Homocysteine and CSRP-3 protein in migrating neural crest cells: A quantitative and qualitative confocal microscopy study

Congenital heart defects are one of the leading causes of infant death. High maternal blood serum levels of the amino acid homocysteine and the frequency of heart defects are correlated, but the immediate cause is unknown. During early embryonic development, a distinctive population of cells arises dorsally as the neural tube closes, forming the brain and spinal cord. These are the neural crest cells, which play a crucial role in the development of the nervous system, head skeletal, certain endocrine and connective tissues, and, notably, the heart’s outflow region. These migratory cells will follow specific pathways and respond to signals encountered in order to further differentiate and arrive at their correct destinations. There is evidence that neural crest cells may be affected in their migration by abnormally high levels of homocysteine. Any failure of cells to migrate efficiently with regard to timing and direction might cause malformations in the developing embryo. A microarray study performed by Rosenquist et al. (2007) revealed that a gene known as CSRP-3, (an acronym for cysteine and glycine rich protein 3) which codes for a member of the LIM (an acronym for the proteins involved: Lin 11, Isl-1, and Mec-3) domain protein group, is over-expressed in neural crest cells exposed to homocysteine. CSRP-3 protein is a nuclear transcription factor but is also involved in cytoskeletal mediation of cell adhesion and migration. CSRP-3 interacts with scaffolding proteins in order to carry out mitotic functions and cytoskeletal specialization for development. This gene’s mRNA was found to be upregulated 177-fold in the presence of homocysteine. I hypothesized that if homocysteine does up-regulate this gene in migrating neural crest cells, it would be detectable by immunostaining using a specific antibody, and could be quantitated, localized and compared with control embryos to provide new information. Neural crest cells, and possibly other cells over-expressing CSRP-3, would show higher intensity of immunofluorescence signal. After injecting homocysteine or saline only (control) into the amnion of stage 10-12 chick embryos and allowing further development, I localized and measured CSRP-3 fluorescence in four regions known to receive migrating neural crest cells: 1) the cardiac pathway region, the brain meninges of the myelencephalon and diencehpalon, 2) the paraxial mesenchyme lateral to the neural tube, 3) the periocular mesenchyme, and 4) the pharynx region. Using confocal microscopy, sectioned and whole mount embryos were examined after undergoing double immunolabeling using an antibody to CSRP-3 protein and an antibody that recognizes neural crest cells. Expressing cells were measured to yield a corrected ratio. Cells identified in the areas of interest were all found to show significantly brighter fluorescence compared to cells in control embryos, supporting and expanding the findings of Rosenquist et al (2007), and providing additional evidence for this mechanism of action of homocysteine