Gabrielle Kardon - US grants

[unreadable] DESCRIPTION (provided by applicant): Development of the vertebrate musculoskeletal system requires the coordinated morphogenesis of muscle, muscle connective tissue, tendon, and skeleton. In the limb, muscle derives from migratory precursors originating from the somites, while the muscle connective tissue, tendons and skeletal elements derive from the mesoderm of the emerging lateral-plate derived limb bud. As the muscle precursors migrate into the limb, they must differentiate into myofibers, become correctly patterned into distinct anataomical muscles, and be assemebled into a functional musculoskeleton. How the over 40 limb muscles are patterned and muscle and muscle connective tissue morphogenesis coordinated is the subject of this proposal. Classical studies had suggested that lateral plate, limb mesodermal signals are important patterning muscle. However, neither the molecular nature of the signal nor the tissue producing it was known. Recently we have identified a population of lateral plate, limb mesodermal cells that expresses the transcription factor Tcf4, a downstream effector of the Wnt/beta-catenin signaling pathway, and that is critical for muscle patterning. Functional studies in the chick suggest that Tcf4-expressing cells establish a prepattern in the limb mesoderm that determines where muscle precursors differentiate and thus where individual muscles will form and the ultimate limb muscle pattern. In addition, preliminary studies indicate that Tcf4-expressing cells are the precursors of the muscle connective tissue, a tissue of fundamental importance to the form and function of the musculoskeleton and whose development has been largely unstudied because of the lack of early molecular markers. We propose to test genetically in the mouse the role of limb mseodermal Tcf4- expressing cells and Wnt/beta-catenin signaling in determining the pattern of limb muscles. In addition, we will determine whether Tcf4-expressing cells are precursors necessary for formation of muscle connective tissue. These experiments will provide important insights into the normal development of muscle and muscle connective tissue and the role of Wnt/beta-catenin signaling in regulating their development. Disruptions in the development of muscle and muscle connective tissues can result in severe musculoskeltal disorders, such as Duchenne's muscular dystrophy and Ullrich congenital muscular dystrophy. Results from our experiments will give us important new insights into the etiology of these diseases. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Development of the vertebrate musculoskeletal system requires the coordinated morphogenesis of muscle, muscle connective tissue, tendon, and skeleton. In the limb, muscle derives from migratory precursors originating from the somites, while the muscle connective tissue, tendons, and skeletal elements derive from the mesoderm of the emerging lateral plate-derived limb bud. As muscle precursors migrate into the limb, they differentiate into myofibers, become correctly patterned into distinct muscles, and are assembled with muscle connective tissue, tendons, and bones into a functional musculoskeleton. How muscles and tendons are patterned and assembled with muscle connective tissue is largely unknown, defective in multiple human genetic syndromes, and the subject of this proposal. The close developmental association of muscle and tendon with connective tissue suggests that interactions between these tissues may be critical for their development. However, study of muscle connective tissue has been hindered by the lack of molecular markers and genetic reagents to label connective tissue fibroblasts. We identified that the transcription factor Tcf4 (Tcf7L2) is strongly expressed in adult connective tissue fibroblasts and in their precursors in the embryo. During the previous grant period, we engineered Tcf4GFPCre and Tcf4CreERT2 mice, the first reagents to allow for genetic manipulation of these fibroblasts. Our preliminary studies of Tcf4 function suggest that the connective tissue regulates muscle and tendon morphogenesis in the developing limb. In addition, we and our collaborators have found that Lmx1b, Tbx3, Tbx4, and Tbx5 are key transcription factors that establish the dorsal/ventral, anterior/posterior, and fore/hindlimb asymmetries of the limb and determine the pattern of limb muscles and tendons. Preliminary studies suggest that these transcription factors regulate muscle and tendon morphogenesis non-cell autonomously via their function in muscle connective tissue. Here we propose to test the hypothesis that connective tissue, via Tcf4, Lmx1b, Tbx3, Tbx4, and Tbx5 function, determines the pattern of limb muscles and tendons. These experiments will elucidate the cell-cell and molecular interactions necessary to coordinate and assemble an integrated and functional musculoskeletal system. Furthermore, mutations in Lmx1b, Tbx3, Tbx4, and Tbx5 have been identified as the genetic causes of human Nail-Patella, Ulnar-Mammary, Small Patella, and Holt-Oram syndromes, characterized by severe abnormalities in limb muscle, tendon, and skeleton. How these genetic mutations cause these musculoskeletal defects is unknown. Our analysis of mouse mutants of these genes, which phenocopy the human syndromes, will elucidate the cellular and molecular defects that lead to these limb musculoskeletal abnormalities.

The diaphragm is an essential mammalian skeletal muscle, as it is vital for respiration and serves as a barrier between the thoracic and abdominal cavities. Development of the diaphragm requires the integration of multiple tissues that derive from several embryonic sources. Defects in diaphragm development are the cause of congenital diaphragmatic hernias (CDHs), a common birth defect (1:3000 births) that results in severe morbidity and 50% mortality. Given the diaphragm's functional importance and the frequency and severity of CDH, an understanding of diaphragm development normally and during herniation is critical. Recently, using mouse genetics, we definitively established that the pleuroperitoneal folds, transient embryonic structures, and the muscle connective tissue fibroblasts derived from them critically regulate development of the diaphragm muscle (Merrell et al. 2015). Furthermore, we showed that mutations in these fibroblasts cause CDH. However, the molecular signals from the fibroblasts that regulate muscle development normally and are defective in CDH are not yet known. Based on preliminary studies, we hypothesize that connective tissue fibroblasts are an important source of secreted signals that recruit muscle progenitors into the developing diaphragm; regulate muscle morphogenesis; and are mis-regulated in CDH. In addition, our mouse studies (Merrell et al. 2015) suggest the novel hypothesis that somatic mosaic mutations in connective tissue fibroblasts are critical for the etiology of CDH ? a hypothesis that may explain the genetic complexity and phenotypic variability of CDH. We propose to use mouse genetic studies and CDH patient samples to test these hypotheses. Our research will elucidate the genetic, molecular, and cellular mechanisms regulating the development of the diaphragm and CDH and provide important insights into potential therapeutic targets to treat CDH.