Transgenic animal models of congenital diaphragmatic hernia: a comprehensive overview of candidate genes and signaling pathways

Congenital diaphragmatic hernia (CDH) is a relatively common and life-threatening birth defect, characterized by incomplete formation of the diaphragm. Because CDH herniation occurs at the same time as preacinar airway branching, normal lung development becomes severely disrupted, resulting almost invariably in pulmonary hypoplasia. Despite various research efforts over the past decades, the pathogenesis of CDH and associated lung hypoplasia remains poorly understood. With the advent of molecular techniques, transgenic animal models of CDH have generated a large number of candidate genes, thus providing a novel basis for future research and treatment. This review article offers a comprehensive overview of genes and signaling pathways implicated in CDH etiology, whilst also discussing strengths and limitations of transgenic animal models in relation to the human condition.


Introduction
Congenital diaphragmatic hernia (CDH) is a developmental abnormality characterized by the presence of a defect in the integrity of the forming diaphragm, affecting between 1.9 and 2.3 cases per 10,000 newborns in the United States [1] and Europe [2]. Defects in the posterolateral diaphragm, commonly referred to as Bochdalek hernias, comprise approximately 90-95% of all CDH cases with about 80% occurring on the left side, 15% on the right and less than 5% bilaterally [3]. Non-posterolateral CDH manifests as anterior conditions such as Morgagni hernias in the anterior retrosternal or peristernal diaphragm and central hernias in the central tendinous portion of the diaphragm [4].
Posterolateral diaphragmatic defects permit protrusion of the abdominal viscera into the thoracic cavity, thus interfering with normal lung development and frequently leading to severe respiratory distress at birth due to the unfortunate combination of pulmonary hypoplasia and persistent pulmonary hypertension of the newborn [3,5].
Over the last decade, CDH remained a life-threatening congenital disorder with mortality rates up to 50% [6][7][8][9]. Treatment usually consists of surgical movement of the abdominal viscera out of the thoracic cavity and closure of the diaphragmatic defect. Large defects may be difficult to repair through direct sutures, requiring the use of a prosthetic patch or abdominal muscle flap [10,11]. Apart from surgical methods, treatment options for CDH are limited due to its poorly understood etiology, thereby motivating the need for better experimental models to elucidate its pathogenesis while also testing new therapeutic approaches. Investigation of novel medical therapies and pharmacological compounds that have the ability to arrest or reverse associated lung hypoplasia in animal models of CDH require the application of standardized research methodologies [12]. In this review article, we discuss the development of and findings associated with transgenic animal models of CDH to highlight the progress made to date in understanding CDH pathogenesis and evolution.

Transgenic animal models of CDH
Both environmental and genetic factors are thought to contribute to the etiology of CDH. To date, genetic causes have been identified in approximately 30% of neonates with CDH [13][14][15]. With the advent of innovative molecular techniques in recent years, transgenic animal models of CDH have become more common, offering new candidate genes and signaling pathways implicated in the pathogenesis and etiology of diaphragmatic defects and associated lung abnormalities (Table 1). So far, 18 mouse models with phenotypic similarities to human CDH have been listed in the Mouse Genome Database (http://www.infor matic s.jax.org).

Retinoid signaling pathway
Several knockout models have originated from gene pathways found to be associated with CDH such as the retinoid signaling pathway [13]. Mice deficient in both subtypes of retinoic acid receptors α and β (Rarα and Rarβ) have been shown to produce offspring with CDH [16-21], consistent with the vitamin A-deficient mouse models observed by Anderson [22,23]. Single Rar null mutation mice did not exhibit the expected anomalies, which were reported in vitamin A-deficient rats [9]. However, when the function of these receptors was suppressed, multiple congenital anomalies were observed, including right-sided CDH in Rarαβ2 mutant mice and left-sided CDH in Rarαβ2 +/− animals. In addition, these mice suffer from severe pulmonary hypertension at birth [9]. Unfortunately, these animals demonstrate a relative low rate of diaphragmatic defects and a high incidence of comorbidities including cranial, vertebral, limb, cardiac, foregut and pulmonary malformations that do not accurately reflect human CDH [20,21]. Nevertheless, mutations in the stimulated by retinoic acid gene 6 (STRA6) and cellular retinoic acid binding protein 1 (CRABP1) on chromosome 15 have been identified in CDH patients [9].

Conclusion and future directions
Experimental animal models of CDH have not only allowed us to study the pathophysiology and etiology of this relatively complex birth defect, but have also provided new insights into the molecular and biochemical basis, thus contributing to advances in the medical and surgical management. Hence, CDH animals in which this malformation occurs naturally are ideal models to investigate disease pathogenesis and associated pulmonary hypoplasia, as there is little or no interference to the animal prior to the study. Additionally, transgenic animal models of CDH not only mimic the natural occurrence of this condition, but also give a better understanding into the genes involved and how their modification might alter the course of the disease. Teratogen-induced CDH models although useful, have in turn the drawback of exposing the animals to a generalized noxious stimulus, which can result in widespread detrimental effects rather than simply targeting a specific organ system. The combination of transgenic animal models with regenerative tissue engineering and stem cell-based therapy may play a role in future CDH research by developing a myogenic patch capable of restoring muscle fraction in fetal diaphragmatic defects and promoting regeneration of hypoplastic lungs [67][68][69][70].
Acknowledgements Open Access funding provided by Projekt DEAL.

Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest.