Review
Myogenesis in the Genomics Era

https://doi.org/10.1016/j.jmb.2015.02.009Get rights and content

Highlights

  • Skeletal myogenesis is a complex biological process regulated in great part at the transcriptional level.

  • Genomics approaches, over the years, have revealed a lot about how myogenesis is orchestrated by transcription factors and chromatin-modifying enzymes.

  • Technical challenges that need to be overcome include integration of diverse genomics data, analysis of purer cell populations, and the study of the non-muscle cell types that support myogenesis.

Abstract

Skeletal myogenesis is the process of formation of the muscles that enable movement and breathing. Muscles form after the fate determination and differentiation of precursor cells. Being an extraordinarily complex process, myogenesis is regulated at multiple levels, and transcriptional regulation naturally plays a big part in the making of muscle. A significant part of what we know today of the transcriptional regulatory networks overseeing myogenesis comes from large-scale functional genomics studies. The objective of this review is to provide an overview of the various genomics techniques that have been employed over the years to understand myogenic regulation, to give a sense of the degree of understanding they have provided us up to now, and to highlight the next challenges to be overcome.

Introduction

At its simplest level, myogenesis is the formation of muscle precursor cells, often referred to as myoblasts, and their subsequent differentiation into contractile cells termed myofibers or myotubes. Two hallmarks of muscle differentiation are that it coincides with the fusion of several myoblasts together to form syncytial myofibers, as well as with an irreversible mitotic exit. In vertebrates, skeletal muscles of the trunk and limbs originate from mesodermal precursors arising in the dermomyotome, the dorsal part of the somites (reviewed in Refs. [1], [2], [3]). Under the influence of morphogens secreted by the surrounding tissues, dermomyotomal cells commit to the myogenic lineage and start their differentiation to acquire a muscle identity. Dermomyotomal precursors eventually migrate ventrally to populate the myotome, the site of primary myogenesis. Later, the dermomyotome undergoes de-epithelialization to allow a second wave of muscle fiber formation. Muscles of the limbs originate from cells at the ventrolateral region of the dermomyotome that delaminate and migrate to the limb buds, after which they undergo terminal differentiation.

Myogenesis also occurs after birth, in postnatal growth, and during regeneration after damage. Muscle stem cells called satellite cells are responsible for both (recently reviewed in Refs. [4], [5]). Satellite cells are undifferentiated precursors committed to the muscle lineage. They are wedged between the basal lamina and the sarcolemma of the myofibers [6]. During the first 3 weeks of a mouse postnatal life, muscle tissue growth occurs predominantly through the fusion of satellite-cell-derived myoblasts with existing fibers [7]. In the adult, after an injury involving myotrauma, satellite cells become activated and leave their state of profound quiescence. After extensive proliferation, the myoblasts produced engage in the differentiation route and fuse to damaged fibers for repair, or they fuse together to form new myofibers. The maintenance of the pool of satellite cells is thought to be enabled by the asymmetric divisions that satellite cells engage in, early in the activation process, or by a return to quiescence of actively proliferating cells.

As in any other cellular system, the profound cell identity changes that occur during the prenatal or postnatal myogenesis programs are put in place by remodeling of the cellular transcriptome, such that the genes required by a given cell type to enable them to carry out their intended functions (e.g., alertness to muscle damage, proliferation, and contraction, to name just a few) are expressed at the appropriate levels. The changes to the complement of genes expressed by muscle cells are quite formidable, both in terms of the amplitude of the expression change that is often witnessed and in terms of the sheer number of genes being regulated concurrently. For example, hundreds of cell cycle effector genes simultaneously go from highly expressed to permanently silenced, when myoblasts differentiate into myotubes [8]. Gene expression changes require to be made in an orderly and concerted fashion and are thus orchestrated by an elaborate transcriptional regulatory network [9]. At its core are sequence-specific transcription factors that control which genes will be expressed and which ones should become or remain silent as cells progress through myogenesis. Because transcription factors perform their duties in the context of a chromatinized template, rather than naked DNA, their activity is tightly connected to the structure of that template: it is thought that transcription factor function can be both a cause and a consequence of specific chromatin states, often referred to as the epigenome [10].

Although a very large number of transcription factors are expressed in muscle cells at some point during development and/or in adulthood, some of them are in fact uniquely expressed, or strongly enriched, in skeletal muscle. The transcription factors most intimately connected to myogenesis are the myogenic regulatory factors (MRFs), a group of four related basic helix–loop–helix factors that are at the center of skeletal muscle formation (reviewed in Ref. [11]). The first member identified, MyoD, was identified by virtue of its ability to convert fibroblasts to the muscle lineage [12], [13]. The MRFs do not act alone but cooperate with many other muscle-enriched factors, notably the homeodomain factors Six1, Six4, and Pbx1 [14], [15]; the MADS box factors of the Mef2 family [16], [17]; and the Rel-homology domain transcription factors of the NFATc family [18], [19]. Other transcription factors are also involved in myogenesis, either by controlling the expression of the MRFs or by other means (reviewed in Ref. [20]). Notable cases include the paired-box transcription factors Pax3 and Pax7 [21], [22], as well as the homeodomain factor Pitx2 [23] and Nfix [24]. Pax7 is especially important in the case of adult muscle myogenesis, as its expression is an essential and defining factor of satellite cells [25] and its absence, just like the absence of satellite cells themselves, abrogates regeneration after injury [26], [27], [28], [29].

