Developmental origin and morphogenesis of the diaphragm, an essential mammalian muscle

Abstract

The diaphragm is a mammalian skeletal muscle essential for respiration and for separating the thoracic and abdominal cavities. Development of the diaphragm requires the coordinated development of muscle, muscle connective tissue, tendon, nerves, and vasculature that derive from different embryonic sources. However, defects in diaphragm development are common and the cause of an often deadly birth defect, Congenital Diaphragmatic Hernia (CDH). Here we comprehensively describe the normal developmental origin and complex spatial-temporal relationship between the different developing tissues to form a functional diaphragm using a developmental series of mouse embryos genetically and immunofluorescently labeled and analyzed in whole mount. We find that the earliest developmental events are the emigration of muscle progenitors from cervical somites followed by the projection of phrenic nerve axons from the cervical neural tube. Muscle progenitors and phrenic nerve target the pleuroperitoneal folds (PPFs), transient pyramidal-shaped structures that form between the thoracic and abdominal cavities. Subsequently, the PPFs expand across the surface of the liver to give rise to the muscle connective tissue and central tendon, and the leading edge of their expansion precedes muscle morphogenesis, formation of the vascular network, and outgrowth and branching of the phrenic nerve. Thus development and morphogenesis of the PPFs is critical for diaphragm formation. In addition, our data indicate that the earliest events in diaphragm development are critical for the etiology of CDH and instrumental to the evolution of the diaphragm. CDH initiates prior to E12.5 in mouse and suggests that defects in the early PPF formation or their ability to recruit muscle are an important source of CDH. Also, the recruitment of muscle progenitors from cervical somites to the nascent PPFs is uniquely mammalian and a key developmental innovation essential for the evolution of the muscularized diaphragm.

Connecting muscle development, birth defects, and evolution: An essential role for muscle connective tissue

“…The diaphragm’s MCT comes from the pleuroperitoneal folds (PPFs), a lateral plate-derived pyramidal tissue located between the thoracic (pleural) and abdominal (peritoneal) cavities (Figure 6A). Histological, immunofluorescent, and genetic lineage studies in mice reveal that the PPFs expand dorsally and ventrally to give rise to the diaphragm’s MCT and central tendon (Figure 6; Babiuk et al, 2003; Merrell et al, 2015; Sefton et al, 2018). The muscle progenitors migrate from the cervical somites to the PPFs, and as the PPFs spread the muscle cells are carried throughout the nascent diaphragm and differentiate into the radially oriented myofibers of the costal diaphragm in close association with the MCT (Figure 6; Merrell et al, 2015; Sefton et al, 2018).…”

“…One way to elucidate the evolutionary origin of the diaphragm is to compare the developmental innovations present in mammals, but absent from birds and reptiles, which do not have a muscularized diaphragm. While cervical-level somites give rise to neck and limb muscles in birds, reptiles, and mammals, it is only in mammals that muscle progenitors migrate to the nascent diaphragm (Hirasawa et al, 2015; Hirasawa and Kuratani, 2013; Sefton et al, 2018). Thus recruitment of muscle progenitors from cervical-level somites to this region is a crucial event uniquely present in mammals.…”

“…Thus recruitment of muscle progenitors from cervical-level somites to this region is a crucial event uniquely present in mammals. In mammals, the PPFs not only are the target of the migrating muscle progenitors, but they subsequently guide muscle morphogenesis (Babiuk et al, 2003; Hitachi et al, 2009; Merrell et al, 2015; Sefton et al, 2018). This suggests that the PPFs, which give rise to the diaphragm’s MCT, may be the developmental innovation that allowed for the formation of a muscularized diaphragm.…”

Prenatal regenerative fetoscopic interventions for congenital anomalies

“…Formation of the diaphragm occurs between the fourth and 10th week of gestation 22. The course of CDH occurs in the setting of aberrant formation of the pleuroperitoneal folds or the post-hepatic mesenchymal plate 2223. The predominant complications associated with CDH are pulmonary hypoplasia and pulmonary arterial hypertension 2425.…”

Exploring use of incorrect terminology used in medical sciences: quest for scientific and academic verity

“…There is some evidence that the PPFs originate in the cervical region (Hirasawa et al, 2016), but nascent PPFs have never been explicitly labelled and their early development tracked. The somites are the source of the diaphragm's muscle, and muscle progenitors emigrate from the somites into the PPFs (Merrell et al, 2015), (Sefton, 2018). The diaphragm thus is better described as having migrated rather than having descended.…”