Homocysteine inhibits cardiac neural crest cell formation and morphogenesis in vivo

Abstract

Elevated homocysteine increases the risk of neurocristopathies. Here, we determined whether elevating homocysteine altered the proliferation or number of chick neural crest cells that form between the midotic and third somite in vivo. Homocysteine increased the number of neural tube cells but decreased neural crest cell number. However, the sum total of cells was not different from controls. In controls, the 5-bromo-2 -deoxyuridine-labeling index was higher in newly formed neural crest cells than in their progenitors, paralleling reports showing these progenitors must pass the restriction point before undergoing epithelial-mesenchymal transition. Homocysteine decreased the labeling index of newly formed neural crest cells, suggesting that it inhibited cell cycle progression of neural crest progenitors or the S-phase entry of newly formed neural crest cells. Homocysteine also inhibited neural crest dispersal and decreased the distance they migrated from the neural tube. These results show neural crest morphogenesis is directly altered by elevated homocysteine in vivo.

Microarray analysis of homocysteine‐responsive genes in cardiac neural crest cells in vitro

“…The present analysis identified 65 genes whose expression was dysregulated by elevated homocysteine, each of which met rigorous criteria for inclusion. Based upon the results of previous studies of cellular function, we hypothesized that the homocysteine-responsive transcriptome of cardiac neural crest cells would be especially well-represented by genes that are important to cell migration (Brauer and Rosenquist, 2002;Tierney et al, 2004;Brauer and Tierney, 2004). The results supported the hypothesis: the largest functional class of genes whose transcription was altered by homocysteine were identified as important to cell migration and adhesion.…”

“…Therefore, understanding the behavior of the cardiac neural crest has become a key to understanding the etiology of conotruncal heart defects. Previously, we have described in detail the impact of elevated homocysteine upon the behavior in vitro of cells from the cardiac neural crest (Brauer and Rosenquist, 2002;Tierney et al, 2004;Brauer and Tierney, 2004). In this study, we have used the same model of cardiac neural crest cells in vitro to obtain RNA for microarray analysis, comparing the gene expression patterns of neural crest cells exposed to elevated homocysteine with untreated cells, taking advantage of the recent publication of the chicken genome (Hillier et al, 2004;Wong et al, 2004), and the consequent availability of high-quality gene chips.…”

Homocysteine enhances cardiac neural crest cell attachment in vitro by increasing intracellular calcium levels

“…These results could explain how Hcys alters cardiac NC motility in vitro (Brauer and Rosenquist, 2002) and account for, at least in part, the decrease in cardiac NC migration seen in Hcystreated chick embryos in vivo (Tierney et al, 2004). chelator, BAPTA, blocked the effect of Hcys on NC attachment, (2) the effect of Hcys was mimicked by the Ca 2ϩ ionophore, ionomycin, (3) Hcys increased the emission from the intracellular Ca 2ϩ -sensitive probe, Fluo-4, and (4) the IP 3 receptor antagonist, 2-APB, blocked Hcys-enhanced NC cell attachment.…”

“…Hcys alters NC migratory behavior in vitro and in vivo and increases NC cell spreading and surface area during migration on fibronectin-coated substrates (Brauer and Rosenquist, 2002;Tierney et al, 2004). To determine whether these effects might be due to Hcys-induced changes in cell attachment to the extracellular matrix, we developed an attachment assay for primary cultures of cardiac NC cells.…”

“…Despite the strong association between neurocristopathies and elevated Hcys, surprisingly little information is known regarding the effects of elevated Hcys and folate deficiencies on NC morphogenic pro-cesses. In the chick embryo, we found elevated Hcys inhibits the entry of newly-formed cardiac NC cells (NC cells derived from the mid-otic to third somite axial levels) into the S-phase of the cell cycle, decreases cardiac NC cell number, and decreases the distance that cardiac NC cells migrate from the neural tube in vivo (Tierney et al, 2004). In addition, elevated Hcys alters cardiac NC cell migratory behavior, cell shape, and differentiation in vitro (Brauer and Rosenquist, 2002;Boot et al, 2003).…”