Pleuroperitoneal Folds Research Articles

Muscle connective tissue controls development of the diaphragm and is a source of congenital diaphragmatic hernias.

The diaphragm is an essential mammalian skeletal muscle, and defects in diaphragm development are the cause of congenital diaphragmatic hernias (CDH), a common and often lethal birth defect. The diaphragm is derived from multiple embryonic sources, but how these give rise to the diaphragm is unknown and, despite the identification of many CDH-associated genes, the etiology of CDH is incompletely understood. Using mouse genetics, we show that the pleuroperitoneal folds (PPFs), transient embryonic structures, are the source of the diaphragm’s muscle connective tissue, regulate muscle development, and their striking migration controls diaphragm morphogenesis. Furthermore, Gata4 mosaic mutations in PPF-derived muscle connective tissue fibroblasts result in the development of localized amuscular regions that are biomechanically weaker and more compliant and lead to CDH. Thus the PPFs and muscle connective tissue are critical for diaphragm development and mutations in PPF-derived fibroblasts are a source of CDH.

The association between congenital diaphragmatic hernia and undescended testes

BackgroundUndescended testes (UDT) is a common abnormality treated by pediatric surgeons. Embryological development of the genitourinary ridge is in close proximity with the pleuroperitoneal fold. The purpose of this paper is to describe the association between congenital diaphragmatic hernia (CDH) and UDT. Materials/methodsAs part of the DHREAMS (Diaphragmatic Hernia Research and Exploration: Advancing Molecular Science) study (www.cdhgenetics.com), all living children had tissue banked and analyzed for common genetic mutations and had a health assessment performed by telephone consultation with the parents at two years of age. The incidence of UDT was then compared to clinical and genetic findings previously identified. ResultsSixty-five males had complete information from their 2year health assessment. Of these, twelve (18%) had a UDT repaired by the time of the 2year assessment. Of the twelve who had a repair, no child had a unilateral UDT which was contralateral to the side of the CDH. There were no differences in rate or number of mutations of any of the genes we checked as part of our study. ConclusionIt appears that a deficiency of diaphragm tissue may affect the first or transabdominal phase of the testicular descent, leading to an increased incidence of UDT.

Myogenin gene expression is not altered in the developing diaphragm of nitrofen-induced congenital diaphragmatic hernia.

Pleuroperitoneal folds (PPFs) represent the only source of muscle precursors cells (MPCs) in the primordial diaphragm. However, the exact pathogenesis of malformed PPFs and congenital diaphragmatic hernia (CDH) remains unclear. The muscle-specific transcription factor myogenin plays a key role during development and muscularization of the fetal diaphragm. Although myogenin knockout mice lack skeletal muscle fibers, the diaphragmatic musculature is intact without any defects. It has further been demonstrated that proliferation and differentiation of MPCs in PPFs and developing diaphragms are normal in rodent CDH models. We hypothesized that myogenin gene expression is not altered in malformed PPFs, developing diaphragms and diaphragmatic musculature in the nitrofen-induced CDH model. Pregnant rats were exposed to nitrofen or vehicle on gestational day 9 (D9). Fetuses were harvested during PPF formation (D13), diaphragmatic development (D14-15) and muscularization (D18-21). Fetal PPFs, developing diaphragms and diaphragmatic musculature were dissected and divided into nitrofen and control groups. Myogenin mRNA levels were analyzed by quantitative real-time polymerase chain reaction, while immunohistochemistry was performed to investigate myogenin protein expression and distribution. Relative mRNA expression of myogenin was not significant different in PPFs (0.30±0.09 vs. 0.48±0.09; P=0.37), developing diaphragms (1.25±0.29 vs. 1.60±0.32; P=0.53) and diaphragmatic musculature (1.08±0.24 vs. 1.59±0.20; P = 0.15) of nitrofen-exposed fetuses compared to controls. Myogenin immunoreactivity was not altered in the muscular components of malformed PPFs, developing diaphragms and diaphragmatic musculature of nitrofen-exposed fetuses compared to controls. Myogenin gene expression is not altered in PPFs, developing diaphragms and diaphragmatic musculature in the nitrofen-induced CDH model, thus suggesting that diaphragmatic defects in this model develop independent of myogenic processes.

