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First published online 23 March 2005
doi: 10.1242/dev.01783

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Shh-dependent formation of the ZLI is opposed by signals from the dorsal diencephalon

Department of Biochemistry and Molecular Biophysics, Columbia University, 701 West 168th Street, New York, NY 10032, USA

View larger version (61K): [in a new window]   Fig. 1. ZLI differentiation in explants. (A-I) Hemisected forebrains viewed from the ventricular surface showing that the dorsal progression of ZLI differentiation in ovo (A-C) is recapitulated in forebrain explants cultured in vitro (D-I), as assessed by whole-mount in situ hybridization with probes for Shh (dark blue) and Lfng (red; A-F), and Ptc2 (G-I). (J) The length of the ZLI as a fraction of the total length of the alar plate (relative ZLI length) was assessed using Shh to mark the ZLI and Lfng to mark the dorsal boundary of the alar plate. (K) Quantitation of ZLI growth rates in ovo (blue line) and in vitro (red line). Error bars indicate s.e.m.

View larger version (77K): [in a new window]   Fig. 2. The ZLI forms from cells in the diencephalic alar plate. (A) Strategy to ascertain whether cells in the basal plate contribute to the ZLI. (B-H) The relative positions of labeled cells along the dorsoventral axis of the diencephalon were assessed immediately following DiI injection (relative initial position in B, and C,F) and after 48 hours in culture (relative final position in B, and D,G). DiI was photo-converted to a brown precipitate and analyzed in relation to the ZLI, as marked by Ptc2 expression (E,H). Cells labeled in the ZLI (B, black circles) and elsewhere in the diencephalon (B, gray circles) remained at the same relative dorsoventral position during the experimental period. (C-E) DiI injected in the basal plate was never observed in the ZLI (n=24). (F-H) Labeling in the ZLI observed after focal injection of DiI into the alar plate (n=11).

View larger version (79K): [in a new window]   Fig. 3. The ventral diencephalic alar plate is sufficient to maintain a program of ZLI differentiation. (A) Strategy to generate stage 13-14 forebrain explants containing the dorsal two-thirds (D2/3) or dorsal half (D1/2) of the diencephalon. Incisions made dorsal to the visible morphological sulcus at the alar-basal plate boundary produced D2/3 and V1/3 explants, whereas D1/2 and V1/2 explants were generated by bisections of the neural tube. ZLI differentiation was monitored by Shh (purple) and Ptc2 (yellow) expression after 48 hours in culture. (B-I) D2/3 explants (D,E), but not D1/2 explants (H,I) can form a ZLI after 48 hours in culture (t=48 hours), as assessed by Shh (D,H) and Ptc2 (E,I) expression. Neither D2/3 explants (B,C) nor D1/2 explants (F,G) contained basal-plate tissue, as assessed by Shh (B,F) and Ptc2 (C,G) expression immediately after dissection (t=0 hours). V1/3 explants, which correspond to the ventral tissue excised from D2/3 explants, expressed the basal-plate markers Shh (J,L) and Ptc2 (K,M) at the time of dissection (J,K) and after 48 hours (L,M). Lfng expression was used to monitor the quality of the explants (red).

View larger version (68K): [in a new window]   Fig. 4. Shh signaling from the basal plate is required for ZLI induction in D1/2 explants. (A) Strategy to test the requirement for Shh signaling from the basal plate. The ventral part of the basal plate was removed and incubated for 1.5 hours at 37°C with or without MAb-5E1. The basal-plate grafts were cultured in conjunction with D1/2 explants for 48 hours and ZLI differentiation was monitored by Shh expression. (B) D1/2 explant controls. The ZLI-inducing activity of the basal plate (C) was blocked by pre-incubation of basal-plate tissue with MAb-5E1 (D).

View larger version (52K): [in a new window]   Fig. 5. Dorsal progression of ZLI differentiation requires continuous Shh signaling. (A) Strategy to test the requirement for Shh signaling during the 48-hour explant culture period. 25 ng/µl MAb-5E1 was added to intact stage 13-14 forebrain explants after a defined period in culture, and the relative length of the ZLI was assessed after 48 hours. (B-G) Addition of MAb-5E1 from the time of explant isolation (C) drastically reduced the length of the ZLI compared with untreated controls (B). MAb-5E1 treatment of stage 11 forebrain explants completely eliminated Shh expression from the ZLI (D). MAb-5E1 addition after 12 (E), 18 (F) and 24 (G) hours in culture arrested the dorsal progression of ZLI differentiation. (H) MAb-5E1 addition at any point during the first 28 hours of the culture period prevented the dorsal progression of the ZLI (blue line) compared with treatment with control serum fixed after corresponding periods in culture (red line). Error bars represent s.e.m.

View larger version (52K): [in a new window]   Fig. 6. ZLI formation in D1/2 explants varies with the levels of Hh signaling applied. Stage 13-14 D1/2 explants were cultured in presence of varying concentrations of Hh-Ag1.3 and the relative length of the ZLI was assessed after 48 hours in culture by Shh (black) and Lfng (red) expression. Exposure to 250 (B), 500 (C) and 1000 (D) nM Hh-Ag1.3 resulted in increasingly longer domains of Shh expression in D1/2 explants than in untreated controls (A). (E) Quantitation of the dose effect of Hh-Ag1.3 addition on the relative length of the Shh domain in the ZLI. Error bars represent s.e.m.

View larger version (53K): [in a new window]   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).

View larger version (97K): [in a new window]   Fig. 8. Grafts of dorsal diencephalic tissue inhibit ZLI propagation. (A,B) Stage 13-14 dorsal diencephalic grafts were excised from a region containing Wnt3a-expressing cells (A), and intermediate telencephalic grafts from a region containing Foxg1-expressing cells (B). (C-F) Expression of Wnt3a (red, C) and Foxg1 (red, D) in relation to Shh (blue, D) after 2 days in culture. The propagation of Shh expression in the ZLI was inhibited adjacent to dorsal diencephalic grafts (E), but not in telencephalic controls (F). Dashed circles indicate grafted tissue.