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Figure 1


Fig. 1. HH signaling is necessary for cell-fate specification in the ventral midbrain. For orientation and embryonic ages, see Materials and methods. (A) At E5, EGFP-electroporated (right side) controls do not show disruptions in the rostral floor plate (rFP; SHH+, brown) or in midbrain arc pattern formation. Midbrain arcs are marked by the homeobox (HX, blue) gene expression of PHOX2A in the first arc (1), PAX6 (P6) and EVX1 (E1). (B) Blockade of FOXA2 (brown) expression following unilateral Ptc1{Delta}loop2 (blue) electroporation (right side). (C) Re-specification of ventral cell fates (marked by HX genes, blue) into dorsal (PAX7+, brown, arrowhead) cell fates. (D,E). Blockade and bi-directional spread of PHOX2A+ (brown) oculomotor complex neurons following bilateral electroporation (E) of Ptc1{Delta}loop2 (blue). Compare E with EGFP-electroporated controls (D). D and E are photographed at the same magnification. (E) Note the lack (caudally, arrowhead) of overlap between PHOX2A and Ptc1{Delta}loop2 transgene expression. Rostral cells (arrow) have extinguished their requirement for HH signaling by this stage (see text). (F,G) Reduced expression and spread of tyrosine hydroxylase (F, dopaminergic neurons), PAX6 (G, brown) and EVX1 (G, blue) following unilateral HH blockade. (H) Cross-section demonstrating the non-autonomous spread of PAX6+ (brown, arrowhead) cells following unilateral Ptc1{Delta}loop2 (blue) electroporation. Note the presence of ectopic PAX6+/Ptc1{Delta}loop2+ cells (arrow, see text). (I,J) E8 whole mounts electroproated at H&H stage 10, demonstrating that, compared to EGFP controls (I), cell spread following HH blockade (J) increases with time (compare with the E5 brains in D and E) and is multidirectional. Blue, TH (arrowheads); brown, ISL1+ motor neurons; 1, first arc; III, third ventricle; bi, bilateral electroporation; E1, EVX1; EP, electroporated; P6, PAX6; HX, homeobox expression of PHOX2A, PAX6, EVX1; MHB, midbrain-hindbrain boundary; rFP, rostral floor plate; TH, tyrosine hydroxylase; tec, tectum.





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