Decreased Expression of GATA4 in the Diaphragm of Nitrofen‐Induced Congenital Diaphragmatic Hernia

The molecular mechanisms underlying the diaphragmatic defect in congenital diaphragmatic hernia (CDH) are still poorly understood. The transcription factor GATA4 is essential for normal development of the diaphragm. Recently, mutations in the GATA4 gene have been linked to human and rodent CDH. We hypothesized that diaphragmatic GATA4 expression is downregulated in the nitrofen CDH model. Pregnant rats received Nitrofen or vehicle on day 9 of gestation (D9). Fetuses were sacrificed on D13, D18, or D21. Pleuroperitoneal folds (n=20) and fetal diaphragms (n=40) were (micro) dissected and divided into CDH group and controls. RNA and protein were extracted. GATA4 mRNA levels were determined by real-time PCR. Protein levels were determined by ELISA and Immunohistochemistry. mRNA levels and Protein levels were significantly decreased in the CDH group compared to controls on D13 (mRNA 15.96±6.99 vs. 38.10±5.01, p<0.05), D18 (mRNA 10.45±1.84 vs. 17.68±2.11, Protein 2.59±0.06 vs. 4.58±0.35 p<0.05) and D21 (mRNA 4.31±0.83 vs. 6.87±0.88, Protein 0.16±0.08 vs. 1.26±0.49, p<0.05). Immunoreactivity of GATA4 was markedly decreased in CDH-diaphragms on D13, D18, and D21. We provide evidence for the first time that diaphragmatic expression of GATA4 is downregulated in the nitrofen model, suggesting that decreased expression of GATA4 may impair diaphragmatic development in nitrofen-induced CDH.

Pax3 gene expression is not altered during diaphragmatic development in nitrofen-induced congenital diaphragmatic hernia

Background/PurposeMalformations of the pleuroperitoneal folds (PPFs) have been identified as the origin of the diaphragmatic defect in congenital diaphragmatic hernia (CDH). Pax3, expressed in muscle precursor cells (MPCs), plays a key role in regulating myogenesis and muscularization in the fetal diaphragm. Pax3 mutant mice display absence of muscular diaphragm. However, the distribution of muscle precursor cells is reported to be normal in the PPF of the nitrofen-CDH model. We designed this study to investigate the hypothesis that Pax3 gene expression is unaltered in the PPF and developing diaphragm in the nitrofen-induced CDH model. MethodsPregnant rats were treated with nitrofen or vehicle on gestational day (D) 9 and sacrificed on D13, D18, and D21. Pleuroperitoneal folds (D13) and developing diaphragms (D18 and D21) were dissected, total RNA was extracted, and real-time quantitative polymerase chain reaction was performed to determine Pax3 messenger RNA levels. Confocal immunofluorescence microscopy was performed to evaluate protein expression/distribution of Pax3. ResultsRelative messenger RNA expression levels of Pax3 in PPFs and developing diaphragms were not significantly different in the nitrofen group compared with controls. Intensity of Pax3 immunofluorescence was also not altered in PPFs and developing diaphragms of the nitrofen group compared with controls. ConclusionPax3 gene expression is not altered in the PPFs and developing diaphragm of nitrofen-CDH model, suggesting that the diaphragmatic defect is not caused by disturbance of myogenesis and muscularization.

