The mammalian respiratory system is unique among living tetrapods in its inclusion of the highly lobated alveolar lung and the diaphragm, both of which contribute to efficiency in oxygen uptake (Perry, 1983, 1998). Although a trade-off between a large surface area for diffusion capacity and the low compliance of the mammalian lung might be expected, the diaphragm – an autapomorphic muscle for the mammals – enables ventilation of the low-compliant lung, thereby circumventing this trade-off (Perry et al. 2009). The evolutionary origin of the diaphragm therefore was crucial for the mammalian lineage, which has benefited from a higher metabolic rate.
The diaphragm is a fairly uniform structure among living mammalian species, in which skeletal muscle fibers span the thoracic cavity transversely at the thoraco-lumbar boundary (Perry et al. 2000, 2010; also see Rommel & Reynolds, 2000, for the manatee diaphragm). The muscle fibers of the diaphragm originate from the lumbar vertebrae, ribs and sternum and insert into the central tendon. This uniformity among mammals and the absence of this muscle from non-mammalian tetrapods have been an obstacle to comparative anatomy in unraveling the evolutionary origin of the diaphragm. However, recent advances in developmental biology have provided new insights into the evolution of the diaphragm. Here we integrate current knowledge regarding the embryonic development of the diaphragm with the evolutionary history of this structure according to the fossil record.
The embryonic development of the diaphragm: an outline
The development of the diaphragm proceeds in two partially overlapping phases: namely the formation of the membranous scaffold and the migration and the differentiation of myogenic cells on the scaffold (Lewis, 1910; Mall, 1910; Wells, 1954; Jinguji & Takisawa, 1983; Greer et al. 1999; Babiuk et al. 2003). The scaffold for the diaphragm is formed by a fusion of protrusions toward the coelomic cavity on the body wall. These protrusions involve the transverse septum and the pleuroperitoneal folds (PPFs, or pulmonary ridges; Lewis, 1910; Fig. 1). Because the embryonic development of the diaphragm has recently been studied using rat embryos, we describe the time sequence of the development of the diaphragm in this animal (Fig. 2).
The PPFs develop bilaterally on the dorsal part of the lateral body wall in the E12.5 embryo (Clugston et al. 2010). At this stage, the PPFs are located just caudal to the common cardinal vein (ductus cuvieri; Kollmann, 1907; Goodrich, 1930). Analyses regarding the migration of the muscle precursor cells and the elongation of the phrenic nerve axons suggest these PPFs are the primordial target of diaphragm (Allan & Greer, 1997a).
The myogenic cells for the diaphragm are likely to be a subpopulation of the migratory cells later entering the forelimb (Babiuk et al. 2003). At E12.5, muscle precursor cells migrate toward the brachial plexus but are still distributed within the lateral body wall (Clugston et al. 2010). At E13.0, the myogenic cells enter the PPFs, and at E13.5, phrenic axons enter the PPFs, passing along the lateral aspect of the ductus cuvieri. Concomitantly, the lung bud grows in the pleuroperitoneal canal, surrounded mainly by the body wall and liver. By E13.5, the caudal end of the lung bud reaches the dorsal aspect of the liver (Gattone & Morse, 1984). As the liver and suprarenal gland become larger, the pleuroperitoneal canal becomes narrower, eventually closing to complete the diaphragm by E15.2. Simultaneously, the body wall is elongated to encapsulate the heart and the diaphragm deep inside the rib cage (Keith, 1905; Jackson, 1909; Mall, 1910; Greer et al. 1999).
Origins of intracoelomic septa
During embryonic development in the gnathostomes, paired protrusions toward the coelomic cavity – the nephric folds – develop, at least transiently, on the medial aspect of the body wall (Goodrich, 1930). The nephric folds are located lateral to the pericardico-peritoneal passage, and therefore are distinguished from other accessory mesenteries, which are located medial to the pericardico-peritoneal passage. While, in the non-amniotes, the nephric folds do not contribute to subdividing the coelomic cavities; in many lineages of amniotes, the cranial parts of the nephric folds fuse with the transverse septum and dorsal mesentery to form complete subdivisions, namely the intracoelomic septa (Goodrich, 1930; Duncker, 1978, 1979; Klein & Owerkowicz, 2006). The PPF of mammals is contiguous with the cranial part of the nephric fold (Goodrich, 1930). Although this mode of development is shared among the amniotes that have intracoelomic septa, the recurrent distribution of intracoelomic septa in the phylogeny indicates that intracoelomic septa were acquired paraphyletically (Duncker, 1979; Klein & Owerkowicz, 2006).
