The zebrafish (Danio rerio) has been used extensively as a model system for developmental studies but, unlike most teleost fish, it grows in a determinate-like manner. A close relative, the giant danio (Devario cf. aequipinnatus), grows indeterminately, displaying both hyperplasia and hypertrophy of skeletal myofibers as an adult. To better understand adult muscle hyperplasia, a postlarval/postnatal process that closely resembles secondary myogenesis during development, we characterized the expression of Pax3/7, c-Met, syndecan-4, Myf5, MyoD1, myogenin, and myostatin during in vitro myogenesis, a technique that allows for the complete progression of myogenic precursor cells to myotubes. Pax7 appears to be expressed only in newly activated MPCs while Pax3 is expressed through most of the myogenic program, as are c-Met and syndecan-4. MyoD1 appears important in all stages of myogenesis, while Myf5 is likely expressed at low to background levels, and myogenin expression is enriched in myotubes. Myostatin, like MyoD1, appears to be ubiquitous at all stages. This is the first comprehensive report of key myogenic factor expression patterns in an indeterminate teleost, one that strongly suggests that Pax3 and/or Myf5 may be involved in the regulation of this paradigm. Further, it validates this species as a model organism for studying adult myogenesis in vitro, especially mechanisms underlying nascent myofiber recruitment.

Although the zebrafish (Danio rerio) has been widely utilized as a model organism for several decades, there is little information available on physiological variation underlying genetic variation among the most commonly used inbred strains. This study evaluated growth performance using physiological and molecular markers of growth in response to fasting in six commonly used zebrafish strains [AB, TU, TL, SJA, WIK, and petstore (PET) zebrafish]. Fasting resulted in a standard decrease in whole blood glucose levels, typical vertebrate glucose metabolism pattern, in AB, PET, TL, and TU zebrafish strains. Alternatively, fasting did not affect glucose levels in SJA and WIK zebrafish strains. Similarly, fasting had no effect on myostatin mRNA levels in AB, PET, TU, and WIK zebrafish strains, but decreased myostatin-1 and -2 mRNA levels in SJA zebrafish. Consistent with previous work, fasting increased myostatin-2 mRNA levels in TL zebrafish. These data demonstrate that variation is present in growth performance between commonly used inbred strains of zebrafish. These data can help future research endeavors by highlighting the attributes of each strain with regard to growth performance so that the most fitting strain may be utilized.

The extraordinary muscle growth potential of teleost fish, particular those of the Salmoninae clade, elicits questions about how the relatively highly conserved transcription factors of the myogenic program are regulated. In addition, the pseudotetraploid nature of the salmonid genome adds another layer of regulatory complexity, and this must be reconciled with epigenetic data to better understand how these fish achieve lifelong muscle growth. To this end, we identified three paralogous pax7 genes (pax7a1, pax7a2, and pax7b) in the rainbow trout genome. During in vitro myogenesis, pax7a1 transcripts remain stable, while pax7a2 and pax7b mRNAs increase in abundance, similarly to myogenin mRNAs and in contrast to the expression pattern of the mammalian ortholog. In addition, we profiled the distribution of repressive H3K27me3 and H3K9me3 and permissive H3K4me3 marks during in vitro myogenesis across these loci, finding that pax7a2 expression was associated with decreased H3K27 trimethylation, while pax7b expression was correlated with decreased H3K9me3 and −K27me3. Altogether, these data link the highly unique differential expression of pax7 paralogs with epigenetic histone modifications in a vertebrate species displaying growth divergent from that of mammals and highlight an important divergence in the regulatory mechanisms of pax7 expression among vertebrates. The system described here provides a more comprehensive picture of the combinatorial control mechanisms orchestrating skeletal muscle growth in a salmonid, leading to a better understanding of myogenesis in this species and across Vertebrata more generally.

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e. teleost fish) and full regeneration following appendage loss (i.e. urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.