COUP-TFII Gene Expression is Upregulated in Embryonic Pleuroperitoneal Folds in the Nitrofen-Induced Congenital Diaphragmatic Hernia Rat Model

The nitrofen model of congenital diaphragmatic hernia (CDH) creates a Bochdalek-type diaphragmatic defect and has been widely used to investigate the pathogenesis of CDH. However, the exact pathogenesis of the diaphragmatic defect in this model is still poorly understood. Chicken ovalbumin upstream promotor-transcription factor II (COUP-TFII) is expressed in the embryonic pleuroperitoneal folds (PPF) in the early stage of development and in the diaphragm in the late days of gestation. COUP-TFII is known to be a strong repressor of the retinoid signaling pathway (RSP), which plays an important role in diaphragm development. Furthermore, it has been recently shown that COUP-TFII is upregulated during early gestation in the nitrofen-induced hypoplastic lung. We designed this study to investigate the hypothesis that COUP-TFII gene expression is upregulated during early diaphragmatic development in the PPF. Timed pregnant rats were exposed to either olive oil (Control) or nitrofen (CDH) on day 9 of gestation (D9). Fetuses were sacrificed on D13, D18 or D21. The PPF was dissected from D13 fetuses using laser capture microdissection. Diaphragms were dissected from D18 and D21 fetuses under the dissection microscope. The relative mRNA expression levels of COUP-TFII were determined using real-time PCR. Immunohistochemistry was performed to evaluate diaphragmatic protein expression and the distribution of COUP-TFII.Results On D13, gene expression levels of COUP-TFII in the PPF were significantly increased in the CDH group (82.93 ± 11.85) compared to Controls (46.22 ± 8.09; p < 0.05), whereas there were no differences at later time points. The immunoreactivity of diaphragmatic COUP-TFII was markedly increased in the PPF in the CDH group compared to controls on D13. No difference in immunoreactivity was observed on D18 and D21. Upregulation of COUP-II gene expression in the PPF may contribute to the diaphragmatic defect in the nitrofen CDH model by inhibiting the RSP.

Pleuroperitoneal Canal Closure and the Fetal Adrenal Gland

Pleuroperitoneal canal (PP canal) closure is generally considered to result from an increase in the height, and subsequent fusion, of the bilateral pleuroperitoneal folds (PP folds). However, the folds develop in the area ventral to the adrenal, in contrast to the final position of the diaphragm, which extends to the dorsal side of the adrenal (the "retro-adrenal" diaphragm). We examined the semiserial histology of 20 human embryos and fetuses (crown-rump length 11-40 mm). We started observations of the canal at the stage through which the lung bud extends far caudally along the dorsal body wall to the level of the future adrenal, and the phrenic nerve has already reached the PP fold. Subsequently, the developing adrenal causes narrowing of the dorsocaudal parts of the canal, and provides the bilateral midsagittal recesses or "false" bottoms of the pleural cavity. However, at this stage, the PP fold mesenchymal cells are still restricted to the ventral side of the adrenal, especially along the liver and esophagus. Thereafter, in accordance with ascent of the lung, possibly due to anchoring of the liver to the adrenal, the PP fold mesenchymal cells seem to migrate laterally along the coelomic mesothelium covering some sheet-like loose mesenchymal tissue behind the adrenal. Final closure of the PP canal by lateral migration to provide the "retro-adrenal" diaphragm is a process quite different from the common dogma. It is likely that the sheet-like loose mesenchymal tissue becomes the caudal part of the pleural cavity through a process involving cell death.

Expression of the Wilm’s tumor gene WT1 during diaphragmatic development in the nitrofen model for congenital diaphragmatic hernia

The nitrofen model of congenital diaphragmatic hernia (CDH) reproduces a typical diaphragmatic defect. However, the exact pathomechanism of CDH is still unknown. The Wilm's tumor 1 gene (WT1) is crucial for diaphragmatic development. Mutations in WT1 associated with CDH have been described in humans. Additionally, WT1(-/-) mice display CDH. Furthermore, WT1 is involved in the retinoid signaling pathway, a candidate pathway for CDH. We hypothesized that diaphragmatic WT1 gene expression is downregulated during diaphragmatic development in the nitrofen CDH model. Pregnant rats received vehicle or nitrofen on gestational day 9 (D9). Embryos were delivered on D13, D18 and D21. The pleuroperitoneal folds (PPFs) were dissected using laser capture microdissection (D13). Diaphragms of D18 and D21 were manually dissected. RNA was extracted and relative mRNA expression of WT1 was determined using real-time PCR. Immunofluorescence was performed to evaluate protein expression of WT1. Statistical significance was considered p<0.05. Diaphragmatic mRNA expression of WT1 was significantly decreased in the nitrofen group on D13, D18 and D21. Intensity of immunofluorescencence of WT1 was markedly decreased in the CDH diaphragms on D13, D18 and D21. Downregulation of diaphragmatic WT1 gene expression may impair diaphragmatic development in the nitrofen CDH model.

