Gata4 is necessary for normal pulmonary lobar development

Abstract

Mutations of Fog2 in mice result in a phenotype that includes pulmonary lobar defects. To determine whether formation of the accessory lobe bronchus is mediated by a Gata family cofactor, we evaluated embryonic lungs from mice carrying missense mutations that cause loss of FOG-GATA protein interaction. Lungs from embryos carrying a missense mutation in Gata6 were structurally normal, while lungs from embryos carrying mutations of either Gata4 or of both Gata4 and Gata6 had a structural phenotype that matched the Fog2 mutant phenotype. Expression analysis showed that Gata4 and Fog2 are expressed in the ventral and medial pulmonary mesenchyme during secondary budding. Although Gata4 has not previously been suspected as playing a role in lung development, we have found that a Fog2-Gata4 interaction is critical for the development of normal pulmonary lobar structure, and this phenotype is not influenced by the additional loss of Gata6 interaction. Fog2 and Gata4 in the early pulmonary mesenchyme participate in patterning the secondary bronchus of the accessory lobe.

Figure 1.

Targeting strategy for knocking the missense mutation, V239G into the Gata6 locus. (A) Partial restriction map of the murine wild-type Gata6 locus, the targeting vector, and the targeted locus. The targeting construct contains the HSV-tk and neomycin resistance genes under the control of the mouse PGK promoter. Homologous recombination replaced wild-type Gata6 with genomic DNA mutagenized to change valine to glycine at position 239, to introduce an MunI cut site, and to incorporate a neomycin cassette for selection. The region used as a probe for Southern blot analysis is shown as a black rectangle. The N-zinc finger motif is in exon 5 and the C-zinc finger motif is in exon 6 (B, BglII; X, XhoI; N, NotI; S, SmaI; Sc, SacI). (B) Southern blot analysis of targeted ES cell DNA. DNA was digested with XhoI and BglII, separated on an agarose gel by electrophoresis followed by Southern blotting and hybridization with the 5′ probe indicated. The knock-in allele yields a fragment of 10.4 kb, and the wild-type allele yields a 8.4-kb fragment. (C) PCR genotyping of mouse pups. Tail DNA was used as template for PCR using the primers flanking the mutation. A representative gel is shown with bands corresponding to Gata6 targeted homozygote (Gata6^ki/ki) mice (lanes 1 and 2), heterozygote (Gata6^ki/+, lane 3), and wild-type mice (lane 4).

Figure 2.

Lungs removed from Gata4 mutant (Gata4^ki/+) intercrossed mice and cultured at e12.5. This litter yielded 10 viable embryos, and all wild-type (A–C) and Gata4^ki/ki mutant (D–E) lungs are shown to illustrate the range of normal and abnormal phenotypes. Gata4^ki/ki mutants range from having minimal (D) to moderate (E) growth delay at e12.5, but distal branching and mesenchymal mass is relatively well preserved after culture for 2 d. Heterozygote mice (Gata4^ki/+) look identical to the wild types. Accessory buds are marked with arrows.

Figure 3.

Lungs removed and cultured from e11.5 mice shown on day of harvest and after 1 d in culture. Photos are representative samples from litters containing Gata4 ki mutants (A and B are littermates), and Gata6 ki mutants or Gata6 and Gata4 compound mutants (C and D are littermates). Mice with loss of Fog–Gata4 interaction (Gata4^ki/ki, B and D) have loss of the accessory lobe. The additional loss of Fog–Gata6 interaction does not change this phenotype (D). For comparison, lungs from Fog2 null mice at the same developmental stage are shown in E. Accessory buds are marked with arrows.

Figure 4.

Gata4 (A–F) and Fog2 (G–H) expression are present in the early developing lung. Transverse sections from wild-type embryos at e10.5 (A–D, G) and e11.5 (E, F, H). A–D are sections through the same embryo (A, most rostral). Sections are counterstained with DAPI. At e10.5, Gata4 expression is evident in the mesothelium ventral to the rostral foregut (A), but is strongest in the mesenchyme ventral to the laryngotracheal diverticulum (B) and primary lung buds (C, lung buds marked with white arrows). Magnification of section of ventral pulmonary mesenchyme from panel C shows nuclear localization of Gata4 in the mesenchymal cells with no staining in the epithelium (D). By e11.5, Gata4 expression is strongest in the pleural and parietal mesothelium (E with DAPI counterstain and F without). Fog2 expression is shown by X-gal staining for β-galactosidase activity in mice carrying a lacZ reporter gene knocked into the Fog2 locus. Transverse sections from embryo at e10.5 (G) shows an expression pattern identical to Gata4 with expression limited to the ventral pulmonary mesenchyme and mesothelium. By e11.5, lung, Fog2 expression becomes diffuse (H) in the mid-lung fields and is also heavily expressed in primordial diaphragmatic tissue of the PPFs (marked with black arrows).