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Fig. 4. Spatiotemporal regulation of HH requirement in the ventral midbrain. (A) Bilateral EGFP (blue) misexpression does not perturb the expression of rFP genes (FOXA2, brown). (B,C) Caudal-medial and lateral, but not antero-medial (arrowhead), regions of the rFP (FOXA2+, brown) can be disrupted following bilateral electroporation of Ptc1^loop2 (blue) at H&H stages 6-9. (C) Cross-section of B at the level indicated by the line in B. (A-C) Note the meager number of Ptc1^loop2+ cells at the midline (arrowheads, B,C) compared with controls (A). (D) HH blockade disrupts lateral rFP specification at H&H stages 15-16. (E) E6 embryo electroporated with Ptc1^loop2 (blue) between H&H stage 9 and 11, demonstrating the uniform blockade of cell-fate specification in all midbrain arcs, assayed by HX gene expression (brown). Note the extensive cell mixing and disruption of the arc pattern. (F) Greater caudal perturbation of the PHOX2A+ (1, brown, arrowhead) first arc following Ptc1^loop2 electroporation (blue) at H&H stages 10-12. The rostral expression of PHOX2A (arrow) is largely unaffected despite the higher bilateral expression of the Ptc1^loop2 transgene in this region. (G) E6 embryo electroporated between H&H stages 17 and 20, demonstrating that midbrain cell fates (brown) are independent of HH signaling, except in lateral regions of the rFP and cells associated with it (e.g. arc 2). (H) Close-up of boxed area in G, demonstrating that midbrain progenitors within the lateral region of the rFP and the cells associated with it (e.g. arc 2; 2) can be re-specified to more-dorsal (PAX6+) cell fates in association with Ptc1^loop2+ cells (arrow). In addition, dorsal cells (PAX6+) can move into this region non-autonomously (arrowhead). 1, first arc; 2, arc 2; III, third ventricle; bi, bilateral electroporation; EP, electroporated; P6, PAX6; H&H, embryonic stages according to Hamburger and Hamilton (Hamburger and Hamilton, 1951); HX, homeobox expression of PHOX2A, PAX6, EVX1; MHB, midbrain-hindbrain boundary; rFP, rostral floor plate.

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