Genetically Modified Mouse Models of Congenital Diaphragmatic Hernia: Opportunities and Limitations for Studying Altered Lung Development.

Congenital diaphragmatic hernia (CDH) is a relatively common and life-threatening birth defect, characterized by an abnormal opening in the primordial diaphragm that interferes with normal lung development. As a result, CDH is accompanied by immature and hypoplastic lungs, being the leading cause of morbidity and mortality in patients with this condition. In recent decades, various animal models have contributed novel insights into the pathogenic mechanisms underlying CDH and associated pulmonary hypoplasia. In particular, the generation of genetically modified mouse models, which show both diaphragm and lung abnormalities, has resulted in the discovery of multiple genes and signaling pathways involved in the pathogenesis of CDH. This article aims to offer an up-to-date overview on CDH-implicated transcription factors, molecules regulating cell migration and signal transduction as well as components contributing to the formation of extracellular matrix, whilst also discussing the significance of these genetic models for studying altered lung development with regard to the human situation.

Introduction

Congenital diaphragmatic hernia (CDH) represents a relatively common and life-threatening birth defect with an estimated global prevalence of 2.3 in 10,000 live births (1, 2). It is characterized by incomplete formation and/or muscularization of the primordial diaphragm, which allows herniation of abdominal viscera into the thoracic cavity, thereby filling space usually reserved to hold the growing lung (3, 4). Hence, pulmonary development is disrupted, leading to immature and hypoplastic lungs (5–7). Today, more than 70% of CDH cases are diagnosed prenatally based on maternal-fetal ultrasound or magnetic resonance imaging in the second trimester of pregnancy, thus potentially altering future outcome (8, 9). Depending on the extent of pulmonary hypoplasia, newborns with CDH often present with severe respiratory distress at birth, requiring immediate and complex treatment (10, 11). Although significant advances have been achieved in postnatal resuscitation and ventilation strategies over the past decades (12, 13), CDH continues to be one of the major challenges in neonatal intensive care with mortality rates ranging between 30 and 50% (14–16). Surgical repair of CDH is generally performed after clinical stabilization either by primary closure or in larger defects by reconstruction using a prosthetic patch or muscle flap (17, 18). While newer therapeutic measures such as gentle ventilation techniques, high-frequency oscillation and extracorporeal membrane oxygenation have improved overall survival rates (19–21), this has led to substantial long-term morbidity in CDH patients (22, 23), including chronic lung disease, gastroesophageal reflux, scoliosis, sensorineural hearing loss and neurodevelopmental deficits (24–26).

A defect in the posterolateral diaphragm (also referred to as Bochdalek hernia) is the most common type of CDH and comprises approximately 80–90% of all cases, with the majority being left-sided (85%), less often right-sided (10%), or bilaterally (<5%) (27). The dual-hit hypothesis explains CDH-associated pulmonary hypoplasia by an initial disruption in bilateral lung organogenesis before diaphragm closure, in combination with a second ipsilateral insult resulting from the intrathoracic herniation and subsequent restriction of fetal breathing movements (28). Typical features of pulmonary hypoplasia in CDH are structural immaturity and smaller lung volume with a significantly reduced number of terminal airways, disrupted alveologenesis, diminished alveolar airspaces, thickened alveolar walls accompanied by increased interstitial tissue and decreased gas-exchange surface area (29). These findings have indicated that the pulmonary anomalies in CDH are at least partially independent of the diaphragmatic defect, suggesting a potential developmental linkage between both organs at a molecular level. Much of our present knowledge on the morphogenetic lung abnormalities in CDH has derived from experimental animal research (30–33). Because diaphragm and pulmonary evolution is remarkably similar between mice and humans, mouse models represent a crucial aspect in advancing our insight into the pathogenic mechanisms underlying CDH and associated lung hypoplasia.

This article aims to offer a comprehensive overview of genetically modified mouse models of CDH, resultant candidate genes and signaling pathways, whilst also discussing new opportunities and limitations for studying altered lung development in relation to the human situation.

