Mef2c factors are required for early but not late addition of cardiomyocytes to the ventricle

During heart formation, the heart grows and undergoes dramatic morphogenesis to achieve efficient embryonic function. Both in fish and amniotes, much of the growth occurring after initial heart tube formation arises from second heart field (SHF)-derived progenitor cell addition to the arterial pole, allowing chamber formation. In zebrafish, this process has been extensively studied during embryonic life, but it is unclear how larval cardiac growth occurs beyond 3 days post-fertilisation (dpf). By quantifying zebrafish myocardial growth using live imaging of GFP-labelled myocardium we show that the heart grows extensively between 3 and 5 dpf. Using methods to assess cell division, cellular development timing assay and Kaede photoconversion, we demonstrate that proliferation, CM addition, and hypertrophy contribute to ventricle growth. Mechanistically, we show that reduction in Mef2c activity (mef2ca+/-;mef2cb-/-), downstream or in parallel with Nkx2.5 and upstream of Ltbp3, prevents some CM addition and differentiation, resulting in a significantly smaller ventricle by 3 dpf. After 3 dpf, however, CM addition in mef2ca+/-;mef2cb-/- mutants recovers to a normal pace, and the heart size gap between mutants and their siblings diminishes into adulthood. Thus, as in mice, there is an early time window when SHF contribution to the myocardium is particularly sensitive to loss of Mef2c activity

What determines growth potential and juvenile quality of farmed fish species?

Enhanced production of high quality and healthy fry is a key target for a successful and competitive expansion of the aquaculture industry. Although large quantities of fish larvae are produced, survival rates are often low or highly variable and growth potential is in most cases not fully exploited, indicating significant gaps in our knowledge concerning optimal nutritional and culture conditions. Understanding the mechanisms that control early development and muscle growth are critical for the identification of time windows in development that introduce growth variation, and improve the viability and quality of juveniles. This literature review of the current state of knowledge aims to provide a framework for a better understanding of fish skeletal muscle ontogeny, and its impact on larval and juvenile quality as broadly defined. It focuses on fundamental biological knowledge relevant to larval phenotype and quality and, in particular, on the factors affecting the development of skeletal muscle. It also discusses the available methodologies to assess growth and larvae/juvenile quality, identifies gaps in knowledge and suggests future research directions. The focus is primarily on the major farmed non-salmonid fish species in Europe that include gilthead sea bream, European sea bass, turbot, Atlantic cod, Senegalese sole and Atlantic halibut

Myogenin promotes myocyte fusion to balance fibre number and size

Each skeletal muscle acquires its unique size before birth, when terminally differentiating myocytes fuse to form a defined number of multinucleated myofibres. Although mice in which the transcription factor Myogenin is mutated lack most myogenesis and die perinatally, a specific cell biological role for Myogenin has remained elusive. We report that loss of function of zebrafish myog prevents formation of almost all multinucleated muscle fibres. A second, Myog-independent, fusion pathway requires Hedgehog signalling. Lack of Myog does not prevent terminal differentiation; the smaller myotome has a normal number of myocytes forming more mononuclear, thin, albeit functional, fast muscle fibres. Mechanistically, Myogenin binds the myomaker promoter and is required for expression of myomaker and other genes essential for myocyte fusion. Adult myog mutants display reduced muscle mass, decreased fibre size and nucleation. Adult-derived myog mutant myocytes show persistent defective fusion ex vivo. Myogenin is therefore essential for muscle homeostasis, regulating myocyte fusion to determine both muscle fibre number and size

Scleraxis genes are required for normal musculoskeletal development and for rib growth and mineralization in zebrafish

Tendons are an essential part of the musculoskeletal system, connecting muscle and skeletal elements to enable force generation. The transcription factor scleraxis marks vertebrate tendons from early specification. Scleraxis-null mice are viable and have a range of tendon and bone defects in the trunk and limbs but no described cranial phenotype. We report the expression of zebrafish scleraxis orthologs: scleraxis homolog (scx)-a and scxb in cranial and intramuscular tendons and in other skeletal elements. Single mutants for either scxa or scxb, generated by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), are viable and fertile as adult fish. Although scxb mutants show no obvious phenotype, scxa mutant embryos have defects in cranial tendon maturation and muscle misalignment. Mutation of both scleraxis genes results in more severe defects in cranial tendon differentiation, muscle and cartilage dysmorphogenesis and paralysis, and lethality by 2-5 wk, which indicates an essential function of scleraxis for craniofacial development. At juvenile and adult stages, ribs in scxa mutants fail to mineralize and/or are small and heavily fractured. Scxa mutants also have smaller muscle volume, abnormal swim movement, and defects in bone growth and composition. Scleraxis function is therefore essential for normal craniofacial form and function and vital for fish development.-Kague, E., Hughes, S. M., Lawrence, E. A., Cross, S., Martin-Silverstone, E., Hammond, C. L., Hinits, Y. Scleraxis genes are required for normal musculoskeletal development and for rib growth and mineralization in zebrafish.</p

Distinct functions of alternatively spliced isoforms encoded by zebrafish mef2ca and mef2cb. Ganassi M, Badodi S, Polacchini A, Baruffaldi F, Battini R, Hughes SM, Hinits Y, Molinari S. Biochim Biophys Acta. 2014 Jul;1839(7):559-70. doi: 10.1016/j.bbagrm.2014.05.003. Epub 2014 May 17.

