Management of Congenital Diaphragmatic Hernia (CDH): Role of Molecular Genetics.

Congenital diaphragmatic hernia (CDH) is a relatively common major life-threatening birth defect that results in significant mortality and morbidity depending primarily on lung hypoplasia, persistent pulmonary hypertension, and cardiac dysfunction. Despite its clinical relevance, CDH multifactorial etiology is still not completely understood. We reviewed current knowledge on normal diaphragm development and summarized genetic mutations and related pathways as well as cellular mechanisms involved in CDH. Our literature analysis showed that the discovery of harmful de novo variants in the fetus could constitute an important tool for the medical team during pregnancy, counselling, and childbirth. A better insight into the mechanisms regulating diaphragm development and genetic causes leading to CDH appeared essential to the development of new therapeutic strategies and evidence-based genetic counselling to parents. Integrated sequencing, development, and bioinformatics strategies could direct future functional studies on CDH; could be applied to cohorts and consortia for CDH and other birth defects; and could pave the way for potential therapies by providing molecular targets for drug discovery.

Defective mesothelium and limited physical space are drivers of dysregulated lung development in a genetic model of congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is a developmental disorder associated with diaphragm defects and lung hypoplasia. The etiology of CDH is complex and its clinical presentation is variable. We investigated the role of the pulmonary mesothelium in dysregulated lung growth noted in the Wt1 knockout mouse model of CDH. Loss of WT1 leads to intrafetal effusions, altered lung growth, and branching defects prior to normal closure of the diaphragm. We found significant differences in key genes; however, when Wt1 null lungs were cultured ex vivo, growth and branching were indistinguishable from wild-type littermates. Micro-CT imaging of embryos in situ within the uterus revealed a near absence of space in the dorsal chest cavity, but no difference in total chest cavity volume in Wt1 null embryos, indicating a redistribution of pleural space. The altered space and normal ex vivo growth suggest that physical constraints are contributing to the CDH lung phenotype observed in this mouse model. These studies emphasize the importance of examining the mesothelium and chest cavity as a whole, rather than focusing on single organs in isolation to understand early CDH etiology.

Pbx1, Meis1, and Runx1 Expression Is Decreased in the Diaphragmatic and Pulmonary Mesenchyme of Rats with Nitrofen-Induced Congenital Diaphragmatic Hernia.

INTRODUCTION: Congenital diaphragmatic hernia (CDH) and associated pulmonary hypoplasia (PH) are thought to originate from mesenchymal defects in pleuroperitoneal folds (PPFs) and primordial lungs. Pre-B-cell leukemia homeobox 1 (Pbx1), its binding partner myeloid ecotropic integration site 1 (Meis1), and runt-related transcription factor 1 (Runx1) are expressed in diaphragmatic and lung mesenchyme, functioning as transcription cofactors that modulate mesenchymal cell proliferation. Furthermore, Pbx1 -/- mice develop diaphragmatic defects and PH similar to human CDH. We hypothesized that diaphragmatic and pulmonary Pbx1, Meis1, and Runx1 expression is decreased in the nitrofen-induced CDH model. MATERIALS AND METHODS: Time-mated rats were exposed to nitrofen or vehicle on gestational day 9 (D9). Fetal diaphragms (n = 72) and lungs (n = 48) were microdissected on D13, D15, and D18, and were divided into control and nitrofen-exposed specimens. Diaphragmatic and pulmonary gene expression levels of Pbx1, Meis1, and Runx1 were analyzed by quantitative real-time polymerase chain reaction. Immunofluorescence-double-staining for Pbx1, Meis1, and Runx1 was combined with mesenchymal/myogenic markers Gata4 and myogenin to evaluate protein expression. RESULTS: Relative mRNA expression of Pbx1, Meis1, and Runx1 was significantly decreased in PPFs (D13), developing diaphragms/lungs (D15), and muscularized diaphragms/differentiated lungs (D18) of nitrofen-exposed fetuses compared with controls. Confocal-laser-scanning-microscopy revealed markedly diminished Pbx1, Meis1, and Runx1 immunofluorescence in diaphragmatic and pulmonary mesenchyme, associated with less proliferating mesenchymal cells in nitrofen-exposed fetuses on D13, D15, and D18 compared with controls. CONCLUSION: Decreased Pbx1, Meis1, and Runx1 expression during diaphragmatic development and lung branching morphogenesis may reduce mesenchymal cell proliferation, causing malformed PPFs and disrupted airway branching, thus leading to diaphragmatic defects and PH in the nitrofen-induced CDH model.