Overview of Genetically Modified Mouse Models of Congenital Diaphragmatic Hernia

A large variety of genetic factors have been found to play key roles during the pathogenesis of CDH and pulmonary hypoplasia. Currently, genetic causes are detected in about 30% of CDH patients (34–36). Through recent advances in genetic engineering technologies, genetically modified mouse models of CDH are now frequently used in basic science research (37), offering several potential genes and signaling pathways involved in the etiology of diaphragmatic defects and allied lung anomalies (Table 1).

TABLE 1

Genetically modified mouse models of congenital diaphragmatic hernia.

Mouse models	Full gene names (functions)	Diaphragmatic defects	Lung abnormalities
ChAT–/–	Choline actelytransferased	Central hernia	–
Chtop–/– or Chtoptr/tr	Chromatin target of Prmt 1a	Posterolateral hernia	Hypoplasia
DNase IIα–/–	Desoxyribonuclease IIαd	Malformed diaphragm with hernia	Lungs not inflated
Eya1–/–;Eya2–/+	Eyes absent homolog 1 and 2a	Absent diaphragm	Hypoplasia, reduced epithelial branching, increased mesenchmal cellularity
Fbn1–/–	Fibrillin 1c	Unspecified hernia with ruptured edges	–
Fbln4–/–	Fibulin 4c	Severe hernia with rupture	Defective distal airways, emphysema
Fras1Q1263*/Q1263* †	Fraser extracellular matrix complex, subunit 1c	Retrosternal hernia (with sac)	–
Frem1eyes2/eyes2 †	Fras1-related extracellular matrix 1c	Retrosternal hernia (with sac)	Long lobulation defects, fused pulmonary lobes
Frem2ne/ne or Frem2b2b3270Clo†	Fras1-related extracellular matrix 2c	Retrosternal hernia (with sac)	–
Fuzb2b1273Clo †	Fuzzy planar cell polarity proteinb	Unspecified hernia	Hypoplasia, single left lung lobe
Gata4+/Δex2	GATA-binging protein 4a	Retrosternal hernia (with sac)	Dilated distal airways, increased saccule size, thickened mesenchyme, abnormal vasculature
Gli2–/–, Gli3–/– or Gli2–/–;Gli3+/–	GLI familiy zinc finger 2 and 3a	Posterior hernia	Hypoplasia, absent right accessory lobe, thickened mesenchyme
Hlx–/–	H2.0-like homeoboxa	Muscular hypoplasia with unspecified hernia	Enlarged lungs with normal structure
Hoxb4 PolII	Homeobox B4a	Unspecified hernia	–
Kif7dd/dd† or Kif7maki†	Kinesin family number 7a, b	Posterior hernia	Hypoplasia, reduced alveolar epithelial cell differentiation
Lox–/– or Loxb2b370.2Clo	Lysyl oxidasec, d	Central hernia with rupture	Hypoplasia, abnormal acini, abnormal elastic fibers
Lrp1b2b1554Clo †	Low density lipoprotein receptor-related protein 1b, d	Unspecified hernia	–
Met–/–	Mesenchymal-epithelial transition factorb	Amascular diaphragm with hernia	Abnormal saccule morphology
Mtpa–/–	Mitochondrial trifunctional enzyme αd	Unspecified lesions	–
MyoR–/– (Msc–/–); Cap–/– (Tcf21–/–)	Myogenic bHLH transcription factor R (Musculin)a; Transcription factor 21 (Capsulin)a	Posterior hernia	Hypoplasia, defective branching morphogenesis, abscence of alveoli, abnormal vasculature
Ndst1 ECKO	N-deacetylase-N-sulfotransferase 1b	Central hernia	Thick interalveolar septa
Nr2f2–/– (Couptf2–/–)	Nuclear receptor subfamily 2, group F, number 2 (Chicken ovalbumin upstream promoter transcription factor 2)a	Posterolateral hernia	Hypoplasia
Pbx1–/–	Pre-B-cell leukemia transcription factor 1a, b	Muscularization and tissue patterning defect	Hypoplasia, alveolar simplification
Pdgfrα–/–	Platelet-derived growth factor receptor, α-polypeptideb	Posterolateral hernia	Hypoplasia, failure of alveogenesis
Pls3W499C	Plastin 3b, c	Posterolateral and anterior muscular thinning, hernia	–
Rarα–/–;Rarβ–/–	Retinoic acid receptor α and βa	Posterior hernia	Hypoplasia, abnormal alveoli, lung agenesis
Robo1–/–;Robo2–/– or Dutt1;Robo1–/–	Roundabout guidance receptor 1 and 2b	Posterior hernia	Irregular bronchioles, reduced terminal air spaces, abnormal alveoli, thick septa
Six1–/–	Six homeobox 1a, b	Absent diaphragm	Hypoplasia, reduced branching morphogenesis, narrow bronchi, arrested expansion of epithelial tubules, dense mesenchymal cellularity, failure of lung maturation
Slit3–/–	Slit guidance ligand 3b	Central hernia (with sac)	–
Sox7+/Δex2	Sex determining region Y-box 7a	Retrosternal hernia (with sac)	–
Wdr35 yet/yet†	WD repeat domain 35d	Unspecified hernia	Hypoplasia
Wt1–/– or conditinal knockouts (e.g., Wt1CreERT2/+;Bcatfx or G2-Gata4Cre;Wt1fl/fl)	Wilms tumor 1a;β-catenina	Posterolateral hernia	Hypoplasia, abnormally fused and malformed lung lobes, collapsed distal air spaces
Zfpm2–/– (Fog2–/–)†	Zinc finger protein, multitype 2 (Friend of GATA-binding protein 2)a	Posterolateral hernia (with sac)	Hypoplasia, absent right middle and accessory lobe

Transcription Factors and Transcriptional (Co-)Activators

Numerous transcription factors and transcriptional (co-)activators have been suggested in the development of the primordial diaphragm and lungs. Many of them are associated with retinoid or sonic hedgehog signaling pathways.

Retinoid Signaling Pathway

Vitamin A (i.e., retinol) and its derivates (i.e., retinoids) are indispensable for various aspects of early embryogenesis. Over the years, several knockout models have indicated a role of the retinoid signaling pathway and its downstream targets in the pathogenesis of CDH (38). For instance, mice lacking both subtypes of retinoic acid receptors α and β (Rarα and Rarβ) have been demonstrated to generate offspring with posterolateral diaphragmatic defects identical to those observed in human patients (39–44), and similar to the vitamin A-deficient CDH mouse model as previously reported by Anderson (45, 46). Surprisingly, single Rar null mutant mice did not display any of the predicted malformations that were seen in rats with vitamin A deficiency (31). Nevertheless, when the activity of several receptors was inhibited, various deformities were noted including right-sided CDH in Rarα/β2 and left-sided CDH in Rarα/β2+/– animals. Moreover, these mice exhibited severe pulmonary hypoplasia (31). Despite the convincing data, these genetically modified mouse models manifest only a comparatively low incidence of diaphragmatic defects and a high rate of additional comorbidities (e.g., cranial, cardiac, vertebral and limb), which do not accurately depict the human situation (39, 42). Still, mutations in the stimulated by retinoic acid gene 6 (STRA6), a membrane receptor that controls the cellular uptake of vitamin A and cellular retinoic acid binding protein 1 (CRABP1), which is located on chromosome 15, have been found to lead to a spectrum of developmental anomalies including CDH and hyperplastic lungs (47, 48).