Characterization of the chromosome 1q41q42.12 region, and the candidate gene DISP1, in patients with CDH.

Cytogenetic and molecular cytogenetic studies demonstrate association between congenital diaphragmatic hernia (CDH) and chromosome 1q41q42 deletions. In this study, we screened a large CDH cohort (N=179) for microdeletions in this interval by the multiplex ligation-dependent probe amplification (MLPA) technique, and also sequenced two candidate genes located therein, dispatched 1 (DISP1) and homo sapiens H2.0-like homeobox (HLX). MLPA analysis verified deletions of this region in two cases, an unreported patient with a 46,XY,del(1)(q41q42.13) karyotype and a previously reported patient with a Fryns syndrome phenotype [Kantarci et al., 2006]. HLX sequencing showed a novel but maternally inherited single nucleotide variant (c.27C>G) in a patient with isolated CDH, while DISP1 sequencing revealed a mosaic de novo heterozygous substitution (c.4412C>G; p.Ala1471Gly) in a male with a left-sided Bochdalek hernia plus multiple other anomalies. Pyrosequencing demonstrated the mutant allele was present in 43%, 12%, and 4.5% of the patient's lymphoblastoid, peripheral blood lymphocytes, and saliva cells, respectively. We examined Disp1 expression at day E11.5 of mouse diaphragm formation and confirmed its presence in the pleuroperitoneal fold, as well as the nearby lung which also expresses Sonic hedgehog (Shh). Our report describes the first de novo DISP1 point mutation in a patient with complex CDH. Combining this finding with Disp1 embryonic mouse diaphragm and lung tissue expression, as well as previously reported human chromosome 1q41q42 aberrations in patients with CDH, suggests that DISP1 may warrant further consideration as a CDH candidate gene.

Early development of the primordial mammalian diaphragm and cellular mechanisms of nitrofen‐induced congenital diaphragmatic hernia

Congenital diaphragmatic hernia (CDH) is a frequently occurring cause of neonatal respiratory distress and is associated with high mortality and long-term morbidity. Evidence from animal models suggests that CDH has its origins in the malformation of the pleuroperitoneal fold (PPF), a key structure in embryonic diaphragm formation. The aims of this study were to characterize the embryogenesis of the PPF in rats and humans, and to determine the potential mechanism that leads to abnormal PPF development in the nitrofen model of CDH. Analysis of rat embryos, and archived human embryo sections, allowed the timeframe of PPF formation to be determined for both species, thus delineating a critical period of diaphragm development in relation to CDH. Experiments on nitrofen-exposed NIH 3T3 cells in vitro led us to hypothesize that nitrofen might cause diaphragmatic hernia in vivo by two possible mechanisms: through decreased cell proliferation or by inducing apoptosis. Data from nitrofen-exposed rat embryos indicates that the primary mechanism of nitrofen teratogenesis in the PPF is through decreased cell proliferation. This study provides novel insight into the embryogenesis of the PPF in rats and humans, and it indicates that impaired cell proliferation might contribute to abnormal diaphragm development in the nitrofen model of CDH.

