N‐Syndecan and HB‐GAM (Heparin‐Binding Growth‐Associated Molecule) associate with early axonal tracts in the rat brain

Abstract

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 ( 1 ) has been isolated as a receptor/coreceptor for the HB‐GAM. We have investigated, whether HB‐GAM and N‐syndecan could have a similar role in neurite outgrowth and axon guidance in early axonal tracts of brain. In the present study N‐syndecan was found to be spatiotemporally associated with the developing axonal tracts already on embryonic day 9 in rat, as revealed by coexpression with class III β‐tubulin, which is one of the earliest neuronal markers ( 2 ; 3 ). Later, N‐syndecan and HB‐GAM were detected in the first afferent 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 expressed in the developing raphe neurones growing neurites towards the diencephalon along HB‐GAM immunoreactive pathways. When rhombencephalic neurones were cultured on decreasing concentrations of substrate‐bound HB‐GAM, E13 neurones showed a significantly better neurite outgrowth response than E11, E16 or E18 neurones. The neurite outgrowth of raphe neurones 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 preferred attaching and growing neurites on pathways containing HB‐GAM as compared with regions containing either laminin or fibronectin alone. Our results suggest that HB‐GAM may function as a developmentally regulated cue for rhombencephalic neurones that possess N‐syndecan on their cell membrane.