GATA4, an essential cardiogenic transcription factor, provides a model for dominant transcription factor mutations in human disease. Dominant GATA4 mutations cause congenital heart disease (CHD), specifically atrial and atrioventricular septal defects (ASDs and AVSDs). We found that second heart field (SHF)-specific Gata4 heterozygote embryos recapitulated the AVSDs observed in germline Gata4 heterozygote embryos. A proliferation defect of SHF atrial septum progenitors and hypoplasia of the dorsal mesenchymal protrusion, rather than anlage of the atrioventricular septum, were observed in this model. Knockdown of the cell-cycle repressor phosphatase and tensin homolog (Pten) restored cell-cycle progression and rescued the AVSDs. Gata4 mutants also demonstrated Hedgehog (Hh) signaling defects. Gata4 acts directly upstream of Hh components: Gata4 activated a cisregulatory element at Gli1 in vitro and occupied the element in vivo. Remarkably, SHF-specific constitutive Hh signaling activation rescued AVSDs in Gata4 SHF-specific heterozygous knockout embryos. Pten expression was unchanged in Smoothened mutants, and Hh pathway genes were unchanged in Pten mutants, suggesting pathway independence. Thus, both the cell-cycle and Hh-signaling defects caused by dominant Gata4 mutations were required for CHD pathogenesis, suggesting a combinatorial model of disease causation by transcription factor haploinsufficiency.Gata4 | ASDs | Hedgehog signaling | second heart field | cell cycle
Rationale Mutations of TBX5 cause Holt–Oram syndrome (HOS) in humans, a disease characterized by atrial or occasionally ventricular septal defects in the heart and skeletal abnormalities of the upper extremity. Previous studies have demonstrated that Tbx5 regulates Osr1 expression in the second heart field (SHF) of E9.5 mouse embryos. However, it is unknown whether and how Tbx5 and Osr1 interact in atrial septation. Objective To determine if and how Tbx5 and Osr1 interact in the posterior SHF for cardiac septation. Methods and Results In the present study, genetic inducible fate mapping showed that Osr1-expressing cells contribute to atrial septum progenitors between E8.0 and E11.0. Osr1 expression in the pSHF was dependent on the level of Tbx5 at E8.5 and E9.5 but not E10.5, suggesting that the embryo stage before E10.5 is critical for Tbx5 interacting with Osr1 in atrial septation. Significantly more atrioventricular septal defects (AVSDs) were observed in embryos with compound haploinsufficiency for Tbx5 and Osr1. Conditional compound haploinsufficiency for Tbx5 and Osr1 resulted in a significant cell proliferation defect in the SHF, which was associated with fewer cells in the G2 and M phases and a decreased level of Cdk6 expression. Remarkably, genetically targeted disruption of Pten expression in atrial septum progenitors rescued AVSDs caused by Tbx5 and Osr1 compound haploinsufficiency. There was a significant decrease in Smo expression, which is a Hedgehog (Hh) signaling pathway modulator, in the pSHF of Osr1 knockout embryos at E9.5, implying a role for Osr1 in regulating Hh signaling. Conclusions Tbx5 and Osr1 interact to regulate posterior SHF cell cycle progression for cardiac septation.