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Pleuroperitoneal folds (PPFs) are the source of the primordial diaphragm's muscle connective tissue (MCT), and developmental mutations have been shown to result in congenital diaphragmatic hernia (CDH). The protein paired-related homeobox 1 (Prx1) labels migrating PPF cells and stimulates expression of transcription factor 4 (Tcf4), a novel MCT marker that controls morphogenesis of the fetal diaphragm. We hypothesized that diaphragmatic Prx1 and Tcf4 expression is decreased in the nitrofen-induced CDH model.Time-mated rats were exposed to either nitrofen or vehicle on gestational day 9 (D9). Fetal diaphragms were microdissected on D13, D15, and D18, and divided into control and nitrofen-exposed specimens. Gene expression levels of Prx1 and Tcf4 were analyzed by qRT-PCR. Immunofluorescence double staining for Prx1 and Tcf4 was performed to evaluate protein expression and localization.Relative mRNA expression of Prx1 and Tcf4 was significantly downregulated in PPFs (D13), developing diaphragms (D15) and fully muscularized diaphragms (D18) of nitrofen-exposed fetuses compared to controls. Confocal laser scanning microscopy revealed markedly diminished Prx1 and Tcf4 expression in diaphragmatic MCT of nitrofen-exposed fetuses on D13, D15, and D18 compared to controls.Decreased expression of Prx1 and Tcf4 in the fetal diaphragm may cause defects in the PPF-derived MCT, leading to development of CDH in the nitrofen model.Level 2c (Centre for Evidence-Based Medicine, Oxford).

Diaphragm development and congenital diaphragmatic hernia

Advances in the understanding of normal diaphragm embryogenesis have provided the necessary foundation for novel insights into the pathogenesis of congenital diaphragmatic hernia (CDH). Although diaphragm formation is still not completely understood, we have identified key structures and periods of development that are clearly abnormal in animal models of CDH. The pleuroperitoneal fold (PPF) is a transient structure which is the target for the neuromuscular component of the diaphragm. The PPF has been shown to be abnormal in multiple animal models of Bochdalek CDH; specifically, a malformation of the nonmuscular component of this tissue is thought to underlie the later defect in the complete diaphragm. Based on data from animal models and the examination of human postmortem tissue, we hypothesize that abnormal PPF development underlies Bochdalek CDH. Further, the understanding of the pathogenesis of rarer subtypes of CDH will be advanced by the study of various new animal models discussed in this review.

Development of the diaphragm and genetic mouse models of diaphragmatic defects

Improving our understanding of diaphragmatic development is essential to making progress in defining the pathogenesis and genetic etiologies of congenital diaphragmatic defects in humans. As mouse genetic technology has given us new tools to manipulate and observe development, a number of mouse models have recently emerged that provide valuable insight to this field. In this article, we review our current understanding of diaphragmatic embryogenesis including the origin of diaphragmatic tissue. We use rodent models to review the muscularization of the diaphragm and review selected genetic models of abnormal muscularization. We also review models of posterior diaphragmatic defects and discuss evidence for the pleuroperitoneal fold (PPF) tissue contributing to the diaphragm. Finally, we discuss models of anterior and central hernias. It may be simplistic to subdivide this review based on anatomic regions of the diaphragm, as evidence is emerging that defects in different regions of the diaphragm in humans and in mice may be etiologically related. However, at this time we do not have enough knowledge to make more mechanistic or genetic classifications though with time, genetic progress in the field of diaphragm development will allow us to do this.

Computer simulation analysis of normal and abnormal development of the mammalian diaphragm.

