Placental and Embryonic Growth Restriction in Mice With Reduced Function Epidermal Growth Factor Receptor Alleles

Embryos lacking an epidermal growth factor receptor (EGFR) exhibit strain-specific defects in placental development that can result in mid-gestational embryonic lethality. To determine the level of EGFR signaling required for normal placental development, we characterized congenic strains homozygous for the hypomorphic Egfr^wa2 allele or heterozygous for the antimorphic Egfr^Wa5 allele. Egfr^wa2 homozygous embryos and placentas exhibit strain-dependent growth restriction at 15.5 days post-coitus while Egfr^Wa5 heterozygous placentas are only slightly reduced in size with no effect on embryonic growth. Egfr^wa2 homozygous placentas have a reduced spongiotrophoblast layer in some strains, while spongiotrophoblasts and glycogen cells are almost completely absent in others. Our results demonstrate that more EGFR signaling occurs in Egfr^Wa5 heterozygotes than in Egfr^wa2 homozygotes and suggest that Egfr^wa2 homozygous embryos model EGFR-mediated intrauterine growth restriction in humans. We also consistently observed differences between strains in wild-type placenta and embryo size as well as in the cellular composition and expression of trophoblast cell subtype markers and propose that differential expression in the placenta of Glut3, a glucose transporter essential for normal embryonic growth, may contribute to strain-dependent differences in intrauterine growth restriction caused by reduced EGFR activity.EPIDERMAL growth factor receptor (EGFR) is the prototypical member of the ERBB family of receptor tyrosine kinases and is known to regulate many aspects of cellular biology including cell proliferation, survival, differentiation, and migration (reviewed in Yarden and Sliwkowski 2001). Eleven known ligands bind the extracellular region of ERBB-family receptors, and activation of the tyrosine kinase domain occurs following receptor homo- or heterodimerization. The resulting biological responses are dependent upon specific signaling cascades initiated by ERBBs and can be influenced by the particular ligand–ERBB combination (Yarden and Sliwkowski 2001). Studies using cultured cells have underscored the importance of EGFR in modulating various cellular processes, while animal models have been able to demonstrate that EGFR is required for numerous developmental and physiological processes (Casalini et al. 2004). In vivo studies have shown that EGFR is particularly important for normal placental development in mice; placentas from Egfr nullizygous (Egfr^{tm1Mag/tm1Mag}) embryos exhibit strain-specific defects that result in differential embryonic lethality (Sibilia and Wagner 1995; Threadgill et al. 1995). Two additional Egfr alleles result in reduced EGFR signaling in mice: the recessive hypomorphic Egfr^wa2 and dominant antimorphic Egfr^Wa5 alleles (Luetteke et al. 1994; Fowler et al. 1995; Du et al. 2004; Lee et al. 2004). These alleles can provide insight into the level of EGFR signaling required for normal placental development.Egfr^wa2 is a classical spontaneous mutation that arose in 1935 that causes a distinct wavy coat phenotype in the homozygote (Figure 1; Keeler 1935). This recessive mutation was subsequently found to be a single nucleotide transversion resulting in a valine → glycine substitution in the highly conserved kinase domain of EGFR (Luetteke et al. 1994; Fowler et al. 1995). Since mice homozygous for the Egfr^tm1Mag null allele die before or shortly after birth depending on genetic background, the hypomorphic Egfr^wa2 allele has been the primary model used to study the effect of attenuated EGFR signaling in a variety of adult physiological and disease states. In addition to eye and hair phenotypes, the adult Egfr^wa2 homozygous mouse exhibits delayed onset of puberty, abnormal ovulation, enlarged aortic valves and cardiac hypertrophy, decreased body size, defects in mammary gland development and lactation, increased susceptibility to colitis, and impaired intestinal adaptation following small bowel resection (Fowler et al. 1995; Helmrath et al. 1997; Chen et al. 2000; Egger et al. 2000; O''Brien et al. 2002; Prevot et al. 2005; Hsieh et al. 2007). Despite the widespread use of the Egfr^wa2 allele, there are limitations in using Egfr^wa2 homozygous mice to clearly define the physiological roles of EGFR. Egfr^wa2 has traditionally been maintained in cis, tightly linked with a hypomorphic Wnt3a allele, Wnt3a^vt (vestigal tail), making phenotypic analysis of reduced EGFR signaling by itself difficult. Furthermore, Egfr^wa2 has also typically been maintained on a mixed genetic background and since the Egfr nullizygous phenotype is similarly influenced by genetic modifiers, a mixed background could mask phenotypes that become evident when Egfr^wa2 mice are inbred.Open in a separate windowFigure 1.