Differential patterns of developing vestibular commissural projection with digestion of chondroitin sulfates

Abstract

Chondroitin sulfate proteoglycans have been attributed with guidance functions in cell migration and process outgrowth. The developing hindbrain was found to be enriched in 6-sulfated isoforms of chondroitin as early as E8.5–9.5 in the mouse; expression levels decreased to undetectable levels in the following two days. To assess if chondroitin sulfates (CS) play a part in determining axonal projections about this period in the early embryonic hindbrain, chondroitinase ABC (ChABC) was delivered into the 4th ventricles of rat embryos (E11.5–13.5) in culture to digest away CS moieties in the nearby hindbrain matrix. Controls received either vehicle or the heat-inactivated enzyme instead. DiI injection was introduced into the vestibular nuclear complex (VNC) near the VIIIth cranial nerve entry zone to map the commissural projections that developed during the treatments. At E11.5 (+1DIV) , few neuritis extended towards the midline in cases of PBS and heatinactivated ChABC. In contrast, with digestion of CSs, robust projections reached the dye-injection site from regions half-way towards the midline. This suggests that CSs limit projection across the hindbrain matrix of early embryos. At E12.5 (+1DIV) , the contralateral-projecting axons assumed fascicles bearing tapered terminals that reached the midline in the controls. In the enzymetreated embryos, the axons however remained unfasciculated as they extended normal to the midline. By E13.5 (+1DIV) , enzyme treatment apparently did not affect the pioneer axons that had advanced normal to the midline and beyond towards the contralateral VNC. However, later axonal outgrowths from the VNC traversed the enzyme-treated matrix as unfasciculated fibres and diverted from the course of the pioneers to cross the midline at various angles and positions along the midline. This suggests that CSs also limit the course of the later projections which otherwise would be attracted to alternative targets. These results provide in vivo evidence for possible contributions of CS to limit stray outgrowth and foster axonal fasciculation as vestibular neurons project across the midline towards the contralateral target.