Zebrafish Embryonic Slow Muscle Is a Rapid System for Genetic Analysis of Sarcomere Organization by CRISPR/Cas9, but Not NgAgo

Zebrafish embryonic slow muscle cells, with their superficial localization and clear sarcomere organization, provide a useful model system for genetic analysis of muscle cell differentiation and sarcomere assembly. To develop a quick assay for testing CRISPR-mediated gene editing in slow muscles of zebrafish embryos, we targeted a red fluorescence protein (RFP) reporter gene specifically expressed in slow muscles of myomesin-3-RFP (Myom3-RFP) zebrafish embryos. We demonstrated that microinjection of RFP-sgRNA with Cas9 protein or Cas9 mRNA resulted in a mosaic pattern in loss of RFP expression in slow muscle fibers of the injected zebrafish embryos. To uncover gene functions in sarcomere organization, we targeted two endogenous genes, slow myosin heavy chain-1 (smyhc1) and heat shock protein 90 α1 (hsp90α1), which are specifically expressed in zebrafish muscle cells. We demonstrated that injection of Cas9 protein or mRNA with respective sgRNAs targeted to smyhc1 or hsp90a1 resulted in a mosaic pattern of myosin thick filament disruption in slow myofibers of the injected zebrafish embryos. Moreover, Myom3-RFP expression and M-line localization were also abolished in these defective myofibers. Given that zebrafish embryonic slow muscles are a rapid in vivo system for testing genome editing and uncovering gene functions in muscle cell differentiation, we investigated whether microinjection of Natronobacterium gregoryi Argonaute (NgAgo) system could induce genetic mutations and muscle defects in zebrafish embryos. Single-strand guide DNAs targeted to RFP, Smyhc1, or Hsp90α1 were injected with NgAgo mRNA into Myom3-RFP zebrafish embryos. Myom3-RFP expression was analyzed in the injected embryos. The results showed that, in contrast to the CRISPR/Cas9 system, injection of the NgAgo-gDNA system did not affect Myom3-RFP expression and sarcomere organization in myofibers of the injected embryos. Sequence analysis failed to detect genetic mutations at the target genes. Together, our studies demonstrate that zebrafish embryonic slow muscle is a rapid model for testing gene editing technologies in vivo and uncovering gene functions in muscle cell differentiation.     

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.     

Overexpression of Myostatin2 in zebrafish reduces the expression of dystrophin associated protein complex (DAPC) which leads to muscle dystrophy

Myostatin, a member of the TGF-β superfamily, is a potent negative regulator of skeletal muscle and growth. Previously, we reported Mstn1 from zebrafish and studied its influence on muscle development. In this study, we identified another form of Myostatin protein which is referred to as Mstn2. The size of Mstn2 cDNA is 1342 bp with 109 and 132 bp of 5′ and 3′-untranslated regions (UTRs), respectively. The coding region is 1101 bp encoding 367 amino acids. The identity between zebrafish Mstn1 and 2 is 66%. The phylogenetic tree revealed that the Mstn2 is an ancestral form of Mstn1. To study the functional aspects, we overexpressed mstn2 and noticed that embryos became less active and the juveniles with bent and curved phenotypes when compared to the control. The RT-PCR and in situ hybridization showed concurrent reduction of dystrophin associated protein complex (DAPC). In cryosection and in situ hybridization, we observed the disintegration of somites, lack of transverse myoseptum and loss of muscle integrity due to the failure of muscle attachment in mstn2 overexpressed embryos. Immunohistochemistry and western blot showed that there was a reduction of dystrophin, dystroglycan and sarcoglycan at translational level in overexpressed embryos. Taken together, these results indicate the suitability of zebrafish as an excellent animal model and our data provide the first in vivo evidence of muscle attachment failure by the overexpression of mstn2 and it leads to muscle loss which results in muscle dystrophy that may contribute to Duchenne syndrome and other muscle related diseases. A. Anusha Amali and Cliff Ji-Fan Lin contributed equally.     

