Supporting conditional mouse mutagenesis with a comprehensive cre characterization resource.

Full realization of the value of the loxP-flanked alleles generated by the International Knockout Mouse Consortium will require a large set of well-characterized cre-driver lines. However, many cre driver lines display excision activity beyond the intended tissue or cell type, and these data are frequently unavailable to the potential user. Here we describe a high-throughput pipeline to extend characterization of cre driver lines to document excision activity in a wide range of tissues at multiple time points and disseminate these data to the scientific community. Our results show that the majority of cre strains exhibit some degree of unreported recombinase activity. In addition, we observe frequent mosaicism, inconsistent activity and parent-of-origin effects. Together, these results highlight the importance of deep characterization of cre strains, and provide the scientific community with a critical resource for cre strain information.

Mechanisms of action of the congenital diaphragmatic hernia-inducing teratogen nitrofen.

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.

Fog2 is required for normal diaphragm and lung development in mice and humans.

Congenital diaphragmatic hernia and other congenital diaphragmatic defects are associated with significant mortality and morbidity in neonates; however, the molecular basis of these developmental anomalies is unknown. In an analysis of E18.5 embryos derived from mice treated with N-ethyl-N-nitrosourea, we identified a mutation that causes pulmonary hypoplasia and abnormal diaphragmatic development. Fog2 (Zfpm2) maps within the recombinant interval carrying the N-ethyl-N-nitrosourea-induced mutation, and DNA sequencing of Fog2 identified a mutation in a splice donor site that generates an abnormal transcript encoding a truncated protein. Human autopsy cases with diaphragmatic defect and pulmonary hypoplasia were evaluated for mutations in FOG2. Sequence analysis revealed a de novo mutation resulting in a premature stop codon in a child who died on the first day of life secondary to severe bilateral pulmonary hypoplasia and an abnormally muscularized diaphragm. Using a phenotype-driven approach, we have established that Fog2 is required for normal diaphragm and lung development, a role that has not been previously appreciated. FOG2 is the first gene implicated in the pathogenesis of nonsyndromic human congenital diaphragmatic defects, and its necessity for pulmonary development validates the hypothesis that neonates with congenital diaphragmatic hernia may also have primary pulmonary developmental abnormalities.

Reductions in the incidence of nitrofen-induced diaphragmatic hernia by vitamin A and retinoic acid.

Congenital diaphragmatic hernia (CDH) is a serious medical condition in which the developing diaphragm forms incompletely, leaving a hole through which the abdominal contents can enter the thoracic space and interfere with lung growth. A perturbation of the retinoid system has been linked to the etiology of CDH. This includes findings that nitrofen, which induces CDH in rodents, inhibits the key enzyme for retinoic acid (RA) production, retinaldehyde dehydrogenase-2 (RALDH2) in vitro. Published studies indicate that antenatal vitamin A administration on gestational day (D) 12 in the nitrofen model of CDH reduced the severity and incidence of right-sided defects and lung hypoplasia. In this study, we administered nitrofen on D8, to include the induction of clinically more prevalent left-sided defects, and examined the efficacy of several vitamin A administration paradigms to gain insights into the developmental stage of susceptibility. Furthermore, we tested the hypothesis that administration of RA, the product of RALDH2 activity, is more potent than administering the substrate, vitamin A, in reducing the incidence of CDH. The incidence of CDH was reduced from approximately 54% (nitrofen alone) to approximately 32% with vitamin A treatment. The efficacy of RA treatment was very marked, with a reduction in the incidence of CDH to approximately 15%. Administration of vitamin A or RA on approximately D10 was most effective. These data lend further support for the potential involvement of retinoid signaling pathways and the etiology of CDH and support data from in vitro studies demonstrating a nitrofen-induced suppression of RALDH2.

A genetic model for a central (septum transversum) congenital diaphragmatic hernia in mice lacking Slit3.

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.

Etiology of congenital diaphragmatic hernia: the retinoid hypothesis.

Congenital diaphragmatic hernia (CDH) is a major life-threatening cause of respiratory failure in the newborn. Although significant efforts have been undertaken to unravel the pathophysiology of CDH, our current understanding of the etiology remains spare. Here we outline recent evidence suggesting that abnormalities linked with the retinoid signaling pathway early in gestation may contribute to the etiology of CDH. These studies include 1) the effect of altering the retinoid system in vitamin A deficient and transgenic animals; 2) disruption of the retinoid system in teratogen-induced CDH in rodents, 3) the effect of co-administration of retinoids in nitrofen-induced CDH on lung and diaphragm development, and 4) clinical evidence suggesting decreased markers of vitamin A status in human CDH. Given the substantial mortality and morbidity associated with this serious developmental anomaly, advancements in this area will be critical. We feel that there is now sufficient circumstantial and direct experimental evidence to warrant further testing of the retinoid-CDH etiology hypothesis, including examination of retinoid-regulated target genes that could be candidates for involvement in CDH.

Retinal dehydrogenase-2 is inhibited by compounds that induce congenital diaphragmatic hernias in rodents.

Currently, the etiology of the serious developmental anomaly congenital diaphragmatic hernia (CDH) is unknown. We have used an animal model of CDH to address this issue. We characterized four separate teratogens that produced diaphragmatic defects in embryonic rats that are similar to those in infants with CDH. We then tested the hypothesis that all these agents share the common mechanism of perturbing the retinoid-signaling pathway. Specifically, inhibition of retinal dehydrogenase-2 (RALDH2), a key enzyme necessary for the production of retinoic acid and that is expressed in the developing diaphragm, was assayed by measuring retinoic acid production in cytosolic extracts from an oligodendrocyte cell line. The following compounds all induce posterolateral defects in the rat diaphragm; nitrofen, 4-biphenyl carboxylic acid, bisdiamine, and SB-210661. Importantly, we demonstrate that they all share the common mechanism of inhibiting RALDH2. These data provide an important component of mounting evidence suggesting that the retinoid system warrants consideration in future studies of the etiology of CDH.

Embryological origins and development of the rat 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.

Diaphragm defects occur in a CDH hernia model independently of myogenesis and lung formation.

Congenital diaphragmatic hernia (CDH) is a significant clinical problem in which a portion of the diaphragmatic musculature fails to form, resulting in a hole in the diaphragm. Here we use animal models of CDH to test two hypotheses regarding the pathogenesis. First, the origin of the defect results from the malformation of the amuscular mesenchymal component of the primordial diaphragm rather than with the process of myogenesis. Second, the defect in the primordial diaphragmatic tissue is not secondary to defects in the developing lung. In c-met(-/-) mouse embryos, in which diaphragm muscle fibers do not form because of a defect in muscle precursor migration, the amuscular substratum forms fully. We show that a defect characteristic of CDH can be induced in the amuscular membrane. In Fgf10(-/-) mouse embryos that have lung agenesis we show that the primordial diaphragm does not depend on signals from lung tissue for proper development and that diaphragmatic malformation is a primary defect in CDH. These data suggest that the pathogenesis of CDH involves mechanisms fundamentally different from previously proposed hypotheses.