BACKGROUND/OBJECTIVES Recent findings have highlighted the detrimental influence of maternal overnutrition and obesity on fetal development and early life development. However, there are no evidence-based guidelines regarding the optimal strategy for dietary intervention before pregnancy. SUBJECTS/METHODS We used a murine model to study whether switching from a high-fat (HF) diet to a normal-fat (NF) diet (H1N group) 1 week before pregnancy could lead to in utero reprogramming of female offspring obesity; comparator groups were offspring given a consistent maternal HF group or NF group until weaning. After weaning, all female offspring were given the HF diet for either 9 or 12 weeks before being killed humanely. RESULTS H1N treatment did not result in maternal weight loss before pregnancy. NF offsprings were neither obese nor glucose intolerant during the entire experimental period. H1N offsprings were most obese after the 12-week postweaning HF diet and displayed glucose intolerance earlier than HF offsprings. Our mechanistic study showed reduced adipocyte insulin receptor substrate 1 (IRS1) and hepatic IRS2 expression and increased adipocyte p-Ser636/639 and p-Ser612 of H1N or HF offspring compared with that in the NF offspring. Among all groups, the H1N offspring had lowest level of IRS1 and the highest levels of p-Ser636/639 and p-Ser612 in gonadal adipocyte. In addition, the H1N offspring further reduced the expression of Glut4 and Glut2, vs those of the HF offspring, which was lower compared with the NF offspring. There were also enhanced expression of genes inhibiting glycogenesis and decreased hepatic glycogen in H1N vs HF or NF offspring. Furthermore, we showed extremely higher expression of lipogenesis and adipogenesis genes in gonadal adipocytes of H1N offspring compared with all other groups. CONCLUSIONS Our results suggest that a transition from an HF diet to an NF diet shortly before pregnancy, without resulting in maternal weight loss, is not necessarily beneficial and may have deleterious effects on offspring.
Although a pre-pregnancy dietary intervention is believed to be able to prevent offspring obesity, research evidence is absent. We hypothesize that a long period of pre-pregnancy maternal diet transition from a high fat (HF)-diet to a normal fat (NF)-diet effectively prevents offspring obesity, and this preventive effect is independent of maternal body weight change. In our study, female mice were either continued on a NF diet (NF-group) or a HF diet (HF-group) until weaning; or switched from a HF to a NF for 1-week (H1N-group), 5-week (H5N-group) or 9- week (H9N-group) before pregnancy. After weaning, the offspring were given the HF diet for 12 weeks to promote obesity. The mothers, regardless of which group, did not display maternal body weight change and glucose intolerance either before pregnancy or after weaning. Compared to the HF group, the H1N and H5N, but not the H9N offspring, developed glucose intolerance earlier, with more severely imbalanced glucose homeostasis. These offspring also displayed hepatocyte degeneration and significant adipocyte hypertrophy associated with higher expression of lipogenesis genes. The molecular mechanistic study showed blunted insulin signaling, overactivated adipocyte Akt signaling and hepatic AMPK signaling with enhanced lipogenesis genes in the H1N and H5N versus the NF offspring. However, maternal H9N diets normalized glucose and lipid metabolism of the offspring, via re-sensitized insulin signaling and normalized Akt and AMPK signaling. In summary, we showed that a long-term maternal diet intervention effectively released the inter-generational obesogenic effect of maternal HF diet, independent of maternal weight management.
Mutations of TBX5 cause Holt-Oram syndrome (HOS) in human, a disease characterized by upper limb and heart defects. Mouse embryos of Osr1 knockout caused similar heart defects, while the upper limb defects have never been reported. By genetically marking Osr1 expressing cells in mice, using Osr1:CreERT2 , we showed that Osr1 expression cells contribute to the atrial septum progenitors between E8.0 and E11.0, and to the forelimb after E9.0. The expression of Osr1 in the forelimb showed a gradient decreasing pattern from the digit 5 to digit 1. Conditional- Tbx5 haploinsuffiency, using Osr1:CreERT2 , compound with Osr1 haploinsuffiency induced more incidence of atrial septal defects (ASDs) and double outlet right ventricle (DORV). Forty percent of these embryos also had digit defects: the digits are either missing, fused or lack normal identity, which were not observed in mouse embryos of either Osr1 or Tbx5 haploinsuffiency. Detailed study of the cardiac progenitors of the compound haploinsufficinecy for Tbx5 and Osr1 showed decreased proliferation in the posterior second heart field, which was associated with lower number of cells transiting from G2 to M phase and less gene expression of Cdk6 and CyclinD2 . In summary, our study demonstrated that interaction of Osr1 and Tbx5 is involved in the mouse limb and heart development and provides a potential mechanism for HOS.
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