IMMUNOHISTOCHEMICAL LOCALIZATION OF CHONDROITIN AND HEPARAN-SULFATE PROTEOGLYCANS IN PRE-SPINA-BIFIDA SPLOTCH MOUSE EMBRYOS

The splotch (Sp) mutation on mouse chromosome I is a genetic model for the neural tube defects spina bifida and exencephaly. Embryos carrying Sp or its allele splotch-delayed (Sp(d)), have been shown to have delays in neural tube closure, and neural crest cell emigration, as well as a reduction in extracellular space around the neural tube. Pre-spina bifida Sp and Sp(d) embryos have abnormalities of notochord, mesoderm and neuroepithelial development. Chondroitin sulphate proteoglycans (CSPG) and heparan sulfate proteoglycans (HSPG) have been shown to play essential roles during neural tube closure and neural crest cell emigration and migration and thus might well be affected by the splotch mutation. Therefore, the effects of Sp and Sp(d) on the temporal and spatial distributions of CSPG and HSPG were studied in pre-spina bifida embryos cytogenetically identified as Sp/Sp (Sp(d)/Sp(d)), Sp/+ (Sp(d)/+) or +/+. Immunohistochemical localization of CSPG by means of the CS-56 monoclonal antibody showed that in Sp/Sp head sections, the neuroepithelial basement membranes stained more intensely at 5-, 10-, and 15-somite stages, whereas similar staining was observed at 16- and 19-somite stages compared with matched +/+ sections. In caudal sections Sp/Sp again showed a more intense stain for CSPG in the neuroepithelial basement membranes in all sections (except one comparison, in which staining was similar) from embryos of 14-, 15-, 16-, and 19-somite stages, compared to matched +/+ sections. Heterozygotes did not differ consistently from the mutant or the normal (+/+) embryos in CS-56 stain intensity. Immunohistochemical staining with antibody to a basement membrane HSPG showed a more intense stain in the two Sp(d)/Sp(d) 10-somite stage embryos tested than in matched +/+ embryos. It is suggested that the greater intensity of CSPG and HSPG staining in Sp and Sp(d) mutant embryos compared to +/+ embryos could be caused by an altered sequence within the Sp gene locus that either directly disrupts the production or degradation of these and other molecules, or does so indirectly through its interaction with neural cell adhesion molecules.