The intracoelomic septa are membranous in most amniotes, but some lineages independently evolved muscle fibers on the edges of (turtles, crocodilians, and birds) or almost throughout (mammals and teiid lizards) the intracoelomic septa (Duncker, 1979; Klein et al. 2003; Klein & Owerkowicz, 2006; Perry et al. 2009). Despite being incapable of voluntary movement, nonmuscular intracoelomic septa may facilitate ventilation by resisting a paradoxical visceral translocation and supporting the caudal portion of the lung. For this reason, intracoelomic septa would have been acquired by and retained in various lineages (Klein & Owerkowicz, 2006). Accordingly, the membranous scaffold for the diaphragm was most likely derived from such an intracoelomic septum (Perry, 1983; Perry et al. 2010).
From the perspective of developmental biology, studies of the congenital diaphragmatic hernia (CDH) suggest the developmental basis of the PPF. Recent studies have shown that the Gata4–Fog2 transcriptional complex, Coup-tfII, and Wt1 are coexpressed in the nonmuscular cell population within the PPF and contribute to the proper formation of the diaphragm (Ackerman et al. 2005; Clugston et al. 2008; Yu et al. 2013). These transcription factors are expressed exclusively in the PPFs and the developing lung, suggesting these tissues develop under shared transcriptional control, unlike the rest of the body wall or the limb bud. This inference is supported by results from the nitrofen-induced CDH model, in which the retinoid signaling pathway (which is upstream of the Gata4–Fog2 transcriptional pathway) is disrupted (Clugston et al. 2006, 2010; Noble et al. 2007). A recent analysis using a whole-transcriptome expression profile identified PBX1, which directs retinoic acid production, as another key factor in the proper formation of the diaphragm (Russell et al. 2012).
Many studies suggest that the Gata4–Fog2 transcriptional pathway controls the bronchoalveolar development of the lung, in addition to the development of the diaphragm (Chinoy, 2002; Ackerman et al. 2005, 2007; Jay et al. 2007; Kantarci & Donahoe, 2007; Morrisey & Hogan, 2010). It is intriguing that the two features specific to the mammalian pulmonary system, namely the bronchoalveolar lung and the diaphragm, may be subject to the same developmental control. Because the other lineage of the amniota (i.e. diapsids) possesses neither bronchoalveolar lung nor the diaphragm, the most parsimonious explanation is that these traits evolved only in the synapsids (Perry, 1983). We hypothesize that the incorporations of the Gata4–Fog2 transcriptional complex into the developments of the lung and the PPF were linked with each other in mammalian respiratory evolution. Investigations into the genetic bases of the intracoelomic septa in other taxa will likely increase our understanding of the marked morphological difference between the PPFs and other intracoelomic septa.
Migratory muscle precursor cells
According to our current understanding, the muscles of the tongue, diaphragm, and limbs in amniotes are derived from cells that migrate a long distance from the ventral part of the dermomyotome while under the control of the SF/HGF and c-Met signaling pathway (Dietrich et al. 1998, 1999; Alvares et al. 2003; Vasyutina & Birchmeier, 2006). Because PAX3 plays an indispensable role during the migration of these myogenic cells (the migratory muscle precursors, MMPs; Tremblay et al. 1998; Li et al. 1999; Buckingham et al. 2006; Buckingham & Relaix, 2007), their mode of migration can be traced by visualizing Pax3 expression. This method was used to show that the muscular part of the diaphragm is likely derived from the subpopulation of cells that later migrates into the forelimb bud (Babiuk et al. 2003). This result is consistent with the previous observation that the ‘diaphragm premuscle mass’ is contiguous with the ‘pectoral premuscle mass’ during embryonic development (Lewis, 1902; Jinguji & Takisawa, 1983). There is no evidence for the contribution of other cell populations, including those of the lateral body wall, to the muscular part of the diaphragm (Babiuk et al. 2003).