Cell culture system to assay candidate genes and molecular pathways implicated in congenital diaphragmatic hernias.

The mammalian muscularized diaphragm is essential for respiration and defects in the developing diaphragm cause a common and frequently lethal birth defect, congenital diaphragmatic hernia (CDH). Human genetic studies have implicated more than 150 genes and multiple molecular pathways in CDH, but few of these have been validated because of the expense and time to generate mouse mutants. The pleuroperitoneal folds (PPFs) are transient embryonic structures in diaphragm development and defects in PPFs lead to CDH. We have developed a system to culture PPF fibroblasts from E12.5 mouse embryos and show that these fibroblasts, in contrast to the commonly used NIH 3T3 fibroblasts, maintain expression of key genes in normal diaphragm development. Using pharmacological and genetic manipulations that result in CDH in vivo, we also demonstrate that differences in proliferation provide a rapid means of distinguishing healthy and impaired PPF fibroblasts. Thus, the PPF fibroblast cell culture system is an efficient tool for assaying the functional significance of CDH candidate genes and molecular pathways and will be an important resource for elucidating the complex etiology of CDH.

Routes of Transdiaphragmatic Migration from the Abdomen to the Chest.

The diaphragm serves as an anatomic border between the abdominal and thoracic cavities. Pathologic conditions traversing the diaphragm are often incompletely described and may be overlooked, resulting in diagnostic delays. Several routes allow abdominal contents or pathologic processes to spread into the thorax, including along normal transphrenic structures, through congenital defects in the diaphragm, through inherent areas of weakness between muscle groups, or by pathways created by tissue destruction, trauma, or iatrogenic injuries. A thorough knowledge of the anatomy of the diaphragm can inform an accurate differential diagnosis. Often, intraperitoneal pathologic conditions crossing the diaphragm may be overlooked if axial imaging is the only approach to this complex region because of the horizontal orientation of much of the diaphragm. Multiplanar capabilities of volumetric CT and MRI provide insight into the pathways where pathologic conditions may traverse this border. Knowledge of these characteristic routes and use of multiplanar imaging are critical for depiction of specific transdiaphragmatic pathologic conditions.©RSNA, 2020.

Congenital Cystic Diaphragm with Diaphragmatic Eventration in a Fetus: A Case Presentation.

Introduction: Congenital diaphragmatic eventration (CDE) is defined as the abnormal elevation of the diaphragm, due to incomplete muscularization of the diaphragm with a thin membranous sheet replacing normal diaphragmatic muscle. Case report: We report a prenatal case with a diaphragmatic mesothelial cyst combined with CDE. Conclusion: A large cystic mass between the thoracic wall and the liver in early pregnancy is highly suggestive of cystic diaphragm.

Ephrin-B1, -B2, and -B4 Expression is Decreased in Developing Diaphragms and Lungs of Fetal Rats with Nitrofen-Induced Congenital Diaphragmatic Hernia.

INTRODUCTION: Congenital diaphragmatic hernia (CDH) is assumed to originate from a malformation of the amuscular mesenchymal component of the primordial diaphragm. Mutations in ephrin-B1, a membrane protein that is expressed by mesenchymal cells, have been found in newborn infants with CDH and associated pulmonary hypoplasia (PH), highlighting its important role during diaphragmatic and airway development. Ephrin-B1, -B2, and -B4 are expressed in fetal rat lungs and have been identified as key players during lung branching morphogenesis. We hypothesized that diaphragmatic and pulmonary expression of ephrin-B1, -B2, and -B4 is decreased in the nitrofen-induced CDH model. MATERIALS AND METHODS: Time-mated rats received nitrofen or vehicle on day 9 (D9). Fetal diaphragms (n = 72) and lungs (n = 72) were harvested on D13, D15, and D18, and divided into control and nitrofen-exposed specimens. Ephrin-B1, -B2, and -B4 gene expression was analyzed by quantitative real-time polymerase chain reaction. Immunofluorescence double staining for ephrin-B1, -B2, and -B4 was combined with mesenchymal and epithelial markers (Gata-4/Fgf-10 and calcitonin gene-related peptide) to evaluate protein expression/localization. RESULTS: Ephrin-B1, -B2, and -B4 gene expression was significantly reduced in pleuroperitoneal folds/primordial lungs (D13), developing diaphragms/lungs (D15), and fully muscularized diaphragms/differentiated lungs (D18) of nitrofen-exposed fetuses compared with controls. Confocal laser scanning microscopy demonstrated markedly diminished ephrin-B1 immunofluorescence in diaphragmatic and pulmonary mesenchyme of nitrofen-exposed fetuses on D13, D15, and D18 compared with controls, whereas ephrin-B2 and -B4 expression was mainly decreased in distal airway epithelium. CONCLUSION: Decreased ephrin-B1, -B2, and -B4 expression may disrupt diaphragmatic development and lung branching morphogenesis by interfering with epithelial-mesenchymal interactions, thus causing diaphragmatic defects and PH.

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

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.

The breadth of the diaphragm: updates in embryogenesis and role of imaging.

The diaphragm is an unique skeletal muscle separating the thoracic and abdominal cavities with a primary function of enabling respiration. When abnormal, whether by congenital or acquired means, the consequences for patients can be severe. Abnormalities that affect the diaphragm are often first detected on chest radiographs as an alteration in position or shape. Cross-sectional imaging studies, primarily CT and occasionally MRI, can depict structural defects, intrinsic and adjacent pathology in greater detail. Fluoroscopy is the primary radiologic means of evaluating diaphragmatic motion, though MRI and ultrasound also are capable of this function. This review provides an update on diaphragm embryogenesis and discusses current imaging of various abnormalities, including the emerging role of three-dimensional printing in planning surgical repair of diaphragmatic derangements.

Congenital diaphragmatic hernia as a part of Nance-Horan syndrome?

Nance-Horan syndrome is a rare X-linked developmental disorder characterized by bilateral congenital cataract, dental anomalies, facial dysmorphism, and intellectual disability. Here, we identify a patient with Nance-Horan syndrome caused by a new nonsense NHS variant. In addition, the patient presented congenital diaphragmatic hernia. NHS gene expression in murine fetal diaphragm was demonstrated, suggesting a possible involvement of NHS in diaphragm development. Congenital diaphragmatic hernia could result from NHS loss of function in pleuroperitoneal fold or in somites-derived muscle progenitor cells leading to an impairment of their cells migration.

Early development of pleuroperitoneal fold of the diaphragm in the rat fetus.

The embryonic diaphragm comprises four major structural components derived from the transverse septum, the dorsal foregut mesentery, the pleuroperitoneal folds (PPFs), and the body wall. In this study, the appearance of PPFs and related factors were investigated using light microscopy of horizontal sections of rat fetuses from embryonic day 12 to 13. In rat fetuses, the sign of PPF projection was noted in the sidewall of the pericardioperitoneal canal at embryonic day 12, and was confirmed as folds at embryonic day 12.25. Expressions of GATA4, COUP-TF2, and FOG2 were detected in PPF at the early stage of formation. Localizations of these factors suggested that COUP-TF2 and FOG2 are the main factors in PPF appearance and that GATA4 is unlikely to be a main factor, although it is necessary for PPF formation.

Gata-6 expression is decreased in diaphragmatic and pulmonary mesenchyme of fetal rats with nitrofen-induced congenital diaphragmatic hernia.

PURPOSE: Congenital diaphragmatic hernia (CDH) and associated pulmonary hypoplasia are thought to be caused by a malformation of the underlying diaphragmatic and airway mesenchyme. GATA binding protein 6 (Gata-6) is a zinc finger-containing transcription factor that plays a crucial role during diaphragm and lung development. In the primordial diaphragm, Gata-6 expression is restricted to mesenchymal compartments of the pleuroperitoneal folds (PPFs). In addition, Gata-6 is essential for airway branching morphogenesis through upregulation of mesenchymal signaling. Recently, mutations in Gata-6 have been linked to human CDH. We hypothesized that diaphragmatic and pulmonary Gata-6 expression is decreased in the nitrofen-induced CDH model. METHODS: Time-mated rats were exposed to either nitrofen or vehicle on gestational day 9 (D9). Fetal diaphragms (n = 72) and lungs (n = 48) were microdissected on selected timepoints D13, D15 and D18, and divided into control and nitrofen-exposed specimens (n = 12 per sample, timepoint and experimental group, respectively). Diaphragmatic and pulmonary gene expression of Gata-6 was analyzed by qRT-PCR. Immunofluorescence-double staining for Gata-6 was combined with the diaphragmatic mesenchymal marker Gata-4 and the pulmonary mesenchymal marker Fgf-10 to evaluate protein expression and localization in fetal diaphragms and lungs. RESULTS: Relative mRNA expression levels of Gata-6 were significantly decreased in PPFs on D13 (0.57 ± 0.21 vs. 2.27 ± 1.30; p < 0.05), developing diaphragms (0.94 ± 0.59 vs. 2.28 ± 1.89; p < 0.05) and lungs (0.56 ± 0.16 vs. 0.71 ± 0.39; p < 0.05) on D15 and fully muscularized diaphragms (1.20 ± 1.10 vs. 2.52 ± 1.86; p < 0.05) and differentiated lungs (0.56 ± 0.05 vs. 0.77 ± 0.14; p < 0.05) on D18 of nitrofen-exposed fetuses compared to controls. Confocal laser scanning microscopy demonstrated markedly diminished immunofluorescence of Gata-6 mainly in diaphragmatic and pulmonary mesenchyme, which was associated with a reduction of proliferating mesenchymal cells in nitrofen-exposed fetuses on D13, D15, and D18 compared to controls. CONCLUSION: Decreased Gata-6 expression during diaphragmatic development and lung branching morphogenesis may disrupt mesenchymal cell proliferation, causing malformed PPFs and reduced airway branching, thus leading to diaphragmatic defects and pulmonary hypoplasia in the nitrofen-induced CDH model.

Genetic specification of left-right asymmetry in the diaphragm muscles and their motor innervation.

The diaphragm muscle is essential for breathing in mammals. Its asymmetric elevation during contraction correlates with morphological features suggestive of inherent left-right (L/R) asymmetry. Whether this asymmetry is due to L versus R differences in the muscle or in the phrenic nerve activity is unknown. Here, we have combined the analysis of genetically modified mouse models with transcriptomic analysis to show that both the diaphragm muscle and phrenic nerves have asymmetries, which can be established independently of each other during early embryogenesis in pathway instructed by Nodal, a morphogen that also conveys asymmetry in other organs. We further found that phrenic motoneurons receive an early L/R genetic imprint, with L versus R differences both in Slit/Robo signaling and MMP2 activity and in the contribution of both pathways to establish phrenic nerve asymmetry. Our study therefore demonstrates L-R imprinting of spinal motoneurons and describes how L/R modulation of axon guidance signaling helps to match neural circuit formation to organ asymmetry.

Abnormal lung development in congenital diaphragmatic hernia.

The outcomes of patients diagnosed with congenital diaphragmatic hernia (CDH) have recently improved. However, mortality and morbidity remain high, and this is primarily caused by the abnormal lung development resulting in pulmonary hypoplasia and persistent pulmonary hypertension. The pathogenesis of CDH is poorly understood, despite the identification of certain candidate genes disrupting normal diaphragm and lung morphogenesis in animal models of CDH. Defects within the lung mesenchyme and interstitium contribute to disturbed distal lung development. Frequently, a disturbance in the development of the pleuroperitoneal folds (PPFs) leads to the incomplete formation of the diaphragm and subsequent herniation. Most candidate genes identified in animal models have so far revealed relatively few strong associations in human CDH cases. CDH is likely a highly polygenic disease, and future studies will need to reconcile how disturbances in the expression of multiple genes cause the disease. Herein, we summarize the available literature on abnormal lung development associated with CDH.

Down-regulation of N-deacetylase-N-sulfotransferase-1 signaling in the developing diaphragmatic vasculature of nitrofen-induced congenital diaphragmatic hernia.

