Placental and embryonic growth restriction in mice with reduced function epidermal growth factor receptor alleles

Abstract

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.

Figure 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.

Figure 2.—

Weights of placentas and embryos from wild-type, Egfr^wa2 heterozygous, and Egfr^wa2 homozygous littermates measured at 15.5 and 18.5 dpc on three genetic backgrounds. All strains included at least five embryos of each genotype. **P < 0.01 compared to wild type; ***P < 0.001 compared to wild type. (A) At 15.5 dpc, Egfr^wa2 homozygous placentas weighed 24% less than wild type on B6, 18% less than wild type on 129, and 39% less than wild type on BTBR. (B) At 18.5 dpc, Egfr^wa2 homozygous placentas weighed 24% less than wild type on B6, 37% less than wild type on 129, and 28% less than wild type on BTBR. (C) At 15.5 dpc, Egfr^wa2 homozygous embryo weights were not significantly different on B6 and 129 compared to wild type but homozygous embryos weighed 17% less than wild type on BTBR. Egfr^wa2 heterozygous embryos on the 129 background weighed 13% more than wild-type embryos (P < 0.01). (D) At 18.5 dpc, Egfr^wa2 homozygous embryo weights were not significantly different on B6 compared to wild type but homozygous embryos weighed 34% less than wild type on 129 and 32% less than wild type on BTBR. (E) Correlation between placenta weight and embryo weight in growth-restricted 18.5-dpc Egfr^wa2 homozygous embryo on 129 and BTBR backgrounds. (F) Growth-restricted 129 Egfr^wa2 homozygous embryo (right) at 18.5 dpc.

Figure 3.—

Weights of placentas and embryos from wild-type and Egfr^Wa5 heterozygous littermates on three genetic backgrounds. All strains included at least 26 embryos of each genotype for the 15.5-dpc time point and 18 embryos of each genotype for 18.5-dpc time point. *P < 0.05 compared to wild type; ***P < 0.001 compared to wild type. (A) At 15.5 dpc, Egfr^Wa5 heterozygous placentas weighed 9% less than wild type on B6, 9% less than wild type on 129, and 5% less than wild type on BTBR. (B) At 15.5 dpc, none of the three genetic backgrounds showed significant differences between Egfr^Wa5 heterozygous and wild-type embryo weights. (C) At 18.5 dpc, 129 Egfr^Wa5 heterozygous placenta and embryo weights did not differ from wild type.

Figure 4.—

Placentas from B6, BTBR, and 129 at 18.5 dpc. sp, spongiotrophoblast; la, labyrinth. (A) PAS-stained wild-type B6 placenta (×1.25 magnification). (B) PAS-stained Egfr^wa2 homozygous B6 placenta (×1.25 magnification). (C) Higher magnification of PAS-stained spongiotrophoblasts in Egfr^wa2 homozygous B6 placenta (×10 magnification). (D) PAS-stained wild-type BTBR placenta. (E) PAS-stained Egfr^wa2 homozygous BTBR placenta. (F) Higher magnification of a very small cluster of PAS-stained spongiotrophoblasts (arrowhead) in Egfr^wa2 homozygous BTBR placenta. (G) PAS-stained wild-type 129 placenta. (H) PAS-stained Egfr^wa2 homozygous 129 placenta. (I) Higher magnification of a small cluster of PAS-stained spongiotrophoblasts (arrowhead) in Egfr^wa2 homozygous 129 placenta. (J) Higher magnification of labyrinth region in wild-type 129 placenta (hematoxylin and eosin stained; ×12 magnification). (K) Higher magnification of labyrinth region in Egfr^wa2 homozygous 129 placenta (hematoxylin and eosin stained stained). (L) PAS-stained Egfr^Wa5 heterozygous 129 placenta.

Figure 5.—

ΔCT gene expression values for Egfr crosses clustered by gene and sample. Red blocks indicate low relative expression and green blocks indicate high relative expression. (A) Cluster analysis and dendrogram of ΔCT values for Egfr^wa2 homozygous, heterozygous, and wild-type samples. Cluster analysis included only genes that were significantly different between wild-type and Egfr^wa2 homozygous placenta. Egfr^wa2 homozygous samples are indicated by red (129) and green (B6) bars on dendrogram. (B) Cluster analysis and dendrogram of ΔCT values for Egfr^wa5 heterozygous and wild-type samples. Analysis included genes that were and were not significantly different between Egfr^wa5 heterozygous and wild-type samples. The 129 samples are indicated by red text; B6 samples are indicated by black text.

Figure 6.—

Wild-type placenta and embryo weights of three inbred strains measured at 15.5 dpc. (A) Placenta weight is dependent on genetic background. The average weight of 15.5-dpc placentas was 98.3 mg for B6 (n = 42), 73.9 mg for 129 (n = 27), and 82.3 mg for BTBR (n = 57). Placental weights were significantly different in all three strains (P < 0.001). (B) Embryo weight is dependent on genetic background. The average weight of 15.5-dpc embryos was 385.0 mg for B6 (n = 29), 318.6 mg for 129 (n = 26), and 332.5 mg for BTBR (n = 59). Embryo weights were significantly different in all three strains (P < 0.001).

Figure 7.—

Summary of strain-dependent late-gestation growth patterns in Egfr^wa2 homozygous placentas and embryos. The percentage reduction in weight compared to wild-type littermates for the placenta and embryo is shown.