Fig. 5. Sulf1^-/-;Sulf2^-/- esophagi have defective GDNF-dependent neurite outgrowth. Esophagi (400 µm) were dissected from E11.5 embryos and plated on collagen gel containing BSA, GDNF or neurotrophins at various concentrations. After 4 days, the whole explant was immunostained with the TuJ1 antibody. Explants that failed to attach to collagen gel were not included in the assay. (A,B) Neurite outgrowth of E11.5 esophageal explants was selectively dependent on GDNF, but not on neurotrophin. Sulf1^-/-;Sulf2^-/- esophagi failed to extend neurites at 10 ng/ml GDNF and showed reduced neurite outgrowth at 20 ng/ml and 50 ng/ml GDNF. (C) Quantification of the neurite outgrowth shown in A and B. The length of the extended neurite was measured along six axes, 30° apart and the average was calculated to represent the neurite outgrowth of one explant. Data represent the mean and the standard deviation of a minimum of four individual cultures. (D,E) Quantification of the total number of neurons in the explants. The neurons in the explants (dark cell-body staining by the TuJ1 antibody, indicated by arrows) were quantified using the bright field at low magnitude. Neurons were scattered, or even migrated out of the control explants in the presence of 10 ng/ml GDNF. In control explants cultured in the presence of BSA or NGF and in GDNF-treated Sulf1^-/-;Sulf2^-/- explants, neurons tended to form clusters. The large clusters of neurons in Sulf1^-/-;Sulf2^-/- explants were quantified by summing the neuronal numbers at different focal planes. ^**, P<0.01 (two-tailed Student's t-test). Scale bars: 250 µm in A,B; 100 µm in D.