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. Congenital diaphragmatic hernia (CDH) is a severe birth defect, characterized by incomplete formation or muscularization of the diaphragm and, as a consequence, herniation of the stomach, intestines, liver or spleen into the pulmonary cavities, leading to pulmonary hypoplasia. CDH occurs approximately in 1 out of 3000 births, accounting for about 8% of all severe congenital anomalies.

80–90% of all the cases are posterolateral hernias, also known as Bochdalek-type CDH, characterized by a defect in the postero (dorsal in mice) lateral area of the diaphragm. In most cases (85%), this defect is located at the left side. The classical hypothesis is that Bochdalek CDH is due to a failure of the fusion of pleuroperitoneal folds (PPFs) with the septum transversum (ST).

PPFs are lateral rims of tissue connecting caudally with the nephric ridges and anteriorly with the ST. Some authors describe that this fusion rather occurs with the posthepatic mesenchymal plate (PHMP), an accumulation of mesenchymal cells derived from the ST and located in the posterodorsal margin of the liver lobes. Although all these tissues form an anatomical continuum, we will refer to the posterior, dorsolateral areas of the liver as PHMP, and PPFs to the tissue folds located in the dorsal part of the coelomic cavity, marking the limit between the peritoneal and the pleural cavities.

The clinical aspects of CDH are well studied but its etiology is still poorly known. Animal models are therefore very valuable in order to investigate the cellular and molecular processes leading to CDH in humans. Recently, it has been described in mice that deletion of GATA4 in the PPFs mesenchyme using a Prrx1 Cre driver leads to the development of amuscular, weak areas in the diaphragm and CDH. This paper assumes that Prrx1 is expressed in the mesenchyme of the pleuroperitoneal folds (PPFs) but not in the ST, and in fact, it suggests that the ST contributes minimally to the definitive diaphragm. However, this report does not distinguish between PPFs and PHMP and the phenotype does not display diaphragmatic discontinuities, neither does it show a prevalence on the left side, which are common features in human CDH.

The retinoic acid (RA) signaling pathway has also been involved in diaphragmatic development. A classical animal model for CDH consists in the treatment of pregnant rats with nitrofen, a substance that inhibits the synthesis of RA. It has been shown that nitrofen treatment leads to reduced size of the PPFs/PHMP, especially in the left side, decreasing cell proliferation in this area (; ) and also decreasing expression of WT1 and GATA4 (, ). RA treatment in this model partially rescues the pulmonary hypoplasia defect (; ). The phenotype of this animal model is thus, quite similar to the human one. To date, only a few examples of CDH in human have been associated to mutations in WT1 locus, such as the Denys-Drash, Meacham and WAGR syndromes (;; ).

WT1 is expressed in the coelomic epithelium of the septum transversum of mouse embryos by the stage E9.0. Loss of function of the Wilms' tumor suppressor gene Wt1 in mice leads to defective diaphragms (; ). However, the mechanism by which WT1 contributes to diaphragm development is still unknown.

To study the role of WT1 in CDH we performed loss-of-function experiments by conditionally inactivating the Wt1 gene in the ST/PHMP/PPFs mesenchyme. The use of WT1 conditional knockout overcomes the early embryonic death caused by systemic deficiency of WT1.

We used a driver based on the G2 enhancer of the Gata4 gene. This enhancer drives expression of Gata4 in the lateral plate mesoderm from the stage E7.5, and by the stage E9.5 is active in the septum transversum and proepicardium, ceasing its activity by E12.5 The activity of this enhancer is completely absent in the intermediate mesoderm. Our findings indicate that WT1 is involved in the generation of the mesenchyme of the ST/PHMP/PPFs continuum through epithelial-mesenchymal transition and they provide a novel perspective on the genesis of the Bochdalek hernia and the evolutionary origin of the diaphragm. In normal E10.5 embryos, the posterior and dorsal margin of the liver shows an accumulation of mesenchymal tissue, which extends from the dorsal mesenterium of the liver to the lateral tips of the lobes. This mesenchymal layer is the PHMP described.

Student Guide

The PPFs, by this developmental stage, appear as a pair of outgrowths of the body wall located at both sides of the lung buds. They are also constituted of mesenchymal cells lined by the coelomic epithelium. The G2- Gata4 enhancer directed LacZ reporter expression in both PPF and PHMP at E10.5 and E11.5 in the mouse embryo. However, no beta galactosidase activity was observed in the posterior and medial part of the septum transversum, where the liver is connected with the digestive tract (asterisk in ), indicating that the G2 enhancer is not active in this specific domain.

( A– D) Wildtype ( A, C) and mutant ( B, D), E10.5 littermates. The larger amount of mesenchymal cells in the posthepatic mesenchymal plate (PHMP) is evident in the wildtype, especially in the right side. Serial sections corresponding to the connection between the PHMP and the pleuroperitoneal folds (PPF) are shown in C. Note the presence of compact mesenchyme in the PHMP and also in the closest part of the PPF (arrow in C). A corresponding section of the mutant in shown in D. Note the lack of PHMP, the coelomic epithelium lying directly on the hepatic tissue (arrows in D) and the limit of the septum transversum (ST) mesenchymal cells (arrowhead in D), which do not extend laterally.

