Search

Congenital diaphragmatic hernia (CDH) is a common birth malformation with a heterogeneous etiology. In this study, we report that ablation of the heparan sulfate biosynthetic enzyme NDST1 in murine endothelium (Ndst1ECKO mice) disrupted vascular development in the diaphragm, which led to hypoxia as well as subsequent diaphragm hypoplasia and CDH. Intriguingly, the phenotypes displayed in Ndst1ECKO mice resembled the developmental defects observed in slit homolog 3 (Slit3) knockout mice. Furthermore, introduction of a heterozygous mutation in roundabout homolog 4 (Robo4), the gene encoding the cognate receptor of SLIT3, aggravated the defect in vascular development in the diaphragm and CDH. NDST1 deficiency diminished SLIT3, but not ROBO4, binding to endothelial heparan sulfate and attenuated EC migration and in vivo neovascularization normally elicited by SLIT3-ROBO4 signaling. Together, these data suggest that heparan sulfate presentation of SLIT3 to ROBO4 facilitates initiation of this signaling cascade. Thus, our results demonstrate that loss of NDST1 causes defective diaphragm vascular development and CDH and that heparan sulfate facilitates angiogenic SLIT3-ROBO4 signaling during vascular development.

This review outlines research that has advanced our understanding of the pathogenesis and etiology of congenital diaphragmatic hernia (CDH). The majority of CDH cases involve incomplete formation of the posterolateral portion of the diaphragm, clinically referred to as a Bochdalek hernia. The hole in the diaphragm allows the abdominal viscera to invade the thoracic cavity, thereby impeding normal lung development. As a result, newborns with CDH suffer from a combination of severe pulmonary hypoplasia and pulmonary hypertension. Despite advances in neonatal intensive care, mortality and serious morbidity remain high. Systematic studies using rat and transgenic mouse models in conjunction with analyses of human tissue are providing insights into the embryological origins of the diaphragmatic defect associated with CDH and abnormalities of developmentally regulated signaling cascades., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Objective: Genetic analyses in humans suggest a role for retinoid-related genes in the pathogenesis of congenital diaphragmatic hernia (CDH). The goal of this study was to investigate the vitamin A status of mothers and their newborns in association with CDH., Methods: We conducted a hospital-based, case-control study with 22 case and 34 control mothers and their newborns. In maternal and cord blood samples, retinol and retinol-binding protein (RBP) levels were measured with high-performance liquid chromatography and an enzyme-linked immunosorbent assay, respectively. Univariate and multivariate logistic regression analyses were performed to determine crude and adjusted risk estimates., Results: Case newborns had significantly lower levels of retinol (0.60 vs 0.76 μmol/L; P=.003) and RBP (5.42 vs 7.11 mg/L; P=.02) than did control newborns. The multivariate logistic regression analysis showed lower levels of retinol and RBP in association with CDH risk; the odds ratio for retinol levels of <15th percentile (<0.61 μmol/L) was 11.11 (95% confidence interval: 2.54-48.66; P=.001), and that for RBP levels of <15th percentile (<4.54 mg/L) was 4.00 (95% confidence interval: 1.00-15.99; P=.05). Retinol and RBP levels were not different between case and control mothers., Conclusions: CDH is strongly associated with low retinol and RBP levels in newborns, independent of maternal retinol status. This is an important finding supporting the idea that human CDH is linked with abnormal retinoid homeostasis.

Congenital diaphragmatic hernia (CDH) is a frequently occurring source of severe neonatal respiratory distress. It has been hypothesized that abnormal retinoid signaling contributes to the etiology of this developmental anomaly. Here, we use rodent models toward specifically understanding the role of retinoid signaling in the developing diaphragm and how its perturbation is a common mechanism in drug-induced CDH. This includes monitoring of retinoic acid (RA) response element (RARE) activation with RARE-lacZ mice, RA supplementation studies, systematic analyses of the expression profile of key elements in the RA signaling pathway within the developing diaphragm, and the in utero delivery of a RA receptor (RAR) antagonist. These data demonstrate the timing of RARE perturbation by CDH-inducing teratogens and the efficacy of RA supplementation. Furthermore, a detailed profile of retinoid binding proteins, synthetic enzymes, and retinoid receptors within primordial diaphragm cells was obtained. The expression profile of RAR-alpha was particularly striking in regard to its overlap with the regions of primordial diaphragm affected in multiple CDH models. Blocking of RAR signaling with the pan-RAR antagonist BMS493 induced a very high degree of CDH, with a marked left-right sidedness that depended on the timing of drug delivery. Collectively, these data demonstrate that retinoid signaling is essential for normal diaphragm development, providing further support to the hypothesis that abnormalities related to the retinoid signaling pathway cause diaphragmatic defects. This study also yielded a novel experimental model that should prove particularly useful for further studies of CDH.

