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P&F Grant Awards

Year 10


Grant # 21

Mechanisms of Mammalian Muscle Morphogenesis

PI: Aaron Johnson, PhD

 

Specific Aims

Congenital myopathies (CM) are a heterogeneous collection of disorders defined by early onset hypotonia, and many myopathy patients will require lifelong mechanical assistance and even surgical interventions to maintain mobility and respiration. Skeletal myogenesis initiates with the specification of myoblasts, which fuse to form a syncytial nascent myotube. The nascent myotube must then elongate and attach to tendon cells to form a functional contractile unit. Our overarching hypothesis is that genetic perturbation to the early myogenic program causes severe developmental defects that result in neonatal mortality, whereas mutations that disrupt myogenesis downstream of myoblast specification underlie the clinical phenotypes associated with CMs.

There remain critical knowledge gaps in our understanding of skeletal muscle development. In particular, the molecules that guide myotube leading edges to their tendon attachment sites, a process we termed myotube pathfinding, remain largely unknown. In addition, the mechanisms by which myotubes respond to extracellular signals are still unclear. Our forward genetic screens and transcriptional profiling studies in Drosophila identified Fibroblast Growth Factors (FGFs) as key guidance molecules for myotube pathfinding. In vertebrates, nascent myotubes express FGF receptors and transduce FGF signals, but a functional role for FGF signaling during myotube morphogenesis has not been characterized. In fact, the mechanisms by which individual mammalian muscles acquire their unique size, shape, and morphology are largely unknown. The specific hypothesis for this application is that FGF ligands act as guidance cues during mammalian myogenesis that target myotube leading edges to tendon attachment sites.

Aim #1: Characterize the in vivo role of the FGF pathway during muscle morphogenesis. FGF signaling directs myoblast proliferation and migration, but the role of the FGF pathway during myotube morphogenesis is unknown. Four FGF receptors (FGFRs) have been identified in vertebrates, and nascent myotubes express FGFR1, FGFR2, and FGFR4. As an entry point to characterize the role of FGF signaling during myotube pathfinding in mice, we will use existing FGFR alleles and a muscle-specific CreER to create triple conditional knockout myotubes (FGFR1floxed/floxed, FGFR2floxed/floxed, FGFR4floxed/floxed, MyogCreERT2+/-; hereafter FGFRTCKO). CreER activity will be induced at two embryonic time points, and we will assess muscle size and morphology in FGFRTCKO neonates. These studies will provide the first functional insights into the roles of FGF signaling during myotube morphogenesis.

Aim #2: Establish an in vitro platform to probe mechanisms of myotube pathfinding. Despite the cellular and molecular parallels between axon guidance and myotube pathfinding, in vitro techniques have yet to be developed to test the function of putative guidance cues in the context of myotube morphogenesis. We will apply microdevice technologies established for the study of axon guidance toward understanding the role of FGF signaling during myotube pathfinding in vitro. These tools will complement our in vivo studies of mammalian myogenesis in the short term, and will serve as a productive screening platform to identify myogenic mechanisms and characterize myopathy disease variants in the long term.