Meeting Abstract

P1.47  Thursday, Jan. 3  Homocysteine Modifies Neural Crest Cell Actin Cytoskeleton and Nuclei CLUBINE, Amanda; WIENS, Darrell J.*; Univ. of Northern Iowa wiens@uni.edu

Elevated levels (> 20 �M) of the natural amino acid homocysteine (Hcy) interfere during development, resulting in neural tube, facial, and cardiac defects. A primary site for Hcy-induced malformations is the neural tube and the neural crest cells (NCCs) that originate from it. These migrate to cranial, cardiac and other destinations where they settle to form important structures. Our experiments tested the effects of Hcy on cardiac neural tube explanted to culture. HH stage 10-11 chick embryos (33-40 hr incubation when NCC migration begins) were isolated from eggs and cardiac segments of the neural tube were microdissected and explanted to culture dishes for 24 hr incubation in medium with or without (control) 100 �M or 300 �M Hcy. During culture, NCCs migrated outward from the explants and neural tube tissue also spread as epithelium. Following culture, explants were fixed, permeabilized, and stained with FITC-phalloidin (binds F-actin). In some experiments cell nuclei were also stained with bis-benzimide. Microscopic fluorescence images of the cells that migrated from the explant were captured and analyzed using OpenLab and NIH Image software. We found that cell size and shape were not affected by Hcy. But F-actin cytoskeletal staining intensity increased 9% over control level with 100 �M Hcy treatment and 40% with 300 �M Hcy. Cells that spread as neuroepithelial sheet did not show this cytoskeletal response to Hcy. We also found that cell nuclei showed reduced bis-benzimide staining when treated with 300 �M Hcy. These findings suggest an effect of Hcy on Ras GTPases or other regulators of the actin cytoskeleton and also suggest that Hcy may cause a change in chromatin methylation resulting in alterations of cell nuclei. Our results imply that Hcy may affect NCC migration and differentiation.