Fig. 3.

Functional assay of Chase/LV properties. Coverslips precoated with poly-D-lysine were coating with CSPGs using a 1-mm thick strip of filter paper saturated with CSPG and then coated with laminin. Coverslips were pretreated for 24 h with one of following supernatants: (1) NPC culture medium; (2) NPC/Chase/LV; (3) ChABC enzyme. DRG from day 7 embryonic chicks were cultured at the border of the CSPG with defined medium, supplemented with 20 ng/ml nerve growth factor and mixed 1:1 with experimental supernatants. Cultures were fixed for 24 h after DRG plating and double stained with anti-tubulin (Tuj1) to identify neurons (green) together with CS-56 to identify CSPG or 3B3 to identify CSPG digestion. Chick DRG grew extensive neurites in all directions except in the CSPG-rich region (A, red area), which remained undigested (D, negative for 3B3 staining) when treated with culture medium. Although few fibers grew into the CSPG area, most of them turned to avoid the CSPG. However, when treated with medium obtained from NPCs infected with Chase/LV, CSPGs were partially digested (B, CS-56 staining, red, and E, 3B3 staining, red), thereby allowing DRG axons to grow into the digested CSPG region. Treatment with ChABC completely digested the CSPG (C, with CS-56 negative staining and strong 3B3 positive staining) and allowed DRG to grow in all directions.