Nr2f2 (Couptf2)

Another important gene that is linked with the retinoid signaling pathway is chick ovalbumin upstream promoter transcription factor II (COUP-TFII), a transcription factor that is affiliated with the nuclear steroid/thyroid hormone receptor superfamily, whose DNA-binding site has been shown to reduce the induction of retinoic acid receptors (49–51). COUP-TFII was recently renamed as nuclear receptor subfamily 2 group F (NR2F2), which is expressed in the diaphragm and lungs during early gestation (34). Mapped to chromosome 15q26 in humans, the NR2F2 gene is situated on a recognized CDH hotspot region, thus making it a likely contributor to the etiology of diaphragmatic defects. On the basis of this observation, You et al. (52) have created a tissue-specific Nr2f2 mouse model that features left-sided Bochdalek-type CDH and pulmonary hypoplasia similar to the human situation. Through targeted ablation of Nr2f2 in the foregut mesenchyme and pleuroperitoneal folds (PPFs), posterolateral diaphragmatic defects presumably arise because of the failure of the posthepatic mesenchymal plate to merge with the lateral body wall, thus enabling stomach and liver to protrude into the chest (52).

Wt1

The creation of genetic mouse models for various other applications has revealed several genes, which one would not necessarily immediately associate with CDH. Initially introduced as a model for the investigation of early urogenital organogenesis (53), Wilm’s tumor 1 (Wt1) null mutant mice die during mid-gestation, displaying posterolateral diaphragmatic defects and lung hypoplasia alongside urogenital abnormalities (54–56). Heterozygous mutations of the WT1 gene, which encodes a transcription factor that contains four zinc finger motifs, is known to produce distinct syndromes with clinical overlap that include CDH (e.g., Denys-Drash syndrome or Meacham syndrome) (57, 58). Wt1, vitamin A-deficient and nitrofen mouse models of CDH each implied a mutual pathomechanism for the formation of diaphragmatic defects with several analogies to the condition in humans (59). More recently, Carmona et al. (60) and Cleal et al. (61) have reported that conditional deletion of Wt1 in the mesenchyme of the septum transversum can cause CDH in mice. Today, it is proven that Wt1 and Couptf2 both interact with the retinoid signaling pathway during embryonic development (3). Surprisingly, Wt1 and Couptf2 are not found in the muscle precursors but in the non-muscular mesenchymal compartment of the PPFs (3). Paris et al. (62) have developed a novel genetically modified mouse model of CDH, demonstrating that Wt1-induced β-catenin loss-of-function produces posterior diaphragmatic defects, bilateral pulmonary hypoplasia and liver herniation, comparable to the phenotypes associated with CDH in human patients. Additionally, a decreased mesothelial proliferation and increased rate of cell death was identified in the posterior diaphragm mesenchyme, and all mouse pups died postnatally with malformed lung lobes and collapsed distal air spaces (62). Loss of Wt1 has also been associated with lung branching defects before diaphragm closure in another genetic model of CDH (63).

Sonic Hedgehog Signaling Pathway

GLI-Kruppel family member 2 (Gli2) and Gli3 and are both members of a highly conserved morphogenetic family, belonging to the sonic hedgehog (Shh) signaling pathway (64). This pathway is thought to be crucial during normal diaphragmatic development (36). A murine model of the VACTERL-like syndrome (i.e., vertebral, anorectal, cardiac, tracheoesophageal, renal and limb anomalies) created by Kim et al. (65) involved Gli2;Gli3 and Gli2;Gli3+/– mice that developed left-sided posterior CDH and pulmonary hypoplasia besides the observed VACTERL components. This was the first experimental model that reproduced the human VACTERL association, indicating that disruptions in Shh signaling might contribute to the pathogenesis of VACTERL syndrome. Likewise, as Gli2, Gli3 and Wt1 all encode important zinc finger proteins, further transcription factors of this type have been hypothesized through the generation of newer genetic animal models of CDH. For example, kinesin family member 7 (Kif7) and pre-B-cell leukemia transcription factor 1 (Pbx1) were recently recognized as indispensable components of the Shh signaling pathway, functioning as regulators during early embryogenesis (66, 67). Kif7 encodes a motor protein that functions downstream of the transmembrane receptor smoothened, and interacts with both Gli2 and Gli3 (68). Furthermore, Kif7 was found to coordinate cell proliferation, central tendon patterning and differentiation of the primordial diaphragm in a genetically modified mouse model of CDH (69). Homozygous Kif7 mutant mice and Pbx1 knockout mice both display left-sided posterior diaphragmatic defects and hypoplastic lungs (36, 69–71). In turn, haploinsufficieny of PBX1 has been associated with various congenital anomalies including CDH (72). Moreover, two predicted variants in the KIF7 gene were recently detected in patients with CDH (73). Additionally, mice lacking chromatin target of protein arginine methyltransferase 1 (Chtop) have numerous developmental abnormalities including posterolateral defects in the diaphragm, pulmonary hypoplasia and liver herniation (74–76). High-resolution 3D imaging further characterized these diaphragmatic defects in Chtop mice embryos (77).