( E) Serial sections of a G2- Gata4 Cre; Wt1 fl/flE11.5 embryo at levels equivalent to those shown in. Despite the presence of normal PHMP in the anterior part, the posterior areas of the liver lack of lateral mesenchymal cells (arrow). The mesenchyme is restricted to the central ST. Renal ridges (RR) appear at a level corresponding to the entrance of the oesophagus (OE) into the ST. MG: mesogastrium. ( F) Wildtype E13.5 embryo showing complete isolation of the pleural cavities by the pleuroperitoneal membranes that constitute the main part of the diaphragm ( D). ( G) G2- Gata4 Cre; Wt1 fl/flE14.5 embryo at eight different levels showing left diaphragmatic defect with herniation of the left liver lobe (LI) into the pleural cavity and severe hypoplasia of the left lung (LL).

Ectopic muscle appear in the mediastinum (MM). Adrenals (AD), mesonephros (MN), gonads (GON) and spleen (SP) appear normal. LU: lungs; H: heart; RL: right lung; STO: stomach. This is a basically descriptive study of the phenotype of mouse embryos with conditional deletion of Wt1 in lateral mesoderm.

Fusion Cdh Student Guide

We have included in the study 36 out 104 mutant embryos detected by genotyping. The rest of them had been previously used for a study of the cardiac phenotype. To understand the role of WT1 positive cells in the development of diaphragm, we conditionally inactivated Wt1 gene using the described G2- Gata4 Cre line. Deletion of WT1 in the PHMP/PPFs continuum, i.e. In the G2 + domain, causes a phenotype characterized by defects in the inflow tract of the heart, coronary vessels and, importantly, Bochdalek's hernia, most frequently located in the left side. The genotypes of the embryos obtained at different ages show the expected percentage of mutants at all embryonic stages (about 25%) except for the stage E15.5, when we only obtained four mutants among 60 embryos.

The number of embryos studied in older stages (42 embryos, nine mutants, 21,4%), was not enough to confirm if the mutation causes some lethality in late gestation.We have included seven embryos from females fed with control diet in the RA-rescue experiment. One embryo showed CDH only at the right side. We analyzed WT1 mutant embryos at different stages of development. Defect in the PHMP in G2- Gata4 Cre;Wt1 fl /fl mutant embryos could be observed as early as E10.5, since less mesenchymal cells are present between the coelomic epithelium and the hepatoblasts when compared with control littermates.

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At later stages, the closure of the pleural cavity by growth of the ST/PPFs crescent is delayed with respect to the controls, and the mesenchymal cells of the PHMP are located more medially, not in the lateral tips of the liver lobes. This is more evident at the left side (arrow in ). Thus, these cells cannot migrate towards the PPFs precluding caudal growth of the lateral commissures of the pleuroperitoneal septa and closure of the pleural cavities. Five E12.5 mutant embryos studied already showed strong reduction of the PHMP size as compared with control littermates, but the diaphragmatic defect of the WT1 conditional knockout embryos became clearly apparent by E13.5, when the pleural cavities are almost completely isolated in wildtype embryos.

Of 23 mutant embryos analyzed by the stages E13.5 or older, only four of them showed normal development of the diaphragm, while the rest of embryos showed different degrees of diaphragmatic defects. Three of the embryos showed thin, membranous diaphragm in the left side and 16 showed a wide defect in the diaphragm, frequently with herniation of the liver into the left pleural cavity and hypoplasia of the left lung. One of them (E13.5) showed the defect only at the right side and the CDH was present in the left side in the rest of them. Thus, about 80% of the E13.5 or older mutant embryos showed abnormal diaphragmatic development. Renal ridges appear well developed in mutant E11.5 embryos at a level corresponding to the entrance of the oesophagus into the ST. The presence of intermediate mesoderm in these thoracic renal ridges was confirmed by ISH of Pax2.

In wildtype embryos of the same age thoracic renal ridges can also be seen, but located more caudally, at the level of the stomach. ( A) Control E11.5 embryo.

Pax2 is expressed in the neural tube (NT). The pleuroperitoneal folds (PPF) lack of Pax2 expression. ( B) G2- Gata4 Cre; Wt1 fl/fl E11.5 embryo. Pax2 is expressed in the PPF at the level of the lung buds (LU). The expression is stronger in the tubular structures that are developing in the left PPF (insert). The sharp boundary between the above explained G2 + and G2 − domains was clearly observed by the immunolocalization of WT1 and GATA4 protein in G2- Gata4 Cre;Wt1 fl/fl mutant embryos.

WT1 immunoreactivity is strong in the PHMP and PPF of the wildtype embryos and localizes in the coelomic epithelium and, with a lower intensity, in mesenchymal cells. However, WT1 is clearly downregulated in the G2 + domain of the mutant embryos indicating an efficient excision of the WT1 floxed allele. Immunolocalization of GATA4 protein marked the border between both G2 domains, confirming the absence of endogenous GATA4 protein and the inactivity of the G2 enhancer in the G2 - domain. The presence of GATA4+ cells in the more cephalic part of the PPF supports a migration of PHMP cells towards the PPFs. Since WT1 is involved in epithelial-mesenchymal transition (EMT) of the epicardium by repression of E-cadherin and activation of Snail1 , we checked the expression of E-cadherin in the developing PHMP of G2- Gata4 Cre;Wt1 fl/fl mutant embryos. Control E11.5 embryos show an E-cadherin negative coelomic epithelium over a layer of mesenchymal cells in the developing PHMP.

However, mutant embryos show expression of E-cadherin in the epithelium of the corresponding area and lack of E-cadherin negative mesenchymal cells.