BackgroundCongenital diaphragmatic hernia (CDH) is a birth defect with significant morbidity and mortality. Knowledge of diaphragm morphogenesis and the aberrations leading to CDH is limited. Although classical embryologists described the diaphragm as arising from the septum transversum, pleuroperitoneal folds (PPF), esophageal mesentery and body wall, animal studies suggest that the PPF is the major, if not sole, contributor to the muscular diaphragm. Recently, a posterior defect in the PPF has been identified when the teratogen nitrofen is used to induce CDH in fetal rodents. We describe use of a cell-based computer modeling system (Nudge++™) to study diaphragm morphogenesis.Methods and resultsKey diaphragmatic structures were digitized from transverse serial sections of paraffin-embedded mouse embryos at embryonic days 11.5 and 13. Structure boundaries and simulated cells were combined in the Nudge++™ software. Model cells were assigned putative behavioral programs, and these programs were progressively modified to produce a diaphragm consistent with the observed anatomy in rodents. Homology between our model and recent anatomical observations occurred under the following simulation conditions: (1) cell mitoses are restricted to the edge of growing tissue; (2) cells near the chest wall remain mitotically active; (3) mitotically active non-edge cells migrate toward the chest wall; and (4) movement direction depends on clonal differentiation between anterior and posterior PPF cells.ConclusionWith the PPF as the sole source of mitotic cells, an early defect in the PPF evolves into a posteromedial diaphragm defect, similar to that of the rodent nitrofen CDH model. A posterolateral defect, as occurs in human CDH, would be more readily recreated by invoking other cellular contributions. Our results suggest that recent reports of PPF-dominated diaphragm morphogenesis in the rodent may not be strictly applicable to man. The ability to recreate a CDH defect using a combination of experimental data and testable hypotheses gives impetus to simulation modeling as an adjunct to experimental analysis of diaphragm morphogenesis.

Bilateral congenital diaphragmatic hernia with absent pleura and pericardium

Bilateral congenital diaphragmatic hernia is a rare form of diaphragmatic hernia. Independently, pericardial defects are an extremely rare phenomenon. In the case presented, we provide the first complete description of an infant with bilateral congenital diaphragmatic hernia with complete agenesis of the pericardium and inferior parietal pleura. A male infant was born at 38 weeks of gestation with a prenatal diagnosis of left-sided congenital diaphragmatic hernia. After 1 week of aggressive management, the patient was taken to the operating room for repair. Intraoperatively, the patient was found to have absence of the diaphragm bilaterally, no pleura inferiorly, and no pericardium. A biological mesh was used to construct a diaphragm. At 6 months of age, the patient is growing normally, requiring only supplemental oxygen without pressure support. Embryologically, this anomaly represents complete lack of development of the pleurocardial folds, pleuroperitoneal folds, and transverse septum, which is previously unreported.

Embryological origins and development of the rat diaphragm.

Textbooks of embryology provide a standard set of drawings and text reflecting the traditional interpretation of phrenic nerve and diaphragm development based on anatomical dissections of embryonic tissue. Here, we revisit this issue, taking advantage of immunohistochemical markers for muscle precursors in conjunction with mouse mutants to perform a systematic examination of phrenic-diaphragm embryogenesis. This includes examining the spatiotemporal relationship of phrenic axon outgrowth and muscle precursors during different stages of myogenesis. Additionally, mutant mice lacking c-met receptors were used to visualize the mesenchymal substratum of the developing diaphragm in the absence of myogenic cells. We found no evidence for contributions to the diaphragm musculature from the lateral body wall, septum transversum, or esophageal mesenchyme, as standard dogma would state. Nor did the data support the hypothesis that the crural diaphragm is of distinct embryological origins. Rather, we found that myogenic cells and axons destined to form the neuromuscular component of the diaphragm coalesce within the pleuroperitoneal fold (PPF). It is the expansion of these components of the PPF that leads to the formation of the diaphragm. Furthermore, we extended these studies to examine the developing diaphragm in an animal model of congenital diaphragmatic hernia (CDH). We find that malformation of the PPF mesenchymal substratum leads to the defect characteristic of CDH. In summary, the data demonstrates that a significant revision of narratives describing normal and pathological development of the diaphragm is warranted.

Structure of the primordial diaphragm and defects associated with nitrofen-induced CDH.