Recent detailed analysis of the mode of migration of MMPs in the pectoral region revealed that, at least in osteichthyes, Tbx5 expression is a prerequisite for the development of the pectoral girdle (Valasek et al. 2011). This developmental control by Tbx5 is known as the ‘forelimb programme’ (Valasek et al. 2011). Tbx5 also is a prerequisite for the diaphragmatic development, leading the claim that the diaphragm develops under the forelimb programme (Valasek et al. 2011). Another insight into the intimate relationship between the diaphragm and forelimb muscles came from an analysis of mutant mice. In addition to SF/HGF and c-Met pathway, Lbx1 also plays a role in the migration of MMPs, particularly regarding lateral rather than ventral distribution (Brohmann et al. 2000; Gross et al. 2000). In Lbx1−/− mice, the forelimb muscles are poorly developed but part of the diaphragm is hypertrophic, compared with the structures in wild-type mice (Gross et al. 2000). The best explanation for the phenotype of the Lbx−/− mice is that some of the cells that normally migrate into the forelimb bud instead enter the PPFs because the lateral migration of MMPs is severely compromised by the absence of Lbx1.
The paths of the phrenic nerves and routes of the MMPs
The diaphragm is innervated by the phrenic nerve, which (in most mammals) arises from spinal nerves C3–C5 (mainly C4 and C5; Nauck, 1939). The phrenic nerve shows only minor interspecific variation, although several exceptions (e.g. incorporation of spinal nerve C6 in the Old World porcupine Hysterix, pangolin Manis, and dromedary camel Camelus dromedarius) have been reported (Kohlbrugge, 1898; Smuts & Bezuidenhout, 1987). Incorporation of the C6 nerve into the phrenic nerve has also been reported as a variation in human anatomy (Kerr, 1918).
During embryonic development, the phrenic nerve grows along the route of the MMPs into the PPFs (Jinguji & Takisawa, 1983; Greer et al. 1999; Babiuk et al. 2003); the path of the phrenic nerve therefore may reflect the route of MMPs. In addition, data regarding the phrenic nerve highlight the intimate developmental relationship between the diaphragm and forelimb muscles (Fig. 2).
In rats, the phrenic and brachial axons emerge from the cervical spinal cord at E11.5 and merge into the brachial plexus at the base of the forelimb bud at E12.5 (Allan & Greer, 1997b). Subsequently, the ‘pioneering’ phrenic axon emerges from the brachial plexus and enters the PPF by E13.0. This pioneering phrenic axon does not branch until the beginning of myotube formation at the PPF (E14.5). After emergence from the brachial plexus, the axonal guidance of the phrenic nerve is under the control of the netrin signaling pathway and is independent of that of the forelimb nerves (Burgess et al. 2006); however, the detailed signaling pathway remains unclear.
Variations of the phrenic nerve in humans comprise mainly three types, showing abnormal communication with the suprascapular nerve (Kodama et al. 1992), the subclavian nerve (Kerr, 1918; Banneheka, 2008) or the cervical ansa (Kikuchi, 1970; Goto et al. 1976; Tanaka et al. 1988). The communications with the suprascapular and subclavian nerves are consistent with the developmental relationship between the diaphragm and forelimb muscles, whereas the communication with the cervical ansa suggests affinity with the infrahyoid (hypobranchial) muscles. It bears mention that the phrenic nerve of monotremes receives two thin contributions from the subclavian nerve (McKay, 1894).
In light of the possible close relationship between the diaphragm and forelimb muscles during embryonic development, we chose to focus on patterns (compositions and topologies) and positions of brachial plexuses, which reflect the routes of forelimb MMPs, to infer the evolutionary scenario of the diaphragm. First, we evaluated the brachial plexuses of extant amniotes to confirm their shared pattern among these species and their position relative to the cervico-thoracic transition in the axial skeleton. Secondly, using fossils, we reconstructed the evolutionary changes of the cervico-thoracic transition at the axial level, to reveal shifts in the position of the brachial plexus during evolution toward mammals. Considering these shifts in position and the mammal-specific pattern of the brachial plexus (including the phrenic nerve), we discuss a possible scenario for the evolution of the diaphragm.