Fetus-derived DLK1 is required for maternal metabolic adaptations to pregnancy and is associated with fetal growth restriction

Abstract

Pregnancy is a state of high metabolic demand. Fasting diverts metabolism to fatty acid oxidation, and the fasted response occurs much more rapidly in pregnant women than in non-pregnant women. The product of the imprinted DLK1 gene (delta-like homolog 1) is an endocrine signaling molecule that reaches a high concentration in the maternal circulation during late pregnancy. By using mouse models with deleted Dlk1, we show that the fetus is the source of maternal circulating DLK1. In the absence of fetally derived DLK1, the maternal fasting response is impaired. Furthermore, we found that maternal circulating DLK1 levels predict embryonic mass in mice and can differentiate healthy small-for-gestational-age (SGA) infants from pathologically small infants in a human cohort. Therefore, measurement of DLK1 concentration in maternal blood may be a valuable method for diagnosing human disorders associated with impaired DLK1 expression and to predict poor intrauterine growth and complications of pregnancy.

Figure 1. The conceptus is the source of elevated maternal plasma DLK1 in late gestation.

(a) Maternal plasma DLK1 levels are elevated during pregnancy. Serum DLK1 levels in non-pregnant WT females (WT NP, n = 8), pregnant WT females crossed to WT males at E15.5 gestation (WT x WT, n = 10), and pregnant Dlk1^-/- females crossed to Dlk1^-/- males at E15.5 (Null x Null, n = 4). (b) Summary of experimental crosses used in the study. (c) Serial plasma DLK1 measurements over the course of pregnancy (n = 4 females/time–point/group). Groups differ significantly by cross, time–point and the interaction between them (each p < 0.001 by Two-way ANOVA). Dotted line represents the detection threshold of the ELISA. (d) Maternal plasma DLK1 at E15.5 (n = 7–8 females/group). Groups differ significantly by cross and DLK1 levels rise only when the conceptus has a functional copy of Dlk1. nd = not detected (below assay threshold). (Inset) DLK1 in maternal plasma at E15.5 normalised to number of Dlk1–expressing embryos in the litter (n = 8 litters/group). Mat embryos in the Null x Mat cross generate significantly more DLK1 than those in other experimental crosses. (e) Litters from Null mothers have more embryos than litters from WT or Mat mothers. Litter size by maternal genotype in WT (n = 8), Null (n = 24) and Mat (n = 16) mothers. Vertical bars show mean ± s. e. m. Groups were compared by Kruskall-Wallis test, with Dunn’s Multiple comparison post-hoc test as indicated (a, d, e), *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2. Fetus not placenta is the source of maternal circulating DLK1.

(a) Schematic of Dlk1 expression in Null-TG conceptuses. (b) DLK1 expression (brown staining) the WT, Null and Null–TG embryo. Scale bar 2mm. (c) DLK1 is not detected in the fetal endothelium of the Null-TG placenta but is retained in cells with large nuclei. Scale bars left 500um, right 50um. (d) Dlk1 expression in the Null-TG, WT levels, and Null placentae (n = 7–8 per group) *** p < 0.001 by Dunnett’s Multiple comparison post-test (vs WT) following One-Way ANOVA. (e) Expression of cleavable (Dlk1A/B) and membrane-bound isoforms (Dlk1C/D) of Dlk1 in WT and Null-TG placentae. (f) DLK1 levels in maternal plasma in Null x Null-TG litters (n = 7–8 females/cross). (g) Schematic of Dlk1 expression in Meox2Cre/Dlk1^fl/fl conceptuses. (h) DLK1 expression in the Meox2Cre/Dlk1^fl/fl embryo compared to a Meox2Cre/Dlk1^+/+ control. Scale bar 2mm. (i–top) Meox2Cre crossed to the mTmG reporter results in GFP+ cells following Cre excision, and mTomato+ in non–recombined cells. (i–bottom) Dlk1 is not detected the fetal endothelium of the Meox2Cre/Dlk1^fl/fl placenta but some labyrinthine expression of DLK1 is retained. Scale bars left 500um, right 50um. (j) and (k) Dlk1 expression in the WT, Meox2Cre/Dlk1^+/+ and Meox2Cre/Dlk1^fl/fl embryo and placenta (5–8 per group), **p < 0.01, ***p < 0.001 as in (d). (l) Maternal plasma DLK1 in Meox2Cre^+/- females crossed with Dlk1^+/+ (control) or Dlk1^fl/fl males (n = 6 females/cross), *p < 0.05 compared by Students’ t–test. All measurements performed at E15.5. Vertical bars show mean ± s. e. m.

Figure 3. Maternal genotype and conceptus-derived DLK1 alters maternal metabolism.

Female weight at E15.5 minus weight on the day of conception (E0.5), divided by the number of live fetuses (a), or total litter mass (sum of all placental and embryonic masses within the litter), (b). Null females gain significantly less weight. (c) Derived abdominal WAT gain during pregnancy –pregnant WAT weight for each cross minus average non–pregnant WAT weight for each genotype, expressed as % WT weight gain. Null females ‘gain’ significantly less abdominal WAT. Fasted 3-hydroxybutyrate, 3-OH (d), glucose (e), insulin (f), total cholesterol (h) and HDL-cholesterol (i) in maternal plasma from Null females crossed to WT or Null males, at E15.5. (g) Relative expression of Hmgcs2 in maternal liver at E15.5, in Null females crossed to WT or Null males, Growth Hormone (GH, j), Estradiol (E2, k) and Corticosterone (l) levels in all groups. Vertical bars show mean ± s. e. m (n = 6–8 females per group). Comparisons (a-c, and j-l) were compared by One-way ANOVA, with Bonferroni's Multiple comparison post-hoc test as indicated, *p < 0.05, **p < 0.01. Pregnant and NP GH and corticosterone samples did not have equal variance, so only pregnant samples were compared. WT NP vs WT x WT were compared to each other using a Student’s t-test with Welch’s correction, ***p < 0.001. (d-i) were compared by Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4. Low DLK1 in human pregnancy is associated with pathological SGA.

(a) Scatter plot of differences in DLK1 levels in maternal plasma of 43 pairs of matched pregnant women, differing by SGA outcome. Pathological = any of the fetal growth restriction (FGR) indicators (UTPI 20wk decile 10, n = 8 pairs; UMPI 36wk decile 10, n = 10 pairs; ACGV 20-36wk decile 1, n = 12 pairs). The short horizontal lines represent means of the differences. (b) Receiver Operating Characteristic (ROC) curve analysis showing the strength of association of maternal plasma DLK1 and SGA comparing cases (n = 45) with the random sample of the cohort (n = 41). AUC = area under curve, 95% confidence interval (CI) and p values for testing the null hypothesis (AUC = 0.5) are shown on respective graphs.