BACKGROUND: Congenital diaphragmatic hernia (CDH) has been attributed to various developmental abnormalities of the underlying tissue components. N-deacetylase-N-sulfotransferase-1 (Ndst1) is a strongly expressed biosynthetic enzyme in endothelial cells, which has recently been identified as an important factor during diaphragmatic vascularization. Loss of endothelial Ndst1 has been demonstrated to cause angiogenic defects in the developing diaphragm and disrupt normal diaphragmatic development. Furthermore, deficiency of Ndst1 diminishes the expression of slit homolog 3 (Slit3), a known CDH-related gene that has been associated with reduced vascular density and muscle defects in the diaphragm of Slit3-/- mice. We hypothesized that expression of Ndst1 and Slit3 is decreased in the diaphragmatic vasculature of fetal rats with nitrofen-induced CDH. METHODS: Time-mated rats received 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 Ndst1 and Slit3 were assessed using qRT-PCR. Immunofluorescence-double-staining for Ndst1 and Slit3 was performed to evaluate protein expression and localization. RESULTS: Relative mRNA expression of Ndst1 and Slit3 was significantly decreased in pleuroperitoneal folds (D13), developing diaphragms (D15) and fully muscularized diaphragms (D18) of nitrofen-exposed fetuses compared to controls. Confocal-laser-scanning-microscopy revealed markedly diminished Ndst1 and Slit3 expression in endothelial cells within the diaphragmatic vasculature on D13, D15 and D18 compared to controls. CONCLUSIONS: Down-regulation of Ndst1 signaling in the developing diaphragm may impair endothelial cell migration and angiogenesis, thus leading to defective diaphragmatic vascular development and CDH. LEVEL OF EVIDENCE: Ib.

Fibrillin-1 Expression Is Decreased in the Diaphragmatic Muscle Connective Tissue of Nitrofen-Induced Congenital Diaphragmatic Hernia.

Introduction Diaphragmatic morphogenesis depends on proper formation of muscle connective tissue (MCT) and underlying extracellular matrix (ECM). Fibrillin-1 is an essential ECM protein and crucial for the structural integrity of MCT in the developing diaphragm. Recently, mutations in the fibrillin-1 gene (FBN1) have been identified in cases of congenital diaphragmatic hernia (CDH), thus suggesting that alterations in FBN1 gene expression may lead to diaphragmatic defects. We designed this study to investigate the hypothesis that the diaphragmatic expression of fibrillin-1 is decreased in the MCT of nitrofen-induced CDH. Materials and Methods Time-mated rats were exposed to nitrofen or vehicle on gestational day 9 (D9). Fetal diaphragms (n = 72) were harvested on D13, D15, and D18, and divided into control and nitrofen-exposed specimens. Laser-capture microdissection was used to obtain diaphragmatic tissue cells. Gene expression levels of FBN1 were analyzed by qRT-PCR. Immunofluorescence-double-staining for fibrillin-1 and the mesenchymal marker Gata4 was performed to evaluate protein expression and localization. Results Relative mRNA expression of FBN1 was significantly decreased in pleuroperitoneal folds on D13 (3.39 ± 1.29 vs. 5.47 ± 1.92; p < 0.05), developing diaphragms on D15 (2.48 ± 0.89 vs. 4.03 ± 1.62; p < 0.05), and fully muscularized diaphragms on D18 (2.49 ± 0.69 vs. 3.93 ± 1.55; p < 0.05) of nitrofen-exposed fetuses compared with controls. Confocal-laser-scanning microscopy revealed markedly diminished fibrillin-1 immunofluorescence mainly in MCT, associated with a reduction of proliferating mesenchymal cells in nitrofen-exposed fetuses on D13, D15, and D18 compared with controls. Conclusions Decreased expression of fibrillin-1 during morphogenesis of the fetal diaphragm may disrupt mesenchymal cell proliferation, causing malformed MCT and thus resulting in diaphragmatic defects in the nitrofen-induced CDH model.

Conditional deletion of WT1 in the septum transversum mesenchyme causes congenital diaphragmatic hernia in mice.

Congenital diaphragmatic hernia (CDH) is a severe birth defect. Wt1-null mouse embryos develop CDH but the mechanisms regulated by WT1 are unknown. We have generated a murine model with conditional deletion of WT1 in the lateral plate mesoderm, using the G2 enhancer of the Gata4 gene as a driver. 80% of G2-Gata4(Cre);Wt1(fl/fl) embryos developed typical Bochdalek-type CDH. We show that the posthepatic mesenchymal plate coelomic epithelium gives rise to a mesenchyme that populates the pleuroperitoneal folds isolating the pleural cavities before the migration of the somitic myoblasts. This process fails when Wt1 is deleted from this area. Mutant embryos show Raldh2 downregulation in the lateral mesoderm, but not in the intermediate mesoderm. The mutant phenotype was partially rescued by retinoic acid treatment of the pregnant females. Replacement of intermediate by lateral mesoderm recapitulates the evolutionary origin of the diaphragm in mammals. CDH might thus be viewed as an evolutionary atavism.