Zfpm2 (Fog2), Gata4 and Sox7

Zinc finger protein 2 (ZFPM2), formerly known as friend of GATA-binding protein 2 (FOG2), encodes another zinc finger-containing protein that regulates the transcriptional activity of GATA4, hereby controlling a number of developmental mechanisms in the forming diaphragm and lung (78–81). In mice, Fog2 was initially found to be expressed in the embryonic septum transversum of the diaphragm (81). In humans, ZFPM2 is located on chromosome 8p23 and has been demonstrated to interact with COUP-TFII (82, 83). However, only a single mutation in the ZFPM2 gene has been identified in isolated patients with non-syndromic CDH to date (31). In a cohort of 275 patients with CDH, Longoni et al. (84) have recently reported the incidence of ZFPM2 mutations to be nearly 5%. In addition, their genetic analysis of a multigenerational family revealed a heritable intragenic ZFPM2 deletion with an approximated penetrance for clinical relevant diaphragmatic defects of around 37.5% (84). On the other side, mice exposed to the chemical mutagen N-ethyl-N-nitrosourea (ENU) generated Fog2–/ offspring with bilateral hypoplastic lungs and a defective posterolateral diaphragm characteristic of CDH (80), while 70% of mice heterozygous for a Gata4 deletion mutation (i.e., Gata4) displayed retrosternal diaphragmatic defects, dilated distal airways and thickened pulmonary mesenchyme (85). Using genetically modified mice, Merrell et al. (86) have shown that Gata4 mosaic mutations in PPF-derived muscle connective tissue fibroblasts led to the development of localized amuscular regions of the diaphragm, which were biomechanically weaker and subsequently caused CDH. GATA4 and ZFPM2 genes have been both found to be absent in humans with CDH (78), emphasizing their roles as possible candidate genes for CDH. Moreover, Zfpm2 is known to interact with Nr2f2, indicating that these two transcription factors together with Gata4 may contribute to diaphragm formation (83). Recurrent microdeletions of 8p23.1, including GATA4 and the sex determining region Y-box 7 (SOX7) gene are accompanied with a significant risk of CDH and cardiovascular anomalies (87). Even though mice lacking the Gata4 gene display both diaphragmatic and cardiac defects, no human patient with cardiac anomalies and GATA4 mutations have been identified with CDH so far (87). However, Wat et al. (87) have recently demonstrated that haploinsufficiency of Sox7 or Gata4 is enough to cause retrosternal diaphragmatic defects in mice and that haploinsufficiency of SOX7 and GATA4 may in turn be involved in the pathogenesis of CDH in patients with 8p23.1 deletions.

Cap (Tcf21) and MyoR (Msc)

Several basic helix-loop-helix transcription factors have been shown to support the development of the primordial diaphragm in mice. Capsulin (Cap) is one of those, which is strongly expressed in the fetal diaphragm and in mesenchymal cells of the lung (88). As one might expect, Cap+/– mice have severe defects in lung morphogenesis and lack alveoli (89). On the other hand, mice homozygous deficient for both cap and the related myogenic bHLH transcription factor R (MyoR lack facial musculature and exhibit posterior diaphragmatic defects. This double mutant mouse model, which was generated at first to study the formation of facial muscles, displayed not only CDH and defective lung branching morphogenesis but also severe facial muscle abnormalities (90). Although these genetically modified mice died soon after birth because of pulmonary and cardiac malformations, the type of diaphragmatic defect seen in this model indicates that both Cap and MyoR are necessary for the integrity of the developing diaphragm. Previously, these genes were referred to as transcription factor 21 (Tcf21) and musculin (Msc), respectively (90, 91).

