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

Abstract

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.

Figure 1. Abnormal Pulmonary and Diaphragmatic Development in the lil Mouse

(A) The mutant hypoplastic lung (right) lacks the development of the accessory lobe and the anterior portion of the right middle lobe (marked with arrows on the control sample on the left). (B) Whole diaphragms show a lack of normal muscularization in the posterolateral regions and the peripheral regions of the mutant diaphragm (control on left and lil diaphragm on the right).

Figure 2. The ENU-Induced lil Mutation in Fog2 is a Splice Site Mutation

(A) RT-PCR revealed a lengthened transcript in mice with the lil phenotype: the first band is a lil mouse, and the second band is a control mouse. (B) Sequencing Fog2 revealed a splice site mutation that causes the insertion of 85 bp of intronic sequence in the mutant mouse. This results in a premature stop codon prior to zinc finger transcription.

Figure 3. Fog2 Is Expressed in the Developing Lung and Diaphragm

Fog2 is expressed in the diffuse pulmonary mesenchyme at E13.5 (A) (arrow shows mesenchyme) and is restricted to the bronchial and vascular smooth muscle (sm) at E16.5 (B). Fog2 is expressed diffusely in the developing diaphragm (Dia) both prior to (E11.5) (C) and after muscularization (E13.5) (D).

Figure 4. Fog2 Is Necessary for Primary Lung Development

Fog2 null lungs removed prior to diaphragmatic muscularization and grown in vitro show no accessory lobe development. Accessory lobe is labeled with black arrow in control littermate lungs.

Figure 5. Fog2 Expression in Embryonic Non-Mutant Lungs

In embryonic non-mutant lungs, Fog2 is most highly expressed at the tips of the accessory (single arrows) and right middle lobes (double arrows) at E11.25 (A) and E11.75 (B), while expression is diffuse by E12.75 (C).

Figure 6. HGF Patterning Is Abnormal in Fog2 Null Mice

In situ hybridization of HGF in E12.5 wild-type (A) and Fog2 ^−/− (B) embryos demonstrates decreased expression in the region where the PPF meets the membranous diaphragm. Li, liver; Lu, lung.

Figure 7. FOG2 Mutation in a Patient with Diaphragm and Lung Abnormalities

Sequencing revealed a de novo heterozygote nonsense mutation in a patient who died at birth with severe pulmonary hypoplasia and a posterior deep diaphragmatic eventration. She was clinically diagnosed with CDH. This nonsense mutation occurs prior to the functional zinc finger domains.