest an inverse correlation between miR-449a levels and HDAC activity, in both SAHA-treated skeletal muscle cells and db/db mice skeletal muscle. Further, in SAHA-treated C2C12 cells, we observed augmented occupancy of acetylated histones on the Cdc20b/miR-449a promoter, which possibly promotes their upregulation. In vivo injection of SAHA to db/db mice significantly restored skeletal muscle miR-449a levels. Our results provide insights into the potential regulatory role of epigenetic histone acetylation of the miR-449a promoter that may regulate its expression in the diabetic skeletal muscle.     

Troponin T3 expression in skeletal and smooth muscle is required for growth and postnatal survival: Characterization of Tnnt3tm2a(KOMP)Wtsi mice

The troponin complex, which consists of three regulatory proteins (troponin C, troponin I, and troponin T), is known to regulate muscle contraction in skeletal and cardiac muscle, but its role in smooth muscle remains controversial. Troponin T3 (TnnT3) is a fast skeletal muscle troponin believed to be expressed only in skeletal muscle cells. To determine the in vivo function and tissue‐specific expression of Tnnt3, we obtained the heterozygous Tnnt3+/flox/lacZ mice from Knockout Mouse Project (KOMP) Repository. Tnnt3^lacZ/+ mice are smaller than their WT littermates throughout development but do not display any gross phenotypes. Tnnt3^lacZ/lacZ embryos are smaller than heterozygotes and die shortly after birth. Histology revealed hemorrhagic tissue in Tnnt3^lacZ/lacZ liver and kidney, which was not present in Tnnt3^lacZ/+ or WT, but no other gross tissue abnormalities. X‐gal staining for Tnnt3 promoter‐driven lacZ transgene expression revealed positive staining in skeletal muscle and diaphragm and smooth muscle cells located in the aorta, bladder, and bronchus. Collectively, these findings suggest that troponins are expressed in smooth muscle and are required for normal growth and breathing for postnatal survival. Moreover, future studies with this mouse model can explore TnnT3 function in adult muscle function using the conditional‐inducible gene deletion approach genesis 51:667–675. © 2013 Wiley Periodicals, Inc.     

Editing of an effector gene promoter sequence impacts plant‐Phytophthora interaction

Pathogen avirulence (Avr) effectors interplay with corresponding plant resistance (R) proteins and activate robust plant immune responses. Although the expression pattern of Avr genes has been tied to their functions for a long time, it is still not clear how Avr gene expression patterns impact plant‐microbe interactions. Here, we selected PsAvr3b, which shows a typical effector gene expression pattern from a soybean root pathogen Phytophthora sojae. To modulate gene expression, we engineered PsAvr3b promoter sequences by in situ substitution with promoter sequences from Actin (constitutive expression), PsXEG1 (early expression), and PsNLP1 (later expression) using the CRISPR/Cas9. PsAvr3b driven by different promoters resulted in distinct expression levels across all the tested infection time points. Importantly, those mutants with low PsAvr3b expression successfully colonized soybean plants carrying the cognate R gene Rps3b. To dissect the difference in plant responses to the PsAvr3b expression level, we conducted RNA‐sequencing of different infection samples at 24 h postinfection and found soybean immune genes, including a few previously unknown genes that are associated with resistance. Our study highlights that fine‐tuning in Avr gene expression impacts the compatibility of plant disease and provides clues to improve crop resistance in disease control management.     

The evolution of skeletal muscle performance: gene duplication and divergence of human sarcomeric α‐actinins

In humans, there are two skeletal muscle α‐actinins, encoded by ACTN2 and ACTN3, and the ACTN3 genotype is associated with human athletic performance. Remarkably, approximately 1 billion people worldwide are deficient in α‐actinin‐3 due to the common ACTN3 R577X polymorphism. The α‐actinins are an ancient family of actin‐binding proteins with structural, signalling and metabolic functions. The skeletal muscle α‐actinins diverged ～250–300 million years ago, and ACTN3 has since developed restricted expression in fast muscle fibres. Despite ACTN2 and ACTN3 retaining considerable sequence similarity, it is likely that following duplication there was a divergence in function explaining why α‐actinin‐2 cannot completely compensate for the absence of α‐actinin‐3. This paper focuses on the role of skeletal muscle α‐actinins, and how possible changes in functions between these duplicates fit in the context of gene duplication paradigms.     