—Congenic 129 Egfr allelic series. Wild-type (left), Egfr^wa2 homozygote (middle), and Egfr^wa5 heterozygote (right) mice. As weanlings and adults, the Egfr^wa2 homozygotes and Egfr^wa5 heterozygotes are grossly indistinguishable.The Egfr^Wa5 allele arose in a large, genomewide N-ethyl-N-nitrosourea mutagenesis screen for dominant visible mutations in the mouse. Egfr^Wa5 heterozygous mice were first identified by their open eyelids at birth and by development of a wavy coat, similar to the phenotype of Egfr^wa2 homozygous mice (Figure 1). Egfr^Wa5 failed to complement the Egfr^tm1Mag null allele and was shown to function as an antimorph since Egfr^Wa5, but not Egfr^tm1Mag, heterozygotes exhibit eyelid and coat phenotypes (Lee et al. 2004). A single nucleotide missense mutation was found in the Egfr^Wa5 allele that results in an Asp → Gly substitution in the highly conserved DFG domain of the EGFR kinase catalytic loop (Du et al. 2004; Lee et al. 2004). Although Egfr^Wa5 heterozygotes are viable, Egfr^Wa5 homozygotes die prenatally and exhibit placental defects identical to those from Egfr^tm1Mag homozygous null embryos. Placentas from Egfr^Wa5 heterozygotes on a mixed background show variable reduction in the spongiotrophoblast layer and minor abnormalities in the labyrinth region, but no effects on embryo survival have been reported.In vitro studies with Egfr^Wa5 suggest that it encodes a kinase-dead EGFR since no phosphorylation of EGFR^Wa5 is detected following stimulation with ligands. In agreement with the genetic data showing that Egfr^Wa5 is an antimorph, in vitro studies have demonstrated that the EGFR^Wa5 receptor can inhibit phosphorylation of EGFR and MAPK in a dose-dependent manner (Lee et al. 2004). In Chinese hamster ovary cells expressing an equimolar ratio of EGFR and EGFR^Wa5 receptors, <10% of wild-type phosphorylation levels were observed by Western blot analysis.The Egfr allelic series available in the mouse has high utility for studying gene function since EGFR is involved in a multitude of developmental processes and human diseases. Although both Egfr^wa2 and Egfr^Wa5 alleles result in reduced EGFR signaling, the activity and phenotypic consequences of Egfr^wa2 homozygosity has not been compared to that of Egfr^Wa5 heterozygosity when both are on the same genetic backgrounds. Adult Egfr^Wa5 heterozygous mice appear highly similar to Egfr^wa2 homozygotes, but crosses with the Apc^Min intestinal tumor model have shown that a more substantial reduction in tumor number occurs when the Apc^Min mutation is bred onto the Egfr^wa2 homozygous vs. Egfr^Wa5 heterozygous background (Roberts et al. 2002; Lee et al. 2004). These results suggest that Egfr^Wa5 heterozygous mice retain higher levels of EGFR activity than Egfr^wa2 homozygous mice; however, the data are confounded by the fact that the crosses were performed using different mixed genetic backgrounds.This study reports a comprehensive genetic analysis of reduced EGFR signaling in Egfr^wa2 homozygotes and Egfr^Wa5 heterozygotes in placental development and embryonic growth for three congenic backgrounds, C57BL/6J (B6), 129S1/SvImJ (129), and BTBR/J-T+, tf/tf (BTBR). Wild-type placenta weight, embryo weight, and mRNA levels of genes selected for their trophoblast-specific expression were found to be highly strain dependent. Egfr^wa2 homozygous placentas are reduced in size in all three strains, and a proportion of 129-Egfr^wa2 homozygotes die before 15.5 days post-coitus (dpc). Egfr^wa2 homozygous embryos also display background-dependent intrauterine growth restriction (IUGR) in late gestation, which is most severe on 129 and BTBR backgrounds and models EGFR-associated IUGR in humans. Egfr^Wa5 heterozygous placentas exhibit a minor reduction in size on all three backgrounds with no impact on embryonic growth. These results suggest that reduced levels of EGFR signaling can interfere with normal placental development and that embryo development is affected only after placental size is sufficiently reduced. In addition, our data show that the level of EGFR signaling in Egfr^Wa5 heterozygous mice is higher than in Egfr^wa2 homozygotes and suggests that different Egfr allele combinations can be generated to “genetically titer” total EGFR activity in vivo.