Eya1 and Six1

Eyes absent (Eya) genes and the transcription factor sine oculis homebox 1 (Six1) form an important signaling network, which plays a central role during embryonic development (92, 93). Eya1 and Six1 together constitute an evolutionary conserved transcriptional complex that coordinates multiple integrated processes needed for normal growth of the primordial diaphragm and lung (94). Further research work has confirmed that the Eya1-Six1 pathway has a key role in lung maturation by regulating its branching morphogenesis (95). Mice deficient in Eya1/ and Eya2/+ have no diaphragm (94), whereas single mutant Eya1 mice die shortly after birth due to respiratory failure, having severely hypoplastic lungs with reduced epithelial branching and increased mesenchymal cellularity (95). In Six1 mice, the diaphragm is also absent, and the Six1 deletion causes pulmonary hypoplasia with greatly reduced epithelial branching, narrow bronchi, dense mesenchyme and obvious failure of normal lung maturation (96, 97). These findings indicate that disruption of the Eya1-Six1 signaling pathway may lead to neonatal lethality as a consequence of an absent diaphragm and hypoplastic lungs.

Hlx and Hoxb4

H2.0-like homeobox (Hlx) is a protein coding gene that is relatively conserved across various species (98). This homeobox transcription factor has been found to be highly expressed during early organogenesis in the septum transversum of the diaphragm and lung mesenchyme (99, 100). Hlx mice suffered early demise and showed diaphragmatic defects (101). Additionally, Farrell et al. (102) reported two human fetuses with multiple congenital anomalies including CDH that were homozygous for a missense variant in the HLX gene. The Hox gene family encodes for multiple transcription factors that have crucial regulatory functions during embryonic development (103). Targeted mutation of the homeobox B4 (Hoxb4) gene in mice resulted in offspring with poorly formed diaphragms and diaphragmatic defects, strikingly similar to the phenotype seen in humans with anterior CDH (104).

Molecules Implicated in Cell Migration, Proliferation and Mesodermal Patterning

Various genes and enzymes involved in cell migration, proliferation and mesodermal patterning have been found to be associated with embryonic diaphragm and lung development.

Slit3, Robo1/2, Ndst1 and Pdgfra

The Slit guidance ligand (Slit) family of proteins comprise a group of molecules with crucial functions in cell migration and adhesion through interaction with roundabout (Robo) receptors. Slit genes are expressed in the mesothelium of the diaphragm during embryogenesis (105). Homozygous Slit3 mice experience faulty detachment of the central tendon region of the diaphragm from the underlying liver due to connective tissue defects, thus causing central-type (i.e., septum transversum) CDH (105, 106). Therefore, this genetically modified model is facing the disadvantage of having the diaphragmatic defect on or near the ventral midline portion of the central tendon as opposed to the posterolateral diaphragm, thus representing less than 5% of CDH cases seen in human patients. Further malformations in this mouse model include ureteric and renal agenesis in combination with intrathoracic herniation of liver and gallbladder (106), which again occurs infrequently in humans with CDH. Until now, no SLIT3 mutations have been identified in CDH patients. Robo genes encode large transmembrane receptors that are involved together with their ligands in numerus developmental mechanisms (107–109). For example, the Slit-Robo signaling pathway has been reported to have various fundamental functions including axon guidance, neural crest cell migration, epithelial cell adhesion, embryonic heart formation as well as diaphragm and kidney development (105–107, 110–114). Inactivation of Robo1 and Robo2 genes in mice has been shown to cause diaphragmatic defects and subsequent herniation of the stomach into the thorax, which leads to poor lung inflation and perinatal death, similar to human CDH cases (107). Homozygous mice with targeted deletion in the Dutt1/Robo1 gene often die at birth due to respiratory failure, demonstrating delayed lung maturation and diaphragmatic defects in some instances (115). More recent studies identified the heparan sulfate proteoglycan as an essential part of the Slit-Robo signaling complex, which stabilizes the Slit-Robo interaction (116). Furthermore, Zhang et al. (117) have noted that absence of the heparan sulfate biosynthetic enzyme N-deacetylase-N-sulfotransferase-1 (Ndst1) in the mouse endothelium interferes with vascular development in the primordial diaphragm, resulting in hypoxia as well as diaphragmatic hypoplasia and central-type CDH. The observed phenotypes in these animals mirror the congenital anomalies seen in Slit3 knockout mice. In addition, implementation of a heterozygous mutation in the Robo4 gene, which encodes the receptor of Slit3, exacerbated the defect in vascular and diaphragmatic formation (117). Thus, these findings suggest that loss of Ndst1 may lead to abnormal vasculogenesis in the diaphragm and CDH and that heparan sulfate in turn promotes the angiogenic Slit3-Robo4 signaling cascade during normal vascular patterning. Apart from this, mice homozygous for null mutations in the platelet-derived growth factor receptor α (Pdgfra) gene exhibit not only posterolateral diaphragmatic defects, they also develop a spectrum of other comorbidities including cardiovascular anomalies, renal and urogenital malformations, facial clefts, lung hypoplasia and failure of alveogenesis (118, 119).