Congenital diaphragmatic hernia (CDH) is a frequently occurring birth defect and a source of potentially fatal neonatal respiratory distress. Recently, through the application of detailed karyotyping methods, several CDH-critical regions within the human genome have been identified. These regions typically contain several genes. Here we focused on genes from 15q26, the best-characterized CDH-critical region, as well as FOG2 and GATA4, genes singled out from CDH-critical regions at 8q22-8q23 and 8p23.1, respectively. We tested the hypothesis that these putative CDH-related genes are expressed within the developing diaphragm at the time of the hypothesized initial defect. Our results show that 15q26 contains a cluster of genes that are expressed in the developing rodent diaphragm, consistent with an association between deletions in this region and CDH. We then examined the protein expression pattern of positively identified genes within the developing diaphragm. Two major themes emerged. First, those factors strongly associated with CDH are expressed only in the nonmuscular, mesenchymal component of the diaphragm, supporting the hypothesis that CDH has its origins in a mesenchymal defect. Second, these factors are all coexpressed in the same cells. This suggests that cases of CDH with unique genetic etiology may lead to a common defect in these cells and supports the hypothesis that these factors may be members of a common pathway. This study is the first to provide a detailed examination of how genes associated with CDH are expressed in the developing diaphragm and provides an important foundation for understanding how the deletion of specific genes may contribute to abnormal diaphragm formation.

Congenital diaphragmatic hernia (CDH) is a developmental anomaly that results in significant mortality and morbidity. The underlying etiology is poorly understood. Insights will arise from an understanding of the mechanisms by which the teratogen nitrofen induces CDH in rodent models. In this study, we use in vitro cell assays in conjunction with whole animal rodent studies to test hypotheses regarding nitrofen's mechanism of action. The first component examined the interaction of nitrofen with various aspects of the retinoid signaling pathway including uptake proteins, binding proteins, receptors, conversion, and degradation enzymes. The second component examined the interactions of nitrofen and vitamins A, C, and E to test the hypothesis that nitrofen was functioning as an antioxidant to interfere with retinoid signaling. Third, we performed a series of experiments examining the interaction of nitrofen and thyroid signaling. Collectively, the data suggest that the primary aspect of retinoid signaling affected by nitrofen is via inhibition of the rate-limiting enzymes controlling retinoic acid synthesis. Retinoid signaling perturbations do not appear to involve oxidative effects of nitrofen. Any substantial roles of nitrofen-induced perturbations of thyroid hormone signaling or receptor function are not supported.

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.

Congenital diaphragmatic hernia (CDH) is a significant cause of pediatric mortality in humans with a heterogeneous and poorly understood etiology. Here we show that mice lacking Slit3 developed a central (septum transversum) CDH. Slit3 encodes a member of the Slit family of guidance molecules and is expressed predominantly in the mesothelium of the diaphragm during embryonic development. In Slit3 null mice, the central tendon region of the diaphragm fails to separate from liver tissue because of abnormalities in morphogenesis. The CDH progresses through continuous growth of the liver into the thoracic cavity. This study establishes the first genetic model for CDH and identifies a previously unsuspected role for Slit3 in regulating the development of the 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., (Copyright 2002 Wiley-Liss, Inc.)

Defects very similar to those seen in infants born with congenital diaphragmatic hernias can be induced in rodents by the administration of the teratogen nitrofen. There is an interest in understanding the biochemical mechanisms of nitrofen's actions in hopes of gaining insights into the etiology of congenital diaphragmatic hernia. In this study, we test the hypothesis that nitrofen is acting to perturb the retinoid signaling pathway by utilizing genetically engineered mice that have the lacZ reporter gene linked to a retinoic acid response element (RARE). We demonstrate a pronounced suppression of RARE-lacZ expression by nitrofen in vitro (by approximately 64%) and in vivo (by approximately 43%)., (Copyright 2003 S. Karger AG, Basel)

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., (Copyright 2000 Wiley-Liss, Inc.)

Congenital diaphragmatic hernia (CDH) is a developmental anomaly characterized by the malformation of the diaphragm and impaired lung development. In the present study, we tested several hypotheses regarding the pathogenesis of CDH, including those suggesting that the primary defect is due to abnormal 1) lung development, 2) phrenic nerve formation, 3) developmental processes underlying diaphragmatic myotube formation, 4) pleuroperitoneal canal closure, or 5) formation of the primordial diaphragm within the pleuroperitoneal fold. The 2,4-dichloro-phenyl-p-nitrophenyl ether (nitrofen)-induced CDH rat model was used for this study. The following parameters were compared between normal and herniated fetal rats at various stages of development: 1) weight, protein, and DNA content of lungs; 2) phrenic nerve diameter, axonal number, and motoneuron distribution; 3) formation of the phrenic nerve intramuscular branching pattern and diaphragmatic myotube formation; and 4) formation of the precursor of the diaphragmatic musculature, the pleuroperitoneal fold. We demonstrated that previously proposed theories regarding the primary role of the lung, phrenic nerve, myotube formation, and the closure of pleuroperitoneal canal in the pathogenesis of CDH are incorrect. Rather, the primary defect associated with CDH, at least in the nitrofen rat model, occurs at the earliest stage of diaphragm development, the formation of the pleuroperitoneal fold.