The goals of this study were to further our understanding of diaphragm embryogenesis and the pathogenesis of congenital diaphragmatic hernia (CDH). Past work suggests that the pleuroperitoneal fold (PPF) is the primary source of diaphragmatic musculature. Furthermore, defects associated with an animal model of CDH can be traced back to the formation of the PPF. This study was designed to elucidate the anatomic structure of the PPF and to determine which regions of the PPF malform in the well-established nitrofen model of CDH. This was achieved by producing three-dimensional renderings constructed from serial transverse sections of control and nitrofen-exposed rats at embryonic day 13.5. Renderings of left- and right-sided defects demonstrated that the malformations were always limited to the dorsolateral portions of the caudal regions of the PPF. These data provide an explanation of why the holes in diaphragmatic musculature associated with CDH are characteristically located in dorsolateral regions. Moreover, these data provide further evidence against the widely stated hypothesis that a failure of pleuroperitoneal canal closure underlies the pathogenesis of nitrofen-induced CDH.

Recent advances in understanding the pathogenesis of nitrofen-induced congenital diaphragmatic hernia

In this review, we discuss recent advances in the study of the pathogenesis of congenital diaphragmatic hernia (CDH). Much of the research has involved the use of an animal model of CDH in which diaphragmatic defects are produced in fetal rats by administering the herbicide nitrofen to dams during mid-gestation. The animal model is described and the relevance to the human condition is discussed. The data derived from the animal studies are critically assessed in the context of commonly cited hypotheses proposed for the pathogenesis of CDH. Finally, experimental strategies are proposed for systematically examining the normal and pathological formation of the pleuroperitoneal fold. We conclude that a malformation of the primordial diaphragm, the pleuroperitoneal fold, underlies the muscle defects associated with CDH. Pediatr Pulmonol. 2000; 29:394–399. © 2000 Wiley-Liss, Inc.

Recent advances in understanding the pathogenesis of nitrofen-induced congenital diaphragmatic hernia.

In this review, we discuss recent advances in the study of the pathogenesis of congenital diaphragmatic hernia (CDH). Much of the research has involved the use of an animal model of CDH in which diaphragmatic defects are produced in fetal rats by administering the herbicide nitrofen to dams during mid-gestation. The animal model is described and the relevance to the human condition is discussed. The data derived from the animal studies are critically assessed in the context of commonly cited hypotheses proposed for the pathogenesis of CDH. Finally, experimental strategies are proposed for systematically examining the normal and pathological formation of the pleuroperitoneal fold. We conclude that a malformation of the primordial diaphragm, the pleuroperitoneal fold, underlies the muscle defects associated with CDH.

An overview of phrenic nerve and diaphragm muscle development in the perinatal rat.

In this overview, we outline what is known regarding the key developmental stages of phrenic nerve and diaphragm formation in perinatal rats. These developmental events include the following. Cervical axons emerge from the spinal cord during embryonic (E) day 11. At approximately E12.5, phrenic and brachial axons from the cervical segments merge at the brachial plexi. Subsequently, the two populations diverge as phrenic axons continue to grow ventrally toward the diaphragmatic primordium and brachial axons turn laterally to grow into the limb bud. A few pioneer axons extend ahead of the majority of the phrenic axonal population and migrate along a well-defined track toward the primordial diaphragm, which they reach by E13.5. The primordial diaphragmatic muscle arises from the pleuroperitoneal fold, a triangular protrusion of the body wall composed of the fusion of the primordial pleuroperitoneal and pleuropericardial tissues. The phrenic nerve initiates branching within the diaphragm at approximately E14, when myoblasts in the region of contact with the phrenic nerve begin to fuse and form distinct primary myotubes. As the nerve migrates through the various sectors of the diaphragm, myoblasts along the nerve's path begin to fuse and form additional myotubes. The phrenic nerve intramuscular branching and concomitant diaphragmatic myotube formation continue to progress up until E17, at which time the mature pattern of innervation and muscle architecture are approximated. E17 is also the time of the commencement of inspiratory drive transmission to phrenic motoneurons (PMNs) and the arrival of phrenic afferents to the motoneuron pool. During the period spanning from E17 to birth (gestation period of approximately 21 days), there is dramatic change in PMN morphology as the dendritic branching is rearranged into the rostrocaudal bundling characteristic of mature PMNs. This period is also a time of significant changes in PMN passive membrane properties, action-potential characteristics, and firing properties.