Fuz, Met and Pls3

Inbred C57BL/6J mice chemically mutagenized with ENU displayed a previously unknown mutation in the fuzzy planar cell polarity protein (Fuz) that was associated with CDH, liver protrusion into the chest cavity and pulmonary hypoplasia with a single left lung lobe (120). The mesenchymal-epithelial transition factor (Met) gene encodes for a receptor tyrosine kinase that is necessary for the migration of muscle precursor cells into the forming diaphragm (121), whereas fibroblast growth factor 10 (Fgf10) is crucial for early organogenesis of the lung (122). Oral administration of the herbicide nitrofen in Met mice with amuscular diaphragms and Fgf10 mice with hypoplastic lungs resulted in CDH in both murine models, indicating that diaphragmatic defects may develop independently of myogenesis and pulmonary development (123). A novel missense variant affecting the actin-binding domains of plastin 3 (PLS3) was recently identified in eight unrelated families, causing X-linked CDH and body wall defects. A genetically modified mouse model of this Pls3 variant resulted in perinatal death and reproduced the main features of the human phenotype, including diaphragmatic and body wall abnormalities (124). An abnormal plastin-actin interaction is the most likely explanation for the observed congenital malformation in both humans and mice.

Components Involved in the Formation of Extracellular Matrix

Normal development of the primordial diaphragm and lung is also dependent on the proper formation of its underlying extracellular matrix (ECM). Today, several components of the ECM are known to be aberrant in CDH and associated lung defects.

Fraser extracellular matrix complex subunit 1 (Fras1), Fras1-related extracellular matrix 1 (Frem1) and Frem2 form a mutually stabilizing ternary complex in the ECM, which plays a critical role in cell adhesion and intercellular signaling (125, 126). After identification of a novel FREM1 deletion in a female infant with isolated left-sided CDH and a membranous sac, Beck et al. (127) developed a Frem1-deficient mouse model that displays a comparable phenotype with retrosternal diaphragmatic defect and reduced levels of cell proliferation in the anterior portion of the growing diaphragm, hereby showing that a deficit of FREM1 can lead to CDH in both humans and mice. Because of the observed phenotypic similarities between Frem1-deficient mice and mice lacking the retinoic acid-responsive transcription factor Gata4, the same author group conducted further studies, revealing that Frem1 interacts not only with Gata4 but also with Slit3 in this mouse model of CDH and concomitant lung lobulation defects (128). More recently, Jordan et al. (129) reported that Frem2 and Fras1 mice developed an almost identical type of anterior midline CDH with herniated viscera covered by a thin membranous sac as seen in Frem1-defcient mice, thus concluding that loss of the Frem1/Frem2/Fras1 complex or its function results in retrosternal CDH in these animals. The cross-linking of collagens and elastin, which is essential for the structural stability of the ECM, is catalyzed by lysyl oxidase (Lox), an extracellular cuproenzyme (130). In turn, Lox mice die at birth, having a ruptured diaphragm as a result of fragmentation in the central tendon (131, 132). However, no human LOX mutations have been reported so far. Another ECM protein associated with the pathogenesis of CDH is fibrillin 1 (Fbn1), an integral part of microfibrils in elastic and non-elastic connective tissues (133). A gene-targeting mutation of the mouse Fbn1 gene has been associated with diaphragmatic defects and histological examination revealed a focal inflammatory infiltrate at the ruptured edges (134). These homozygous Fbn1 mutant mice died postnatally due to pulmonary insufficiency, exhibiting CDH and herniation of abdominal viscera into the thoracic cavity (134). Fibulin 4 (Fbln4) also belongs to a family of ECM proteins, which controls fiber assembly and is known to bind Lox (135). Fbln4 null mutant mice die just after birth with severe CDH and rupture of the diaphragm, in addition to defective distal airways and lung emphysema (136).

