Fig. 2. Sox9 induces HNK-1 expression and neural crest differentiation. (A-I) Neural tubes electroporated with Sox9, 6 (A-C), 12 (D-F) and 24 (G-I) hours post transfection (hpt) analysed for HNK-1 expression. High level expression of the transfected construct was detected within 6 hpt (A-C) and robust ectopic induction of HNK-1 was detected by 12 hpt (D-F) and continued at 24 hpt (G-I) following Sox9 electroporation (EP). Confocal images indicate that the effect of Sox9 on HNK-1 expression is cell autonomous (C,F,I and data not shown). (G-I) An increase in the number of neural crest cells delaminating dorsally was evident and several transfected cells were observed delaminating from the neural tube in the intermediate and ventral region of the neural tube at 24 hpt (white arrows). (J) Dorsal view of the trunk neural tube electroporated with Sox9 at 40 hpt and assayed for HNK-1 expression by whole-mount immunofluorescence (K). Sox9-expressing cells are observed in the neural tube and in the delaminating neural crest cells (J, white brackets). An increase in the amount of delaminating HNK-1-expressing neural crest cells is detected on the transfected side of the embryo (K, white brackets). (L) Sox9-expressing cells are observed delaminating from the pial surface of the dorsal and intermediate region (white arrowhead) of the neural tube, corresponding to regions where laminin production is lost. (M) Sox9-expressing cells with ectopic HNK-1 expression are observed delaminating from the ventral neural tube (white arrowhead). (N-U) Induction of neural crest cells by Sox9 in neural plate explants. Vectors encoding either Sox9 and GFP or GFP alone were electroporated into the open neural plate region of the HH stage 10 chick embryos, [i] regions dissected and cultured for 48 hours in vitro before examining HNK-1 expression. (N-P) Explants of [i] regions transfected with GFP alone do not induce HNK-1 expression (O,P) and the DAPI image indicates cells remain confined to the explant (Q). By contrast, ectopic HNK-1 expression is detected in [i] explants transfected with Sox9 and many cells emigrate from the explants (R-U). A, anterior; P, posterior.