Decreased Desmin expression in the developing diaphragm of the nitrofen-induced congenital diaphragmatic hernia rat model.

PURPOSE: Congenital diaphragmatic hernia (CDH) is presumed to originate from defects in the primordial diaphragmatic mesenchyme, mainly comprising of muscle connective tissue (MCT). Thus, normal diaphragmatic morphogenesis depends on the structural integrity of the underlying MCT. Developmental mutations that inhibit normal formation of diaphragmatic MCT have been shown to result in CDH. Desmin (DES) is a major filament protein in the MCT, which is essential for the tensile strength of the developing diaphragm muscle. DES -/- knockout mice exhibit significant reductions in stiffness and elasticity of the developing diaphragmatic muscle tissue. Furthermore, sequence changes in the DES gene have recently been identified in human cases of CDH, suggesting that alterations in DES expression may lead to diaphragmatic defects. This study was designed to investigate the hypothesis that diaphragmatic DES expression is decreased in fetal rats with nitrofen-induced CDH. METHODS: Time-mated Sprague-Dawley rats were exposed to either nitrofen or vehicle on gestational day 9 (D9). Fetuses were harvested on selected time-points D13, D15 and D18, and dissected diaphragms (n = 72) were divided into control and nitrofen-exposed specimens (n = 12 per time-point and experimental group, respectively). Laser-capture microdissection was used to obtain diaphragmatic tissue elements. Diaphragmatic gene expression of DES was analyzed by quantitative real-time polymerase chain reaction. Immunofluorescence double staining for DES was combined with the mesenchymal marker GATA4 to evaluate protein expression and localization in developing fetal diaphragms. RESULTS: Relative mRNA expression levels of DES were significantly decreased in pleuroperitoneal folds on D13 (1.49 ± 1.79 vs. 3.47 ± 2.32; p < 0.05), developing diaphragms on D15 (1.49 ± 1.41 vs. 3.94 ± 3.06; p < 0.05) and fully muscularized diaphragms on D18 (2.45 ± 1.47 vs. 5.12 ± 3.37; p < 0.05) of nitrofen-exposed fetuses compared to controls. Confocal laser scanning microscopy demonstrated markedly diminished immunofluorescence of DES mainly in diaphragmatic MCT, which was associated with a reduction of proliferating mesenchymal cells in nitrofen-exposed fetuses on D13, D15 and D18 compared to controls. CONCLUSION: Decreased expression of DES in the fetal diaphragm may disturb the basic integrity of myofibrils and the cytoskeletal network during myogenesis, causing malformed MCT and leading to diaphragmatic defects in the nitrofen-induced CDH model.

Expression of Prx1 and Tcf4 is decreased in the diaphragmatic muscle connective tissue of nitrofen-induced congenital diaphragmatic hernia.

BACKGROUND/PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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 OF EVIDENCE: Level 2c (Centre for Evidence-Based Medicine, Oxford).

Polygenic Causes of Congenital Diaphragmatic Hernia Produce Common Lung Pathologies.

Congenital diaphragmatic hernia (CDH) is one of the most common and lethal congenital anomalies, and significant evidence is available in support of a genetic contribution to its etiology, including single-gene knockout mice associated with diaphragmatic defects, rare monogenetic disorders in humans, familial aggregation, and association of CDH with chromosomal abnormalities. Structural lung defects in the form of lung hypoplasia are almost invariably seen in patients with CDH and frequently in animal models of this condition. Better understanding of the mechanisms of pulmonary defects in CDH has the potential for creating targeted therapies, particularly in postnatal stages, when therapeutics can have maximum clinical impact on the surviving cohorts. Successful treatment of CDH is dependent on the integration of human genomic and genetic data with developmental expression profiling, mouse knockouts, and gene network and pathway modeling, which have generated a large number of candidate genes and pathways for follow-up studies. In particular, defective alveolarization appears to be a common and potentially actionable phenotype in both patients and animal models.