Rbfox-regulated alternative splicing is critical for zebrafish cardiac and skeletal muscle functions

Rbfox RNA binding proteins are implicated as regulators of phylogenetically-conserved alternative splicing events important for muscle function. To investigate the function of rbfox genes, we used morpholino-mediated knockdown of muscle-expressed rbfox1l and rbfox2 in zebrafish embryos. Single and double morphant embryos exhibited changes in splicing of overlapping sets of bioinformatically-predicted rbfox target exons, many of which exhibit a muscle-enriched splicing pattern that is conserved in vertebrates. Thus, conservation of intronic Rbfox binding motifs is a good predictor of Rbfox-regulated alternative splicing. Morphology and development of single morphant embryos were strikingly normal; however, muscle development in double morphants was severely disrupted. Defects in cardiac muscle were marked by reduced heart rate and in skeletal muscle by complete paralysis. The predominance of wavy myofibers and abnormal thick and thin filaments in skeletal muscle revealed that myofibril assembly is defective and disorganized in double morphants. Ultra-structural analysis revealed that although sarcomeres with electron dense M- and Z-bands are present in muscle fibers of rbfox1l/rbox2 morphants, they are substantially reduced in number and alignment. Importantly, splicing changes and morphological defects were rescued by expression of morpholino-resistant rbfox cDNA. Additionally, a target-blocking MO complementary to a single UGCAUG motif adjacent to an rbfox target exon of fxr1 inhibited inclusion in a similar manner to rbfox knockdown, providing evidence that Rbfox regulates the splicing of target exons via direct binding to intronic regulatory motifs. We conclude that Rbfox proteins regulate an alternative splicing program essential for vertebrate heart and skeletal muscle functions.     

Hsp27 is persistently expressed in zebrafish skeletal and cardiac muscle tissues but dispensable for their morphogenesis

Constitutive expression of Hsp27 has been demonstrated in vertebrate embryos, especially in developing skeletal and cardiac muscle. Results of several previous studies have indicated that Hsp27 could play a role in the development of these tissues. For example, inhibition of Hsp27 expression has been reported to cause defective development of mammalian myoblasts in vitro and frog embryos in vivo. In contrast, transgenic mice lacking Hsp27 develop normally. Here, we examined the distribution of Hsp27 protein in developing and adult zebrafish and effects of suppressing Hsp27 expression using phosphorodiamidate morpholino oligonucleotides (PMO) on zebrafish development. Consistent with our previous analysis of hsp27 messenger RNA expression, we detected the protein Hsp27 in cardiac, smooth, and skeletal muscle of both embryonic and adult zebrafish. However, embryos lacking detectable Hsp27 after injection of antisense hsp27 PMO exhibited comparable heart beat rates to that of control embryos and cardiac morphology was indistinguishable in the presence or absence of Hsp27. Loss of Hsp27 also had no effect on the structure of the skeletal muscle myotomes in the developing embryo. Finally, embryos injected with antisense hsp27 and scrambled control PMO displayed equal motility. We conclude that Hsp27 is dispensable for zebrafish morphogenesis but could play a role in long-term maintenance of heart and muscle tissues. Tucker and Ustyugov contributed equally to this work.     

Delta-sarcoglycan is required for early zebrafish muscle organization

Mutations in sarcoglycans (alpha-, beta-, gamma-, and delta-) have been linked with limb girdle muscular dystrophy (LGMD) types 2C-F in humans. We have cloned the zebrafish orthologue encoding delta-sarcoglycan and mapped the gene to linkage group 21. The predicted zebrafish delta-sarcoglycan protein is highly homologous with its human orthologue including conservation of two of the three predicted glycosylation sites. Like other members of the dystrophin-associated protein complex (DAPC), delta-sarcoglycan localizes to the sarcolemmal membrane of the myofiber in adult zebrafish, but is more apparent at the myosepta in developing embryos. Zebrafish embryos injected with morpholinos against delta-sarcoglycan were relatively inactive at 5 dpf, their myofibers were disorganized, and swim bladders uninflated. Immunohistochemical and immunoblotting experiments show that delta-, beta-, and gamma-sarcoglycans were all downregulated in the morphants, whereas dystrophin expression was unaffected. Whereas humans lacking delta-sarcoglycan primarily show adult phenotypes, our results suggest that delta-sarcoglycan plays a role in early zebrafish muscle development.     