Additional Genes and Enzymes Participating in Signal Transduction and Apoptosis

Several other genes and enzymes responsible for signal transduction, intracellular signaling and apoptosis have been discovered in association with diaphragmatic defects in genetically modified mouse models.

The acetylcholine-synthesizing enzyme choline acetyltransferase (ChAT) has been reported to be implicated in various morphogenetic processes during embryonic development (137, 138). In fact, cross-sections of the diaphragm from ChAT mice showed liver herniation through the tendinous center of the diaphragm (139), presumably as a consequence of impaired muscle formation. Deoxyribonuclease IIα (DNase IIα) belongs to a large group of endonucleases involved in DNA digestion during apoptosis. DNase IIα mice displayed a malformed diaphragm with hernia and non-inflated lungs, suggesting that these animals suffer perinatal lethality because of a dysfunctional diaphragm and associated respiratory insufficiency (140). Low-density lipoprotein receptor-related protein 1 (Lrp1) is crucial for proper embryonic development through regulation of intracellular signaling cascades (141). ENU-induced mutation in the Lrp1 gene of mice resulted in body wall closure defects with CDH and liver protruding outside of the abdominal cavity (142). Mitochondrial trifunctional protein (Mtp) is a multi-enzyme complex of four α and four β subunits that catalyzes oxidation of long-chain fatty acids, which is essential for normal embryogenesis (143). Mtpα knockout mice suffer neonatal death with cardiac and diaphragmatic defects, indicating that deficiency of Mtpa may cause dysfunction of the diaphragm and subsequent respiratory insufficiency (143). WD repeat domain 35 (Wdr35) is a protein coding gene, which participates in intracellular trafficking, cargo recognition and binding during embryonic development (144). Following a recessive ENU mutagenesis screen for genes affecting embryogenesis, Mill et al. (144) noticed that mutant Wdr35 mice embryos died before birth, displaying diaphragmatic defects and hypoplastic lungs.

Conclusion and Future Directions

Over the years, experimental animal models of CDH have not only permitted us to investigate the pathogenesis of this relatively common but complex birth defect in more detail, they have also led to a better understanding of the molecular genetic basis of the underlying tissue defects. Therefore, animals with CDH in which this congenital anomaly develops naturally represent the ideal research models to study disease pathomechanisms and related lung abnormalities, as there is minimal interference to the animal before the examination. Furthermore, genetically modified animal models of CDH not only resemble the natural development of this malformation, they also provide new insights into the participating genes and signaling pathways, and how their modification can potentially change the course of this life-threatening condition. With the recent advent of novel molecular techniques including biomedical engineering and ENU mutagenesis screens, we hopefully may identify additional CDH-related mutations that are linked with abnormal diaphragm and lung development in other genetic mouse models (145–147).

Author Contributions

FF, UR, and PP critically revised the initial manuscript draft for important intellectual content and performed the literature search for the work. FF outlined and wrote the initial manuscript draft. All authors approved the final version to be published and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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