Maternal high-fat diet interacts with embryonic Cited2 genotype to reduce Pitx2c expression and enhance penetrance of left-right patterning defects

Abstract

Deficiency of the transcription factor Cited2 in mice results in cardiac malformation, adrenal agenesis, neural tube, placental defects and partially penetrant cardiopulmonary laterality defects resulting from an abnormal Nodal->Pitx2c pathway. Here we show that a maternal high-fat diet more than doubles the penetrance of laterality defects and, surprisingly, induces palatal clefting in Cited2-deficient embryos. Both maternal diet and Cited2 deletion reduce embryo weight and kidney and thymus volume. Expression profiling identified 40 embryonic transcripts including Pitx2 that were significantly affected by embryonic genotype-maternal diet interaction. We show that a high-fat diet reduces Pitx2c levels >2-fold in Cited2-deficient embryos. Taken together, these results define a novel interaction between maternal high-fat diet and embryonic Cited2 deficiency that affects Pitx2c expression and results in abnormal laterality. They suggest that appropriate modifications of maternal diet may prevent such defects in humans.

Figure 1.

Effect of maternal high-fat diet on maternal weight, fasting glucose and insulin. (A) Experimental design. C57BL6/J.Cited2^+/− females were weaned at 3 weeks, and at 6 weeks (day 0) were switched from chow to either a control diet (CD) or to a high-fat diet (HFD). Timed matings with C57BL6/J.Cited2^+/− males were started at day 56, and embryos for MRI collected at 15.5 dpc, or for microarray and quantitative reverse-transcriptase–polymerase chain reaction (QRT–PCR) at 8.5 dpc. (B–D) Weight, fasting glucose and insulin by diet (CD or HFD) and time-point day 0 or day 56. Data are shown as mean ± standard error. The number of observations is given in Supplementary Material, Table S1. P is the probability of a type I error. NS = not significant.

Figure 2.

Phenotypic abnormalities in Cited2 deficient embryos at 15.5 dpc analysed using MRI. (A) Transverse section of a wild-type embryonic heart showing normal anatomy. The right and left atria (RA and LA), right and left ventricles (RV and LV), interventricular septum (IVS) are indicated. (B) Transverse section of Cited2^+/− heart from a high-fat diet mother. A large ventricular septal defect (VSD) is indicated. (C) 3D reconstruction of a wild-type embryonic heart. The LV gives rise to the ascending aorta (AAo) which continues as the aortic arch (AoA) and the descending aorta (DAo). The RV gives rise to the pulmonary artery (PA) which communicates with the descending aorta via the ductus arteriosus. The trachea (Tr) is indicated. (D) 3D reconstruction of a Cited2^+/− embryonic heart from a high-fat diet mother. The aortic arch is interrupted, and does not join the descending aorta. (E) Transverse section of wild-type embryonic heart, with the systemic venous sinus (SVS) opening into the right atrium (RA). The left atrium (LA), primary atrial septum (PAS), right and left ventricles (RV, LV), interventricular septum (IVS) and mitral and tricuspid valves (MV, TV) are indicated. (F) Transverse section of Cited2^−/− heart from a high-fat diet mother. The PAS is absent resulting in a common atrium (A), which has bilateral systemic venous sinuses (LSVS, RSVS). A common atrioventricular valve (AVV) straddles a ventricular septal defect (VSD). These appearances are consistent with right atrial isomerism. (G) Volumes of the transverse and ascending aorta (TA and AA), in Cited2^+/− embryos from control and high-fat diet groups (CD, HFD). The volume (µl/g) is corrected for embryo weight. Data are shown as mean ± standard error, with nine embryos in each group. P is the probability of a type I error. NS, not significant. (H) Minimum diameter of the transverse aorta in Cited2^+/− embryos from control and high-fat diet groups (CD, HFD). The diameter (μm/g) is corrected for embryo weight. Data are shown as mean ± standard error, with nine embryos in each group. P is the probability of a type I error. (I) Sagittal section of a Cited2^+/+ HFD embryo. The palate (P) and tongue (T) are indicated. (J) Sagittal section of a Cited2^−/− HFD embryo. The palate is absent. (K) Coronal section of a Cited2^+/+ HFD embryo. The palate (P) and tongue (T) are indicated. (L) Coronal section of a Cited2^−/− HFD embryo. The palate is absent. Scale bars 0.5 mm. Axes: D, dorsal; V, ventral; R, right; L, left; A, anterior; P, posterior.

Figure 3.

Effect of genotype and maternal diet on embryo and placental weight and organ volumes. Genotypes are Cited2^+/+ (WT), Cited2^+/− (HET) and Cited2^−/− (NULL). The significance of genotype, diet and genotype*diet interaction is indicated for each data set. P is the probability of a type I error. NS, not significant. R is Pearson's coefficient of correlation. N is the number. (A and B) Embryo and placenta weight by genotype. (C) Correlation of embryo and placental weight for all genotypes. Analysis by genotype indicated that this correlation was strongest in wild-type and in Cited2^−/− embryos (N = 41; R = 0.3158; P = 0.04 for wild-type, and N = 41; R = 0.3585; P = 0.02 Cited2^−/− embryos). The correlation was weaker in Cited2^+/− embryos (N = 87; R = 0.1447; P = 0.18). (D–F) Kidney, adrenal and thymus volumes by genotype. Data for control diet (CD, open circles) and for high-fat diet (HFD, closed circles) are shown as mean ± standard error, with the number of observations in each group indicated either above (CD) or below (HFD). Adrenal glands were absent in all Cited2^−/− embryos (43 from CD, and 52 from HFD), and volumes were taken as 0.

Figure 4.

Effect of diet on Pitx2c expression. (A) Quantitative reverse transcriptase–polymerase chain reaction (QRT–PCR) for Pitx2c mRNA in Cited2^+/+ embryos at 8.5 dpc on control and on high-fat diets (CD-wt and HFD-wt). Data show mean ± standard error of Ro values, and individual data points are shown. P is the probability of a type I error. NS, not significant. (B) QRT–PCR for Pitx2c mRNA in Cited2^−/− embryos at 8.5 dpc on control and on high-fat diets (CD-null and HFD-null).