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular dystrophies^1-3, congenital myopathies^4,5, and disruptions in sarcomeric assembly^6,7, due to high genomic and structural conservation with mammals⁸. Muscular disorganization and locomotive impairment can be quickly assessed in the zebrafish over the first few days post-fertilization. Two assays to help characterize skeletal muscle defects in zebrafish are birefringence (structural) and touch-evoked escape response (behavioral).Birefringence is a physical property in which light is rotated as it passes through ordered matter, such as the pseudo-crystalline array of muscle sarcomeres⁹. It is a simple, noninvasive approach to assess muscle integrity in translucent zebrafish larvae early in development. Wild-type zebrafish with highly organized skeletal muscle appear very bright amidst a dark background when visualized between two polarized light filters, whereas muscle mutants have birefringence patterns specific to the primary muscular disorder they model. Zebrafish modeling muscular dystrophies, diseases characterized by myofiber degeneration followed by repeated rounds of regeneration, exhibit degenerative dark patches in skeletal muscle under polarized light. Nondystrophic myopathies are not associated with necrosis or regenerative changes, but result in disorganized myofibers and skeletal muscle weakness. Myopathic zebrafish typically show an overall reduction in birefringence, reflecting the disorganization of sarcomeres.The touch-evoked escape assay involves observing an embryo''s swimming behavior in response to tactile stimulation^10-12. In comparison to wild-type larvae, mutant larvae frequently display a weak escape contraction, followed by slow swimming or other type of impaired motion that fails to propel the larvae more than a short distance¹². The advantage of these assays is that disease progression in the same fish type can be monitored in vivo for several days, and that large numbers of fish can be analyzed in a short time relative to higher vertebrates.     

Smyd1b_tv1, a key regulator of sarcomere assembly, is localized on the M-line of skeletal muscle fibers

Background

Smyd1b is a member of the Smyd family that plays a key role in sarcomere assembly during myofibrillogenesis. Smyd1b encodes two alternatively spliced isoforms, smyd1b_tv1 and smyd1b_tv2, that are expressed in skeletal and cardiac muscles and play a vital role in myofibrillogenesis in skeletal muscles of zebrafish embryos.

Methodology/Principal Findings

To better understand Smyd1b function in myofibrillogenesis, we analyzed the subcellular localization of Smyd1b_tv1 and Smyd1b_tv2 in transgenic zebrafish expressing a myc-tagged Smyd1b_tv1 or Smyd1b_tv2. The results showed a dynamic change of their subcellular localization during muscle cell differentiation. Smyd1b_tv1 and Smyd1b_tv2 were primarily localized in the cytosol of myoblasts and myotubes at early stage zebrafish embryos. However, in mature myofibers, Smyd1b_tv1, and to a small degree of Smyd1b_tv2, exhibited a sarcomeric localization. Double staining with sarcomeric markers revealed that Smyd1b_tv1was localized on the M-lines. The sarcomeric localization was confirmed in zebrafish embryos expressing the Smyd1b_tv1-GFP or Smyd1b_tv2-GFP fusion proteins. Compared with Smyd1b_tv1, Smyd1b_tv2, however, showed a weak sarcomeric localization. Smyd1b_tv1 differs from Smyd1b_tv2 by a 13 amino acid insertion encoded by exon 5, suggesting that some residues within the 13 aa insertion may be critical for the strong sarcomeric localization of Smyd1b_tv1. Sequence comparison with Smyd1b_tv1 orthologs from other vertebrates revealed several highly conserved residues (Phe223, His224 and Gln226) and two potential phosphorylation sites (Thr221 and Ser225) within the 13 aa insertion. To determine whether these residues are involved in the increased sarcomeric localization of Smyd1b_tv1, we mutated these residues into alanine. Substitution of Phe223 or Ser225 with alanine significantly reduced the sarcomeric localization of Smyd1b_tv1. In contrast, other substitutions had no effect. Moreover, replacing Ser225 with threonine (S225T) retained the strong sarcomeric localization of Smyd1b_tv1.

Conclusion/Significance

Together, these data indicate that Phe223 and Ser225 are required for the M-line localization of Smyd1b_tv1.     

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see ¹). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel² or sapje³) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes⁴. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy⁵. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally¹. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo⁶, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells⁷.Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail^{8, 9}. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish^{10, 11}.     

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.¹ These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.     

