Loss of Erk3 function in mice leads to intrauterine growth restriction, pulmonary immaturity, and neonatal lethality

Abstract

Extracellular signal-regulated kinase 3 (Erk3) is an atypical member of the mitogen-activated protein (MAP) kinase family. No function has yet been ascribed to this MAP kinase. Here we show that targeted disruption of the Mapk6 gene (encoding Erk3) leads to intrauterine growth restriction, associated with marked pulmonary hypoplasia, and early neonatal death during the first day of life. Around 40% of Erk3(-/-) neonates die within minutes after birth from acute respiratory failure. Erk3-deficient mice have normal lung-branching morphogenesis, but show delayed lung maturation characterized by decreased sacculation, atelectasis, and defective type II pneumocyte differentiation. Interestingly, in utero administration of glucocorticoid promoted fetal lung maturity and rescued differentiation of type II cells, but failed to alter the neonatal lethality. We observed that loss of Erk3 retards intrauterine growth, as reflected by a marked reduction in fetal lung, heart, and liver weights, and by low body weight at birth. Importantly, we found that insulin-like growth factor (IGF)-2 levels are decreased in the serum of Erk3-deficient mice. Our findings reveal a critical role for Erk3 in the establishment of fetal growth potential and pulmonary function in the mouse.

Fig. 1.

Targeted disruption of the Mapk6 gene. (A) Schematic representation of the Mapk6 locus, targeting vector and recombinant allele. The targeting vector carries a neomycin resistance gene (Neo^r) and the LacZ gene fused in frame with the Mapk6 coding sequence 12 amino acids downstream of the initiation codon. Exons 2 to 6 and restriction sites are shown (E, EcoR1; K, KpnI; S, SpeI; A, ApaI). Open boxes correspond to UTRs and dark boxes indicate coding regions. Introns are shown as bars. (B) Southern blot analysis of EcoRI-digested genomic DNA from two correctly targeted ES clones indicating the wild-type (9.1 kb) and mutant (7.5 kb) alleles. (C) Quantitative RT-PCR analysis of Erk3 mRNA isolated from the lungs of wild-type and Mapk6 null embryos at E18.5. Results are expressed relative to WT1. (D) Immunoblot analysis of Erk3 protein expression in cellular extracts prepared from wild type and Mapk6^−/− mouse embryonic fibroblasts.

Fig. 2.

Deletion of Mapk6 leads to neonatal lethality. (A) Representative photograph of a cyanotic Erk3^−/− pup and wild-type littermate immediately after birth. (B) Lungs isolated from a wild-type newborn are well inflated as compared to those of a dead Erk3^−/− mouse. (C) Erk3^−/− newborns surviving acute respiratory failure lack milk (*) in their stomach. Note the carpoptosis (arrow) in the Erk3^−/− mouse. (D) Body weight distribution of Erk3^+/+, Erk3^+/−, and Erk3^−/− newborns after birth. The mean body weight is indicated for each genotype: Erk3^+/+, 1.34 ± 0.07 g (n = 18); Erk3^+/−, 1.27 ± 0.09 g (n = 31); Erk3^−/−, 1.18 ± 0.08 g (n = 21). *, P < 0.05; **, P < 0.01.

Fig. 3.

Histological analysis of the developing lung in wild-type and Erk3^−/− mice. (A) Lung sections from littermate Erk3^+/+ and Erk3^−/− embryos were prepared at the indicated developmental stages and stained with hematoxylin and eosin. Magnification, 20×. (B) Morphometric analysis of lung saccular airspace in E18.5 wild-type and Erk3^−/− embryos (n = 5–6). *, P < 0.01.

Fig. 4.

Defective maturation of the distal lung epithelium in Erk3-deficient mice. (A) Sections of E18.5 Erk3^+/+ and Erk3^−/− lungs inflated with fixative were stained with hematoxylin and eosin. Magnification, 5×. Immunostaining for T1α (B) and SP-C (C) in lungs from littermate wild-type and Erk3^−/− embryos at E18.5. Magnification, 40×. (D) Ultrastructure of E18.5 Erk3^+/+ and Erk3^−/− lungs. Mature type II pneumocytes present characteristic villi (black arrow) and contain lamellar bodies (open arrow). Type II cells from Erk3-deficient lungs contain an abundant pool of glycogen (*) and present attenuated villi. Secreted surfactant can be observed occasionally in the airspace of Erk3^−/− embryos (black arrow). (E) PAS staining (arrows) indicating cytoplasmic glycogen in lung sections from E18.5 wild-type and Erk3^−/− embryos. Magnification, 40×. (F) Quantification of PAS staining (n = 5–6). *, P < 0.01.

Fig. 5.

Antenatal dexamethasone rescues the lung maturation defect of Erk3-deficient mice. (A) Pregnant females from Erk3^+/− intercrosses were treated with dexamethasone or saline at E16.5 and E17.5 of gestation. Embryos were collected by caesarean section at E18.5 and lung sections were stained with hematoxylin and eosin. Magnification, 40×. (B) Quantification of PAS staining in lung sections from E18.5 Erk3^+/+ and Erk3^−/− embryos (n = 4–5) treated as in A. *, P < 0.01.

Fig. 6.

Growth restriction, attenuated lung cell proliferation, and reduced IGF-2 levels in Erk3-deficient mice. (A) Body weight of littermate Erk3^+/+ (filled bars) and Erk3^−/− (open bars) embryos at various developmental stages (n = 6–12). *, P < 0.05, **, P < 0.01. (B) Lungs and heart from wild-type and Erk3^−/− E18.5 embryos. (C) Ratio of lung, heart and liver weight relative to body weight in wild-type and Erk3^−/− E18.5 embryos (n = 8–13). * P < 0.05, **, P < 0.01. (D) Quantification of cell proliferation assessed by Ki67 immunostaining in lung sections from Erk3^+/+ (filled bars) and Erk3^−/− (open bars) embryos at various developmental stages (n = 3–6). *, P < 0.05, **, P < 0.001. (E) IGF-1 and IGF-2 serum levels in E18.5 wild-type and Erk3^−/− embryos (n = 8–13). *, P < 0.01.