A detailed understanding of how myogenesis is regulated by these and other transcription factors, as well as by the epigenome, would be beneficial for multiple reasons, beyond simply improving our general knowledge: understanding of regulatory principles that may also prevail in other biological systems, elucidating the etiology of muscular diseases, and obtaining important clues on how we may be able to treat the afflicted patients. For instance, reprogramming of pluripotent stem cells (embryonic or induced) in view of cell therapy requires an in-depth knowledge of the myogenic regulatory network [30], [31], [32], [33], [34], [35]. Beyond the obvious involvement of skeletal muscle in breathing and movement, this tissue also plays an important role in energy metabolism due to its high total mass and it being responsible of about 20% of the body's resting energy expenditure. Skeletal muscle takes care of the bulk of glucose uptake in response to insulin, and deregulation in muscle composition or homeostasis is intimately connected to energy metabolism. For instance, obese insulin-resistant (type 2) diabetic patients tend to have a lower proportion of type I oxidative muscle fibers [36] and have malfunctioning skeletal muscle mitochondria [37]. Likewise, diet-resistant and diet-sensitive obese patients tend to have different muscle characteristics, with diet-sensitive patients having more type I oxidative muscle fibers compared to diet-resistant individuals [38]. Thyroid hormone signals to skeletal muscle to help establish the basal metabolic rate and to elicit adaptive thermogenesis (reviewed in Ref. [39]). Understanding the mechanisms controlling muscle formation, adaptation, and homeostasis can therefore inform us on the regulation of energy balance and on how metabolic changes are brought about by variations in physical activity, endocrine status, mode of life, or disease states.

Thus, we can identify three broad goals to be achieved for an understanding of the myogenesis transcriptional regulatory network: (1) to obtain an accurate representation of transcriptomes of cells at the successive stages of myogenesis, (2) to identify the transcription factors and their target loci involved in establishing these changes, and (3) to determine the state of the epigenome at those stages and identify its relationship to transcription factor function. Over the last 10–15 years, one arguably successful way of achieving these goals has been the use of a systems biology approach, where genomics tools are employed to provide us with a global picture of the myogenic regulatory network. The next sections summarize the progress made and highlight areas for future research.

Section snippets

Genomics Phase I: Gene Expression Profiling

The invention of gene expression profiling by DNA microarrays [40] revolutionized the way we study genome regulation, and in fact, it was instrumental to the emergence of systems biology as an experimental science [41]. The first microarray studies of gene expression in skeletal muscle were performed using the murine C2C12 myoblast cell line, an in vitro model of myogenesis. Derived from a hindlimb muscle, the cells proliferate rapidly under high-growth-factor conditions and differentiate when

Genomics Phase II: Genome-Wide Location Analysis of Transcription Factors

The development of methods that allow us to identify the genomic sites of binding of transcription factors has been of tremendous accelerator of our understanding of myogenesis. Experiments using chromatin immunoprecipitation of transcription factors followed by identification of the associated genomic DNA fragments by microarrays (ChIP-on-chip [69]) or by high-throughput sequencing (ChIP-seq [70], [71]) tell investigators where their factor of interest locates in the genome, essentially

Genomics Phase III: Contribution of the Epigenome

Transcription factors function on a chromatin template. Histone proteins are subject to a myriad of covalent modifications that correlate with, and sometimes regulate, the transcriptional state of genes. The Dynlacht laboratory has used ChIP-seq to map in C2C12 myoblasts and myotubes an impressive number of histone marks associated to gene activity or silencing and to identify the binding sites of the protein complexes that write these marks to control myogenesis [84], [85], [86], [87]. In

Challenges and Opportunities Ahead

Despite the terrific progress that functional genomics approaches have collectively generated to understand the myogenic regulatory network, there is obviously a lot that remains to be discovered. Some areas of research, despite being challenging, have enormous potential.

The vast amount of genomic data that have been garnered so far on the myogenic regulatory network is truly impressive; each study puts a new piece of the puzzle into place, such that we understand a little bit better every time

Conclusion

From the discovery of MyoD to mapping of its transcriptional targets and to elucidation of epigenetic marks associated to muscle transcription factor function, the field of skeletal myogenesis research has made tremendous progress. Every novel finding sheds some new light on the transcriptional regulatory network that controls this process. The advent of new technologies, with increased analytical resolution, sensitivity, and/or precision has heightened the pace of discoveries, and there is

Acknowledgements

The author would like to acknowledge the help of laboratory members for insightful discussions. Research in the author's laboratory is supported by an operating grant from the Canadian Institutes of Health Research (MOP 119458).

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