Fig. 7. Removal of dorsal tissue increases the sensitivity of the prospective ZLI region to Hh signaling. (A) Scheme for explant dissections. The dorsal fifth of stage 13-14 dorsal explants was removed to generate intermediate explants, or restored to generate D^* explants. The ventral boundaries of the explants described in this figure were shifted to a position between the D1/2 and D2/3 explants to improve survival. (B) Quantitation of the effects of exposure to Hh-Ag1.3 on ZLI length. Error bars represent s.e.m. Shh expression extended to within 10% of the dorsal edge of the explant in 38% (13/34) of intermediate explants exposed to 250 nM Hh-Ag1.3; this extension was not seen in any (0/14) of the dorsal explants similarly treated. (C-E) Wnt3a expression is maintained in the dorsal neural tube and in the thalamus after two days in culture in intact (C) and dorsal (D) explants, but only the thalamic domain is present in intermediate explants (E). Although the narrow intermediate explants may bend during the culture period, the orientation with respect to the axis of the neural tube is maintained. (F-H) Exposure to 250 nM Hh-Ag1.3 produced longer domains of Shh (black) expression in the ZLI of intermediate explants (G), when compared with dorsal (F) and D^* (H) explants after 48 hours. (I-K) Electroporation of mShh-CD4 and GFP in stage 13-14 explants, assessed by staining for Shh (red) and GFP (green) after 48 hours. Shh was not detected in dorsal explants grown in control medium (I), but exposure to 250 nM Hh-Ag1.3 supported Shh induction exclusively in cells neighboring the electroporated region (K). Removal of dorsal tissue in intermediate explants supported the induction of Shh within the electroporated region and in cells dorsal to it (J).