Mapping of chondroitin sulfate glycoform and chondroitin 6-sulfotransferase in neuroepithelial boundaries of mouse embryos

Abstract

As chondroitin sulfate (CS) isoforms have been found in the hindbrain region of mouse embryos during early development, we hypothesized that these isoforms are involved in patterning hindbrain segmentation. We chose to study the segmentation pattern in E 8.5 to E 11.5 embryos. Immunostaining with the monoclonal antibody CS-56 revealed immunopositivity in the neuroepithelial boundaries of both tissue sections and whole-mount embryos. The signals were found stronger in E 8.5 and E 9.5 embryos than in later stages. By neutralization with CS isoforms, we found that CS-56 was more sensitive to chondroitin 6-sulfate (C6S) than other CS glycoforms. As C6S is likely a product of chondroitin 6-sulfotransferase (C6ST)-catalyzed reaction, we reasoned that upregulation of C6ST gene expression took place in the boundary regions. A riboprobe specific for the C6ST gene was synthesized and used for in situ hybridization. Signals were found between rhombomeric boundaries in E 8.5 and E 9.5 mouse embryos but not in E 10.5 and E 11.5 embryos. These results imply that C6S was produced by C6ST in the formation or maintenance of the boundaries. Further work will be performed to identify signals that trigger the in situ positive cells to change in C6ST expression and to test the consequence of this change to the formation of rhombomeric boundaries.