Gabrielle Kardon

SUMMARY: The function of many organs, including skeletal muscle, depends on its three-dimensional structure. Muscle regeneration therefore requires not only reestablishment of myofibers, but restoration of tissue architecture. Resident muscle stem cells (SCs) are essential for regeneration, but how SCs regenerate muscle architecture is largely unknown. We address this problem using genetic labeling of SCs and whole mount imaging to reconstruct in three dimensions muscle regeneration. Unexpectedly, we found that the residual basement membrane of necrotic myofibers is critical for promoting fusion and orienting regenerated myofibers and myofibers form via two distinct phases of fusion. Furthermore, the centralized myonuclei characteristic of regenerated myofibers are associated with myofibrillogenesis and permanently mark regenerated myofibers. Finally, we elucidate a new cellular mechanism for the formation of branched myofibers, a pathology characteristic of diseased muscle. We provide a new synthesis of the cellular events of regeneration and show these differ from those used during development.

The diaphragm is a domed muscle between the thorax and abdomen essential for breathing in mammals. Diaphragm development requires the coordinated development of muscle, connective tissue, and nerve, which are derived from different embryonic sources. Defects in diaphragm development cause the common and often lethal birth defect, congenital diaphragmatic hernias (CDH). HGF/MET signaling is required for diaphragm muscularization, but the source of HGF and the specific functions of this pathway in muscle progenitors and effects on phrenic nerve have not been explicitly tested. Using conditional mutagenesis in mice and pharmacological inhibition of MET, we demonstrate that the pleuroperitoneal folds (PPFs), transient embryonic structures that give rise to the connective tissue in the diaphragm, are the source of HGF critical for diaphragm muscularization. PPF-derived HGF is directly required for recruitment of MET+ muscle progenitors to the diaphragm and indirectly (via its effect on muscle development) required for phrenic nerve primary branching. In addition, HGF is continuously required for maintenance and motility of the pool of progenitors to enable full muscularization. Localization of HGF at the diaphragm’s leading edges directs dorsal and ventral expansion of muscle and regulates its overall size and shape. Surprisingly, large muscleless regions in HGF and Met mutants do not lead to hernias. While these regions are likely more susceptible to CDH, muscle loss is not sufficient to cause CDH.

Vertebrate skeletal muscle is composed of multinucleate myofibers that are surrounded by muscle connective tissue. Following injury, muscle is able to robustly regenerate because of tissue-resident muscle stem cells, called satellite cells. In addition, efficient and complete regeneration depends on other cells resident in muscle – including fibro-adipogenic progenitors (FAPs). Increasing evidence from single-cell analyses and genetic and transplantation experiments suggests that satellite cells and FAPs are heterogeneous cell populations. Here, we review our current understanding of the heterogeneity of satellite cells, their myogenic derivatives and FAPs in terms of gene expression, anatomical location, age and timing during the regenerative process – each of which have potentially important functional consequences.

The diaphragm is a domed muscle between the thorax and abdomen essential for breathing in mammals. Diaphragm development requires the coordinated development of muscle, connective tissue, and nerve, which are derived from different embryonic sources. Defects in diaphragm development cause the common and often lethal birth defect, Congenital Diaphragmatic Hernias (CDH). HGF/MET signaling is required for diaphragm muscularization, but the source of HGF and the specific functions of this pathway in muscle progenitors or potentially the phrenic nerve have not been explicitly tested. Using conditional mutagenesis and pharmacological inhibition of MET, we demonstrate that the pleuroperitoneal folds (PPFs), transient embryonic structures that give rise to the connective tissue, are the source of HGF critical for diaphragm muscularization and phrenic nerve primary branching. HGF not only is required for recruitment of muscle progenitors to the diaphragm, but is continuously required for maintenance and motility of the pool of progenitors to enable full muscularization. Thus, the connective tissue fibroblasts and HGF coordinately regulate diaphragm muscularization and innervation. Defects in PPF-derived HGF result in muscleless regions that are susceptible to CDH. Summary Statement: Fibroblast-derived HGF signals to Met+ muscle progenitors and nerve to control the expansion of diaphragm muscle and primary branching of phrenic nerve axons - structures critical for breathing in mammals.

The diaphragm is a mammalian muscle critical for respiration and separation of the thoracic and abdominal cavities. Defects in the development of the diaphragm are the cause of congenital diaphragmatic hernia (CDH), a common birth defect. In CDH, weaknesses in the developing diaphragm allow abdominal contents to herniate into the thoracic cavity and impair lung development, leading to a high neonatal mortality. The genetic etiology of CDH is complex. Single nucleotide variants (SNVs), insertion/deletions (indels), and structural/copy number variants in more than 150 genes have been associated with CDH, although few genes are recurrently mutated in multiple patients and recurrently mutated genes can be incompletely penetrant. This suggests that multiple genetic variants in combination, other not yet investigated classes of variants, and/or nongenetic factors contribute to CDH susceptibility. However, to date no studies have comprehensively investigated the contribution of all possible classes of variants throughout the genome to the etiology of CDH. In our study, we used a unique cohort of four patients with isolated CDH with samples from blood, skin, and diaphragm connective tissue and parental blood samples and deep whole genome sequencing to assess germline and somatic de novo and inherited variants of various sizes (SNVs, indels, and structural variants) in exons, introns, UTRs, and intergenic regions. In each patient we found a different mutational landscape that included germline de novo, and inherited SNVs and indels in multiple genes. We also found in two patients an inherited 343 bp deletion interrupting an annotated enhancer of the CDH associated gene, GATA4 , and we hypothesize that this common deletion (found in 1-2% of the population) acts as a sensitizing allele for CDH. Overall, our comprehensive reconstruction of the genetic architecture of four CDH individuals demonstrates that the etiology of CDH is heterogeneous and multifactorial. AUTHOR SUMMARY: Deep whole genome sequencing of family trios shows that etiology of congenital diaphragmatic hernias is heterogeneous and multifactorial.

The mammalian muscularized diaphragm is essential for respiration and defects in the developing diaphragm cause a common and frequently lethal birth defect, congenital diaphragmatic hernia (CDH). Human genetic studies have implicated more than 150 genes and multiple molecular pathways in CDH, but few of these have been validated because of the expense and time to generate mouse mutants. The pleuroperitoneal folds (PPFs) are transient embryonic structures in diaphragm development and a critical cellular source of CDH. We have developed a system to culture PPF fibroblasts from E12.5 mouse embryos and show that these fibroblasts, in contrast to the commonly used NIH 3T3 fibroblasts, maintain expression of key genes in normal diaphragm development. Using pharmacological and genetic manipulations that result in CDH in vivo , we also demonstrate that differences in proliferation provide a rapid means of distinguishing healthy and impaired PPF fibroblasts. Thus, the PPF fibroblast cell culture system is an efficient tool for testing the functional significance of CDH candidate genes and molecular pathways and will be an important resource for elucidating the complex etiology of CDH. SUMMARY STATEMENT: Multiple candidate genes and molecular pathways have been identified as potentially contributing to the etiology of congenital diaphragmatic hernias. We describe a cell culture system to efficiently functionally test these candidates.