Cited2 is a cAMP-responsive element-binding protein (CBP)͞p300 interacting transcriptional modulator and a proposed negative regulator for hypoxia-inducible factor (HIF)-1␣ through its competitive binding with HIF-1␣ to CBP͞p300. Disruption of the gene encoding Cited2 is embryonic lethal because of defects in the development of heart and neural tube. Morphological and Doppler echocardiographic analyses of Cited2 ؊/؊ embryos reveal severe cardiovascular abnormalities, including pulmonic arterial stenosis and ventricular septal defects accompanied by high peak outflow velocities, features of the human congenital cardiac defect termed tetralogy of Fallot. The mRNA levels of several HIF-1␣-responsive genes, such as vascular endothelial growth factor (VEGF), Glut1, and phosphoglycerate kinase 1, increased in the Cited2 ؊/؊ hearts. The increase of VEGF levels is significant, because defects in the Cited2 ؊/؊ embryos closely resemble the major defects observed in the VEGF transgenic embryos. Finally, compared with wild-type, cultured fibroblasts from Cited2 ؊/؊ embryos demonstrate an enhanced expression of HIF-1␣-responsive genes under hypoxic conditions. These observations suggest that functional loss of Cited2 is responsible for defects in heart and neural tube development, in part because of the modulation of HIF-1 transcriptional activities in the absence of Cited2. These findings demonstrate that Cited2 is an indispensable regulatory gene during prenatal development.
Vitamin A is essential for normal embryonic cardiogenesis. The vitamin A-deficient phenotype in the avian embryo includes an abnormal heart tube closed at the sinus venosus and the absence of large vessels that normally connect the embryonic heart to the developing circulatory system. In vitamin A-deficient embryos the expression of cardiomyocyte differentiation genes, including atrial-specific myosin heavy chain, ventricular-specific myosin, and sarcomeric myosins as well as the putative cardiomyocyte specification gene Nkx-2.5, is not altered. However, the expression of transcription factor GATA-4 is severely decreased in the heart-forming regions of vitamin A-deficient stage 7-10 embryos. Significantly, GATA-4 transcripts are completely lacking in the lateral mesoderm posterior to the heart, in the area of the developing cardiac inflow tract that later displays prominent morphological defects, including a closed nonseptated heart lacking a sinus venosus. The administration of retinol to the vitamin A-deficient embryo restores GATA-4 expression and completely rescues the vitamin A-deficient phenotype. Our results indicate that GATA-4 is a component of the retinoid-mediated cardiogenic pathway unlinked to cardiomyocyte differentiation, but involved in the morphogenesis of the posterior heart tube and the development of the cardiac inflow tract.
Vitamin A-deficient (VAD) quail embryos have severe abnormalities, including a high incidence of reversed cardiac situs. Using this model we examined in vivo the physiological function of vitamin A in the left/right (L/R) cardiac asymmetry pathway. Molecular analysis reveals the expression of early asymmetry genes activin receptor IIa, sonic hedgehog, Caronte, Lefty-1, and Fgf8 to be unaffected by the lack of retinoids, while expression of the downstream genes nodal-related, snail-related (cSnR), and Pitx2 is altered. In VAD embryos nodal expression in left lateral plate mesoderm (LPM) is severely downregulated and the expression domain altered during neurulation. Similarly, the expression of cSnR in the right LPM and of Pitx2 in the left side posterior heart-forming region (HFR) is downregulated in the VAD embryos. The lack of retinoids does not cause randomization or ectopic expression of nodal, cSnR, or Pitx2. At the six- to eight-somite stage nodal is expressed transiently in the left posterior HFR of normal quail embryos; this expression is missing in VAD embryos and may be linked to the loss of Pitx2 expression in this region of VAD quail embryos. Administration of retinoids to VAD embryos prior to the six-somite stage rescues the expression of nodal, cSnR, and Pitx2 as well as the randomized VAD cardiac phenotype. There is an absolute requirement for retinoids at the four- to five-somite developmental window for cardiogenesis and cardiac L/R specification to proceed normally. We conclude that retinoids do not regulate the left/right-specific sidedness assignments for expression of genes on the vertebrate cardiac asymmetry pathway, but are required during neurulation for the maintenance of adequate levels of their expression and for the development of the posterior heart tube and a loopable heart. Cardiac asymmetry may be but one of several critical events regulated by retinoid signaling in the retinoid-sensitive developmental window.
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