The diaphragm is an essential skeletal muscle, playing a critical role in respiration and serving as a barrier that separates the thoracic and abdominal cavities. Development of the diaphragm requires the integration of muscle, connective tissue, tendon, bone, and nerves that arise from different embryonic sources. Defects in muscularization of the diaphragm cause congenital diaphragmatic hernias (CDH), a common birth defect (1 in 3,000 births) where the liver herniates into the thorax, impeding lung development and resulting in a 50% neonatal mortality rate. Diaphragm development entails the migration of muscle precursors from the somites and the extension phrenic nerve axons (the sole source of innervation) to connective tissue progenitors. However, in spite of the high incidence of CDH and functional importance of the diaphragm, the cellular interactions and molecular signals directing muscle migration and axon guidance required for diaphragm muscularization and innervation are largely unknown and the subject of this proposal. Our lab has recently demonstrated that interactions between muscle and connective tissue are crucial for normal development of the diaphragm muscle and defects in this interaction are a source of CDH. The muscle connective tissue originates from transient embryonic structures, termed the pleuroperitoneal folds (PPFs). This project focuses on the role of PPFs and, in particular, Hepatocyte Growth Factor (HGF) secreted by the PPFs, in guiding muscle progenitors and the phrenic nerve to the developing diaphragm. Preliminary data showing the expression of Met in the diaphragm's muscle progenitors and Hgf in PPF fibroblasts suggests that PPF-derived HGF is important for recruiting MET+ muscle progenitors into the developing diaphragm. However, an earlier MET requirement for delamination of progenitors from the somites has precluded an explicit test of this hypothesis.
Aim 1 will determine which somites are the source of diaphragm muscle and test the hypothesis that HGF is an important PPF-derived secreted signal that critically regulates migration of muscle into and throughout the diaphragm. By conditional deletion of Hgf in PPFs after muscle progenitors have emigrated from the somite, I will test the role of HGF/MET signaling in muscle progenitor migration into the diaphragm. Preliminary data suggest that loss of PPF-derived Hgf disrupts normal muscularization and guidance of phrenic nerve axons into the diaphragm.
Aim 2 will test the hypothesis that PPF and muscle derived signals are required for guiding phrenic nerve axons to and throughout the diaphragm. I will determine whether the pleuroperitoneal folds have chemoattractant properties in culture and characterize how the depletion of secreted signals, including Hgf, affect the guidance of phrenic nerve axons. Taken together, these studies exploit the powerful genetics of mice to define the role of connective tissue progenitors in muscle migration and axon guidance to the developing diaphragm and establish a basis for understanding how connective tissue defects can contribute to congenital diaphragmatic hernias.

Public Health Relevance

Defects in the development of the diaphragm, a vital skeletal muscle required for respiration, are the source of a common defect with a 50% mortality rate termed congenital diaphragmatic hernias (CDH). This study uses the mouse as a model system to characterize the role of connective tissue in coordinating the migration of muscle progenitors and the guidance of axons to properly form and innervate the diaphragm. Our findings will provide new directions for understanding the genetic and cellular basis of diaphragm development and CDH.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Postdoctoral Individual National Research Service Award (F32)
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Special Emphasis Panel (ZRG1)
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Toyama, Reiko
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University of Utah
Schools of Medicine
Salt Lake City
United States
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Sefton, Elizabeth M; Gallardo, Mirialys; Kardon, Gabrielle (2018) Developmental origin and morphogenesis of the diaphragm, an essential mammalian muscle. Dev Biol 440:64-73