Chondroitin sulfates restrict axonal growth along the projection path of vestibular nuclear (VN) neurons across the hindbrain of prenatal rats

Abstract

In our study of chondroitin sulfate proteoglycan (CSPG) deposition in rhombomere boundaries of the developing hindbrain, we found chondroitin 6-sulfotransferase (C6ST)- expressing cells within rhombomeres of E8.5 to E10.5 mouse embryos. This is coincident with the growth of axons from vestibular nuclear (VN) neurons across the hindbrain. The spatio-temporal concurrence of axonal growth and CS-expressing cells in the hindbrain suggests a role of CSPGs in determining the axonal trajectory of VN neurons. To perturb the possible effect of hindbrain CS in axonal development from the VN neurons, chondroitinase ABC (ChABC) was injected into the fourth ventricle of Sprague–Dawley rats at E12.5, comparable to E9.5 in mouse. The embryos were maintained in vitro for 24 h. Axons from the VN neurons were then traced with DiI labeling. Successful removal of CS chains was confirmed when the fixed embryos were immunostained for CSPG stubs revealed by the enzyme. Controls were injected instead with PBS vehicle. Results showed that with the control injection, the axons of the VN neurons advanced towards the midline and barely crossed the midline to the contralateral side of the neurotube. In embryos injected with ChABC, the axons were defasciculated when compared with the PBS-treated embryos, even though there was no significant change in the normal trajectories of the axons. These results suggested that the CS components were important in confining the axonal path of the VN neurons as they crossed the midline. Further examination of the axonal projection pattern of VN neurons as perturbed by ChABC treatment in earlier stages of embryos is in progress to determine the period in which CS moieties contribute to the restriction of growing axons to their projection path. Acknowledgement: Supported by HK RGC grant HKU 7294/01M.