N-SYNDECAN AND HB-GAM (HEPARIN-BINDING GROWTH-ASSOCIATED MOLECULE) ASSOCIATE WITH EARLY AXONAL TRACTS IN THE RAT-BRAIN

Heparin-Binding Growth-Associated Molecule (HB-GAM)/pleiotrophin is an 18 kDa extracellular matrix-and cell-surface-associated protein shown to enhance neurite outgrowth of perinatal forebrain neurones in vitro , The heparan sulphate proteoglycan N-syndecan (Raulo et al., 1994) ha s been isolated as a receptor/coreceptor for the HB-GAM. We have inves tigated, whether HB-GAM and N-syndecan could have a similar role in ne urite outgrowth and axon guidance in early axonal tracts of brain. In the present study N-syndecan was found to be spatiotemporally associat ed with the developing axonal tracts already on embryonic day 9 in rat , as revealed by coexpression with class III P-tubulin, which is one o f the earliest neuronal markers (Easter et al., 1993; Brittis et al., 1995). Later, N-syndecan and HB-GAM were detected in the first afferen t serotonergic projections arising from the pontine raphe nuclei. The expression pattern of HB-GAM peaked in the developing rhombencephalon at embryonic stage (E) 13-14. At the same time, N-syndecan was express ed in the developing raphe neurones growing neurites towards the dienc ephalon along HB-GAM immunoreactive pathways. When rhombencephalic neu rones were cultured on decreasing concentrations of substrate-bound HB -GAM, E13 neurones showed a significantly better neurite outgrowth res ponse than Ell, E16 or E18 neurones. The neurite outgrowth of raphe ne urones in vitro was inhibited by adding soluble heparin or N-syndecan into the culture medium, whereas addition of chondroitin sulphate had no effect. In a simple pathway assay, E13 raphe neurones selectively p referred attaching and growing neurites on pathways containing HB-GAM as compared with regions containing either laminin or fibronectin alon e. Our results suggest that HB-GAM may function as a developmentally r egulated cue for rhombencephalic neurones that possess N-syndecan on t heir cell membrane.