In vitro whole-organ imaging: 4D quantification of growing mouse limb buds.

Quantitative mapping of the normal tissue dynamics of an entire developing mammalian organ has not been achieved so far but is essential to understand developmental processes and to provide quantitative data for computational modeling. We developed a four-dimensional (4D) imaging technique that can be used to quantitatively image tissue movements and dynamic GFP expression domains in a growing transgenic mouse limb by time-lapse optical projection tomography (OPT).

The dynamics of spleen morphogenesis.

The mammalian spleen has important functions in immunity and haematopoiesis but little is known about the events that occur during its early embryonic development. Here we analyse the origin of the cells that gives rise to the splenic mesenchyme and the process by which the precursors assume their position along the left lateral side of the stomach. We report a highly conserved regulatory element that regulates the Nkx2-5 gene throughout early spleen development. A transgenic mouse line carrying this element driving a reporter gene was used to show that morphogenesis of the spleen initiates bilaterally and posterior to the stomach, before the splenic precursors grow preferentially leftward. In addition the transgenic line was used in an organ culture system to track spleen precursor cells during development. Spleen cells were shown to move from the posterior mesenchyme and track along the left side of the stomach. Removal of tissue from the anterior stomach resulted in splenic cells randomly scattering suggesting a guidance role for the anterior stomach. Using a mouse line carrying a conditional Cre recombinase to mark early precursor cell populations, the spleen was found to derive from posterior mesenchyme distinct from the closely adjacent stomach mesenchyme.

Homocysteine induces endothelial cell detachment and vessel wall thickening during chick embryonic development.

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 micromol/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.

Ets-1 and Ets-2 transcription factors are essential for normal coronary and myocardial development in chicken embryos.

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.

Cardiac outflow tract malformations in chick embryos exposed to homocysteine.

OBJECTIVE: Increased homocysteine concentrations have been associated with cardiac outflow tract defects. It has been hypothesized that cardiac neural crest cells were the target cells in these malformations. Cardiac neural crest cells migrate from the neural tube and contribute to the condensed mesenchyme of the aorticopulmonary septum and outflow tract cushions of the heart. The aim of this study is to investigate the effects of homocysteine on cardiac neural crest cells in relation to heart malformations. METHODS: Homocysteine was injected either into the neural tube lumen (30 micromol/l), or into the circulatory system (30 or 300 micromol/l) of chick embryos. LacZ-retroviral labeling was used to study cardiac neural crest cell migratory pathways after exposure to homocysteine. RESULTS: Cardiac neural crest cells contributed to the aorticopulmonary septum of both control and homocysteine-treated embryos. However, the outflow tract of homocysteine-neural tube injected embryos displayed 60% less apoptosis and 25% reduced myocardialization. A subarterial ventricular septal defect was observed in 83% of the embryos. None of these abnormalities were observed in homcysteine-circulatory system injected embryos. CONCLUSION: This study demonstrates that homocysteine disturbs apoptosis and myocardialization of the outflow tract, probably by affecting the cardiac neural crest cells.

Spatiotemporally separated cardiac neural crest subpopulations that target the outflow tract septum and pharyngeal arch arteries.

We used lacZ-retrovirus labeling combined with neural crest ablation in chick embryos to determine whether the cardiac neural crest cells constitute one group of multipotent cells, or they emigrate from the neural tube in time-dependent groups with different fates in the developing cardiovascular system. We demonstrated that early-migrating cardiac neural crest cells (HH9-10) massively target the aorticopulmonary septum and pharyngeal arch arteries, while the late-migrating cardiac neural crest cells (HH12) are restricted to the proximal part of the pharyngeal arch arteries. These results suggest a prominent role for early-migrating cells in outflow tract septation, and a function for late-migrating cells in pharyngeal arch artery remodeling. We demonstrated in cultures of neural tube explants an intrinsic difference between the early and late populations. However, by performing heterochronic transplantations we showed that the late-migrating cardiac neural crest cells were not developmentally restricted, and could contribute to the condensed mesenchyme of the aorticopulmonary septum when transplanted to a younger environment. Our findings on the exact timing and migratory behavior of cardiac neural crest cells will help narrow the range of factors and genes that are involved in neural crest-related congenital heart diseases.

The myth of ventrally emigrating neural tube (VENT) cells and their contribution to the developing cardiovascular system.

The cardiac neural crest cells are a group of cells that emigrate from the dorsal side of the neural tube during a specific time window and contribute to the pharyngeal arch arteries and the aorticopulmonary septum of the heart. Recent publications have suggested that another group of cells emigrating from the ventral side of the neural tube also contributes to the developing cardiovascular system. The first aim of our study was to define the specific time window of cardiac neural crest cell migration by injecting a retrovirus containing a lacZ reporter gene into a chick embryo at different stages during development. The second aim was to study the contribution of the supposed ventrally emigrating neural tube cells to the cardiovascular system using three approaches. One approach was to inject a lacZ retrovirus into the lumen of the chick hindbrain. Secondly, we injected the retrovirus into the neural tube at the position of the 10-12 somite pair. Finally, we used the chimera technique in which we transplanted a quail neural tube segment into a chick embryo. Cardiac neural crest cells were shown to emigrate from the dorsal side of the neural tube between HH9 and HH13(-). The HH13(+) neural tube has ceased to produce cardiac neural crest cells between the level of the otic placode and the fourth pair of somites. Retroviral injection directly into the chick hindbrain at HH14 resulted in 50% of the embryos with minimal labeling of the hindbrain and intense labeling of the adjacent mesenchyme, suggesting that virus was spilled. This implies that this technique is not useful for confirming the existence of ventrally emigrating cells. Both retroviral injections into the neural tube lumen at HH14 at the position of the 10-12 somite pair and the chimeras showed no signs of ventrally emigrating neural tube cells. We conclude that there is no contribution of ventral neural tube cells to the developing cardiovascular system.

Connexin43 levels are increased in mouse neural crest cells exposed to homocysteine.

BACKGROUND: Elevated homocysteine levels during embryonic development can result in neural tube and cardiovascular defects. The mechanisms that underlie the toxic effect of homocysteine are largely unknown. METHODS: We cultured mouse neural tube explants to study the effects of homocysteine on the migratory behavior of neural crest cells and on the levels of the gap junction protein Connexin43 (Cx43) and the actin- and Cx43-interacting protein ZO-1. RESULTS: Homocysteine exposure resulted in a significantly augmented maximal migration distance (MMD). The level of Cx43 immunolabeling was 2 times higher in the cytoplasm and cell protrusions of neural crest cells in homocysteine-treated cultures than in control cultures. Furthermore, colocalization of Cx43 and ZO-1 was increased in neural crest cell protrusions by this treatment. CONCLUSION: Increased Cx43 levels were previously shown to result in abnormal embryonic development. Our data raises the hypothesis that the embryotoxic effects of homocysteine may be mediated in part by its effects on Cx43 expression level and gap junction function in neural crest cells.

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, DiGeorge 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.

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.