In mammals, an array of MEF2C proteins is generated by alternative splicing (AS), yet specific functions have not been ascribed to each isoform. Teleost fish possess two MEF2C paralogues, mef2ca and mef2cb. In zebrafish, the Mef2cs function to promote cardiomyogenic differentiation and myofibrillogenesis in nascent skeletal myofibers. We found that zebrafish mef2ca and mef2cb are alternatively spliced in the coding exons 4-6 region and these splice variants differ in their biological activity. Of the two, mef2ca is more abundantly expressed in developing skeletal muscle, its activity is tuned through zebrafish development by AS. By 24hpf, we found the prevalent expression of the highly active full length protein in differentiated muscle in the somites. The splicing isoform of mef2ca that lacks exon 5 (mef2ca 4-6), encodes a protein that has 50% lower transcriptional activity, and is found mainly earlier in development, before muscle differentiation. mef2ca transcripts including exon 5 (mef2ca 4-5-6) are present early in the embryo. Over-expression of this isoform alters the expression of genes involved in early dorso-ventral patterning of the embryo such as chordin, nodal related 1 and goosecoid, and induces severe developmental defects. AS of mef2cb generates a long splicing isoform in the exon 5 region (Mef2cbL) that predominates during somitogenesis. Mef2cbL contains an evolutionarily conserved domain derived from exonization of a fragment of intron 5, which confers the ability to induce ectopic muscle in mesoderm upon over-expression of the protein. Taken together, the data show that AS is a significant regulator of Mef2c activit

Maternal Larp6 controls oocyte development, chorion formation and elevation.

La-related protein 6 (Larp6) is a conserved RNA-binding protein found across eukaryotes that has been suggested to regulate collagen biogenesis, muscle development, ciliogenesis, and various aspects of cell proliferation and migration. Zebrafish have two Larp6 family genes: larp6a and larp6b Viable and fertile single and double homozygous larp6a and larp6b zygotic mutants revealed no defects in muscle structure, and were indistinguishable from heterozygous or wild-type siblings. However, larp6a mutant females produced eggs with chorions that failed to elevate fully and were fragile. Eggs from larp6b single mutant females showed minor chorion defects, but chorions from eggs laid by larp6a;larp6b double mutant females were more defective than those from larp6a single mutants. Electron microscopy revealed defective chorionogenesis during oocyte development. Despite this, maternal zygotic single and double mutants were viable and fertile. Mass spectrometry analysis provided a description of chorion protein composition and revealed significant reductions in a subset of zona pellucida and lectin-type proteins between wild-type and mutant chorions that paralleled the severity of the phenotype. We conclude that Larp6 proteins are required for normal oocyte development, chorion formation and egg activation

Oesophageal and sternohyal muscle fibres are novel Pax3-dependent migratory somite derivatives essential for ingestion

Striated muscles that enable mouth opening and swallowing during feeding are essential for efficient energy acquisition, and are likely to have played a fundamental role in the success of early jawed vertebrates. The developmental origins and genetic requirements of these muscles are uncertain. Here, we determine by indelible lineage tracing in mouse that fibres of sternohyoid muscle (SHM), which is essential for mouth opening during feeding, and oesophageal striated muscle (OSM), which is crucial for voluntary swallowing, arise from Pax3-expressing somite cells. In vivo Kaede lineage tracing in zebrafish reveals the migratory route of cells from the anteriormost somites to OSM and SHM destinations. Expression of pax3b, a zebrafish duplicate of Pax3, is restricted to the hypaxial region of anterior somites that generate migratory muscle precursors (MMPs), suggesting that Pax3b plays a role in generating OSM and SHM. Indeed, loss of pax3b function led to defective MMP migration and OSM formation, disorganised SHM differentiation, and inefficient ingestion and swallowing of microspheres. Together, our data demonstrate Pax3-expressing somite cells as a source of OSM and SHM fibres, and highlight a conserved role of Pax3 genes in the genesis of these feeding muscles of vertebrates

The histone methyltransferase Set7/9 promotes myoblast differentiation and myofibril assembly

Set7 associates with the MyoD transcription factor to enhance expression of genes required for muscle differentiation