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Body movements are mainly provided by mechanical function of skeletal muscle. Skeletal muscle is composed of numerous bundles of myofibers that are sheathed by intramuscular connective tissues. Each myofiber contains many myofibrils that run longitudinally along the length of the myofiber. Myofibrils are the contractile apparatus of muscle and they are composed of repeated contractile units known as sarcomeres. A sarcomere unit contains actin and myosin filaments that are spaced by the Z discs and titin protein. Mechanical function of skeletal muscle is defined by the contractile and passive properties of muscle. The contractile properties are used to characterize the amount of force generated during muscle contraction, time of force generation and time of muscle relaxation. Any factor that affects muscle contraction (such as interaction between actin and myosin filaments, homeostasis of calcium, ATP/ADP ratio, etc.) influences the contractile properties. The passive properties refer to the elastic and viscous properties (stiffness and viscosity) of the muscle in the absence of contraction. These properties are determined by the extracellular and the intracellular structural components (such as titin) and connective tissues (mainly collagen) ^1-2. The contractile and passive properties are two inseparable aspects of muscle function. For example, elbow flexion is accomplished by contraction of muscles in the anterior compartment of the upper arm and passive stretch of muscles in the posterior compartment of the upper arm. To truly understand muscle function, both contractile and passive properties should be studied.The contractile and/or passive mechanical properties of muscle are often compromised in muscle diseases. A good example is Duchenne muscular dystrophy (DMD), a severe muscle wasting disease caused by dystrophin deficiency ³. Dystrophin is a cytoskeletal protein that stabilizes the muscle cell membrane (sarcolemma) during muscle contraction ⁴. In the absence of dystrophin, the sarcolemma is damaged by the shearing force generated during force transmission. This membrane tearing initiates a chain reaction which leads to muscle cell death and loss of contractile machinery. As a consequence, muscle force is reduced and dead myofibers are replaced by fibrotic tissues ⁵. This later change increases muscle stiffness ⁶. Accurate measurement of these changes provides important guide to evaluate disease progression and to determine therapeutic efficacy of novel gene/cell/pharmacological interventions. Here, we present two methods to evaluate both contractile and passive mechanical properties of the extensor digitorum longus (EDL) muscle and the contractile properties of the tibialis anterior (TA) muscle.     

Dissociated flexor digitorum brevis myofiber culture system--a more mature muscle culture system

Considerable knowledge regarding skeletal muscle physiology and disease has been gleaned from cultured myoblastic cell lines or isolated primary myoblasts. Such muscle cultures can be induced to differentiate into multinucleated myotubes that become striated. However they in general do not fully mature and therefore do not model mature muscle. Contrastingly, fresh and cultured dissociated adult mouse flexor digitorum brevis (FDB) myofibers have been studied for many years. We aimed to investigate the possibility of using the FDB myofiber culture system for drug screening and thus long-term cultures of enzymatically dissociated FDB myofibers were established in 96-well plates. Ca2+ handling experiments were used to investigate the functional state of the myofibers. Imaging of intracellular Ca2+ during electric field stimulation revealed that calcium handling was maintained throughout the culture period of at least 8 days. Western blot and immunostaining analysis showed that the FDB cultures maintained expression of mature proteins throughout the culture period, including alpha-sarcoglycan, dystrophin, fast myosin heavy chain and skeletal muscle alpha-actin. The high levels of the fetal proteins cardiac alpha-actin and utrophin, seen in cultured C2C12 myotubes, were absent in the FDB cultures. The expression of developmentally mature proteins and the absence of fetal proteins, in addition to the maintenance of normal calcium handling, highlights the FDB culture system as a more mature and perhaps more relevant culture system for the study of adult skeletal muscle function. Moreover, it may be a useful system for screening therapeutic agents for the treatment of skeletal muscle disorders.     

Visualizing normal and defective bone development in zebrafish embryos using the fluorescent chromophore calcein.

Zebrafish have recently become a model of choice among developmental biologists. This unique model enables both modern molecular and genetic studies to be carried out to identify genes involved in a wide variety of developmental processes. The success of the genetic approach depends largely on the application of an easy and effective screening method to identify interesting mutants. In order to develop a method for visualizing skeletal structures in zebrafish embryos that would be suitable for screening skeletal mutants, we investigated the use of the fluorescent chromophore calcein, which binds specifically to calcified skeletal structures. By using this method, we followed the development of the skeletal structures in zebrafish embryos from day 1 to day 21 postfertilization, and analyzed the effect of bone morphogenetic protein-2 (BMP2) on axial skeleton development. We found the development of the calcified skeletal structure to appear in a progressive fashion from head to tail. Calcified structures in the head (i.e., the jaw) developed first, which were then followed by the axial skeleton in the trunk. Interesting to note was that there appeared to be two domains in the calcification of vertebrae within the axial skeleton. The first three vertebrae were in the first domain; the rest being in the second domain. Compared with Alcian blue staining, we found that calcein staining indeed labels calcified skeletal structures, and, moreover, it is a more sensitive and inclusive method for visualizing skeletal structures. To determine whether calcein staining could also be used to detect abnormal bone development, we ectopically expressed BMP2 in zebrafish notochord cells. We demonstrated that ectopic expression of BMP2 in notochord cells inhibited the development of the axial skeleton. Together, these results clearly demonstrated the sensitivity of calcein staining for visualizing bone structures in developing zebrafish embryos and its effectiveness for screening for mutants that have bone structure defects.