A Tie2‐driven BAC‐TRAP transgenic line for in vivo endothelial gene profiling

Recent technological innovations including bacterial artificial chromosome‐based translating ribosome affinity purification (BAC‐TRAP) have greatly facilitated analysis of cell type‐specific gene expression in vivo, especially in the nervous system. To better study endothelial gene expression in vivo, we have generated a BAC‐TRAP transgenic mouse line where the L10a ribosomal subunit is tagged with EGFP and placed under the control of the endothelium‐specific Tie2 (Tek) promoter. We show that transgene expression in this line is widely, but specifically, detected in endothelial cells in several brain regions throughout pre‐ and postnatal development, as well as in other organs. We also show that this line results in highly significant enrichment of endothelium‐specific mRNAs from brain tissues at different stages. This BAC‐TRAP line therefore provides a useful genetic tool for in vivo endothelial gene profiling under various developmental, physiological, and pathological conditions. genesis 54:136–145, 2016. © 2016 Wiley Periodicals, Inc.     

DNA methylation regulates gabrb2 mRNA expression: developmental variations and disruptions in l‐methionine‐induced zebrafish with schizophrenia‐like symptoms

Single nucleotide polymorphisms (SNPs) in the human type A gamma‐aminobutyric acid (GABA) receptor β₂ subunit gene (GABRB2) have been associated with schizophrenia and quantitatively correlated with mRNA expression in the postmortem brain tissue of patients with schizophrenia. l ‐Methionine (MET) administration has been reported to cause a recrudescence of psychotic symptoms in patients with schizophrenia, and similar symptoms have been generated in MET‐induced mice. In this study, a zebrafish animal model was used to evaluate the relationship between the gabrb2 mRNA expression and its promoter DNA methylation in developmental and MET‐induced schizophrenia‐like zebrafish. The results indicated developmental increases in global DNA methylation and decreases in gabrb2 promoter methylation in zebrafish. A significant increase in gabrb2 mRNA levels was observed after GABA was synthesized. Additionally, the MET‐triggered schizophrenia‐like symptoms in adult zebrafish, involving social withdrawal and cognitive dysfunction analyzed with social interaction and T‐maze behavioral tests, were accompanied by significantly increased DNA methylation levels in the global genome and the gabrb2 promoter. Furthermore, the significant correlation between gabrb2 mRNA expression and gabrb2 promoter methylation observed in the developmental stages became non‐significant in MET‐triggered adult zebrafish. These findings demonstrate that gabrb2 mRNA expression is associated with DNA methylation varies by developmental stage and show that these epigenetic association mechanisms are disrupted in MET‐triggered adult zebrafish with schizophrenia‐like symptoms. In conclusion, these results provide plausible epigenetic evidence of the GABA_A receptor β₂ subunit involvement in the schizophrenia‐like behaviors and demonstrate the potential use of zebrafish models in neuropsychiatric research.     

A polymorphism in the porcine miR‐208b is associated with microRNA biogenesis and expressions of SOX‐6 and MYH7 with effects on muscle fibre characteristics and meat quality

MicroRNAs (miRNAs) encoded by the myosin heavy chain (MHC) genes are muscle‐specific miRNAs (myomiRs) and regulate the expression of MHC isoforms in skeletal muscle. These miRNAs have been implicated in muscle fibre types and their characteristics by affecting the heterogeneity of myosin. In pigs, miR‐208b and miR‐499 are embedded in introns of MYH7 and MYH7b respectively. Here, we identified a novel single nucleotide polymorphism (SNP) in intron 30 of MYH7 by which porcine miR‐208b is encoded. Based on the association study using a total of 487 pigs including Berkshire (n = 164), Landrace (n = 121) and Yorkshire (n = 202), the miR‐208b SNP (g.17104G>A) had significant effects on the proportions of types I and IIb fibre numbers (P < 0.010) among muscle fibre characteristics and on drip loss (P = 0.012) in meat quality traits. Moreover, the SNP affected the processing of primary miR‐208b into precursor miR‐208b with a marginal trend towards significance (P = 0.053), thereby leading to significant changes in the levels of mature miR‐208b (P = 0.009). These SNP‐dependent changes in mature miR‐208b levels were negatively correlated with the expression levels of its target gene, SOX‐6 (P = 0.038), and positively associated with the expression levels of its host gene, MYH7 (P = 0.046). Taken together, our data suggest that the porcine miR‐208b SNP differentially represses the expression of SOX‐6 by regulating miRNA biogenesis, thereby affecting the expression of MYH7 and the traits of muscle fibre characteristics and meat quality.