In vivo studies of the roof plate of the spinal cord and midline optic tectum in rodent and the developing subplate in the telencephalon of the chick showed that two glycosaminoglycans, keratan sulfate and chondroitin sulfate, possibly in the proteoglycan form (KS-PG, CS-PG, or KS/CS-PG), were present at times when axons approach closely but do not invade these territories. To address the question of whether KS/CS-PG actively inhibits growth cone elongation and to determine which component(s) of the proteoglycan may be critical to this phenomenon, we used a technique employing nitrocellulose-coated petri dishes onto which stripes of various purified macromolecules were attached. Isolated E9 chick dorsal root ganglia were grown on lanes of KS/CS-PG in alternation with lanes of the growth-promoting molecule laminin (LN). Neurite outgrowth was abundant along stripes of LN. In contrast, upon encountering a stripe containing KS/CS-PG, neurites either stopped abruptly or turned and traveled along the KS/CS-PG stripe border. The effect was dependent upon the concentration of the proteoglycan with intermediate concentrations producing intermittent patterns of crossing. We mixed LN with the KS/CS-PG, where the LN was in concentrations which alone support outgrowth, and observed that the KS/CS-PG was still inhibitory when such a growth-promoting molecule was present. A 10-fold higher concentration of LN was able to overcome the inhibitory effect of the KS/CS-PG. These results suggest that the interaction of inhibitory and growth-promoting molecules can interact to produce a wide spectrum of neurite patterns ranging from complete inhibition to totally unimpeded outgrowth. Selective enzymatic removal of the KS or CS from the KS/CS-PG permitted various degrees of neurite outgrowth to occur across the previously inhibitory lanes, and digestion of both glycosaminoglycan moieties, leaving only the protein core of the molecule, resulted in a complete lack of inhibition. These assays demonstrated that KS/CS-PG is inhibitory to embryonic dorsal root ganglia neurites in vitro and that complete inhibition requires contributions from both KS and CS moieties.
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