Marit J. Boot

Ets-1 and Ets-2 Transcription Factors Are Essential for Normal Coronary and Myocardial Development in Chicken Embryos

&NA; —In the development of a functional myocardium and formation of the coronary vasculature, epicardium‐derived cells play an essential role. The proepicardial organ contributes to the developing coronary system by delivering mural cells to the endothelium‐lined vessels. In search of genes that regulate the behavior of (pro)epicardial cells, the Ets‐1 and Ets‐2 transcription factors stand out as strong candidates. In the present study, the hypothesis that Ets transcription factors have a role in proper coronary and myocardial development was tested via antisense technology, by targeting Ets‐1 and Ets‐2 mRNAs to downregulate protein expression in chicken embryos. The results suggest that hereby the development of the coronary system is hampered, primarily by defects in the process of epithelial‐mesenchymal transformation of the mesothelia of the primary and secondary heart fields. This was indicated by a lack of periarterial and epicardial mesenchyme, of peripheral coronary smooth muscle cells, and changes in myocardial morphology. A defect in myocardial perfusion caused by the absence of one or both coronary ostia seems to be “solved” by the development of numerous small fistulae connecting the ventricular lumen with the subepicardially located coronary vessels. The presence of coronary vascular aberrations in the antisense‐Ets phenotype enabled us for the first time to study abnormal coronary development in a model that is not lethal to the embryo. (Circ Res. 2003;92:749–756.)

Folic acid and homocysteine affect neural crest and neuroepithelial cell outgrowth and differentiation in vitro.

The beneficial effect of additional folic acid in the periconceptional period to prevent neural tube defects, orofacial clefts, and conotruncal heart defects in the offspring has been shown. Folate shortage results in homocysteine accumulation. Elevated levels of homocysteine have been related to neural tube defects. We studied the behavior of neuroepithelial cells and cranial and cardiac neural crest cells in vitro. Neural tube explants were cultured for 24 and 48 hr in medium after addition of folic acid and/or homocysteine. Folic acid addition increased neuroepithelial cell outgrowth and increased neural crest cell differentiation into nerve and smooth muscle cells. Addition of homocysteine increased neural crest cell outgrowth and migration from the neural tube and inhibited neural crest cell differentiation. Our findings suggest that neural tube defects caused by folate deficiency and hyperhomocysteinemia develop due to increased neuroepithelial to neural crest cell transformation. This increased transformation leads to a shortage of neuroepithelial cells in the neural tube. Defects in orofacial and conotruncal development are explained by abnormal differentiation of neural crest cells in the presence of high homocysteine concentrations. Our findings supports a critical role for folic acid and homocysteine in the development of neural tube defects and neural crest related heart malformations. Developmental Dynamics 227:301–308, 2003.

Homocysteine Induces Endothelial Cell Detachment and Vessel Wall Thickening During Chick Embryonic Development

Abstract— Homocysteine affects the migration and differentiation of neural crest cells in vitro and can result in neural tube defects in vivo. Furthermore, homocysteine has been described as an important determinant in vascular disease in human adults. However, little is known about the effects of homocysteine on the development of embryonic vessels. In this study, we injected homocysteine (30 &mgr;mol/L) into the neural tube lumen of chick embryos at the time point of neural crest cell emigration, and analyzed the effects on the neural crest–derived pharyngeal arch arteries, like the brachiocephalic arteries, and the mesoderm-derived arteries, such as the dorsal aorta. By stage HH35, we observed detachment of the endothelium, decreased expression of the extracellular matrix proteins fibrillin-2, and fibronectin in the pharyngeal arch arteries, whereas the dorsal aorta was identical in homocysteine-neural tube–injected and control embryos. No effect of homocysteine on endothelin-1 mRNA expression was observed. By stage HH40, the brachiocephalic arteries of homocysteine-neural tube–injected embryos displayed a decreased lumen diameter, an increased intima- and media-thickness, and an increased number of actin layers compared with the brachiocephalic arteries in control embryos. We propose that homocysteine affects the neural crest–derived smooth muscle cells and their extracellular matrix proteins in the pharyngeal arch arteries, resulting in an abnormal smooth muscle to endothelial cell interaction, leading to endothelial cell detachment. We suggest that, as in adult life, increased homocysteine concentrations lead to vascular damage in the embryo. This prenatal damage might increase the susceptibility to develop vessel pathology later in life.

A Chicken Model for DGCR6 as a Modifier Gene in the DiGeorge Critical Region

DGCR6 is the most centromeric gene in the human DiGeorge critical region and is the only gene in the region with a second functional copy on a repeat localized more distally on chromosome 22. We isolated the chicken ortholog of DGCR6 and showed an embryonic expression pattern that is initially broad but becomes gradually restricted to neural crest cell derivatives of the cardiovasculature. Retrovirus based gene transduction was used to deliver sense and antisense messages to premigrating neural crest cells in vivo. Embryos in which DGCR6 expression was attenuated revealed cardiovascular anomalies reminiscent of those found in DiGeorge syndrome. Moreover, the expression profiles of three other genes from the DiGeorge critical region, TBX-1, UFD1L, and HIRA, were shown to be altered in this model. TBX-1 and UFD1L levels were increased, whereas HIRA was decreased in the hearts and pharyngeal arches of embryos treated with antisense or partial sense constructs, but not with sense constructs for DGCR6. The expression changes were transient and followed the normal DGCR6 expression profile. These data show that neural crest cells might have a role in the distribution of modulator signals to the heart and pharyngeal arches. Moreover, it shows a repressor function for DGCR6 on the expression of TBX-1 and UFD1L. For the first time, Di-George syndrome is shown to be a contiguous gene syndrome in which not only several genes from the critical region, but also different cell types within the embryo, interact in the development of the phenotype.

The embryology of the common arterial trunk

Abstract The developmental background of common arterial trunk still remains to be elucidated. In this anomaly both the coronary, systemic and pulmonary circulation arise from a common arterial stem with a common orifice. The contribution of the embryonic ectomesenchymal neural crest seems to be essential for a normal separation of the aorta, pulmonary trunk and the adjoining myocardium lined outflow tract. Experimental neural crest ablation studies in chicken have proved that there is a role for neural crest cells. Several molecular biological studies, however, have shown that we are not solely dealing with the influence of neural crest. Many genes and gene cascades have been manipulated in mice and have shown that e.g. the endothelin cascade, Foxc1 and Foxc2, and more recently Sema3C and neuropilin-1 are factors, that if disturbed, can lead to common arterial trunk often in combination with aortic arch anomalies such as interruption type B. In humans common arterial trunk is one of the characteristics of the 22q11 deletion syndrome. In the mouse several of these genes, located on the syntenic chromosome 16 have been studied for this effect, but most result in an embryolethal phenotype. Neural crest tracing studies in both chicken–quail chimeras and transgenic neural crest reporter mice have revealed that there is a difference in neural crest contribution to the aortic and pulmonary truncal wall. The orifice of the common arterial trunk reflects this finding in that the often abnormally high positioned coronary orifices reside in the aortic part of the orifice. Combining current data the common denominator for common arterial trunk seems to be a defective interaction of neural crest, endothelial cells and the surrounding mesenchyme. Disturbances of genes in any of the three cell types can lead to the malformation.