Fig. 7. Coordinate regulation of medial arc pronuclei by SHH manipulations. (A,B) Expanded gene expression for SHH (A) and its downstream transcriptional targets FOXA2 and ISL1 (B) is seen at E5, after electroporation of pXeX-SHH into chick E2 ventral midbrain. In this example, the electroporated side of the brain is the right side; the left side serves as an internal control. In the hindbrain the expanded ISL1 gene expression identifies the trochlear (Tr) nucleus. (C-F) An enlarged SHH source elicits a strictly coordinate expansion of the OMC and the pRN, as demonstrated by two-color gene expression for PHOX2A and BRN3A (C,D), and PHOX2A and EMX2 (E,F). The E5 wholemounts shown in C and E were cut at the level of the asterisks to prepare the sections illustrated in D and F. The OMC and pRN territories, although expanded, maintain their spatial relationship in all three dimensions: co-extensive along the mediolateral axis (D,F), partially overlapping along the anteroposterior axis (C,E) and completely segregated along the ventricular-pial axis (D,F). (G) E5 wholemount of EMX2 and PHOX2A gene expression shows that SHH in lateral tegmentum elicits a properly patterned ectopic medial arc (1^*). Arrows indicate EMX2 labeling of the ectopic pRN, which partially overlaps the PHOX2A labeling of the ectopic OMC and lies pial to it. (H) Wholemount of E5 midbrain hemitegmentum (below dotted line) and tectum (above dotted line) demonstrating that SHH misexpression in dorsal midbrain also produces an ectopic medial arc (1^*) containing an ectopic PHOX2A+ OMC and an ectopic BRN3A+ pRN. Note that SHH misexpression in dorsal midbrain results in the loss of BRN3A labeling of the tectum, which is found in the inner part of the mantle layer and is thereby readily distinguished from pRN-like BRN3A labeling, which is found more pially. (H, inset) Cross-section through an ectopic medial arc in dorsal midbrain documents the ventricular-pial segregation of the PHOX2A+ (brown) and BRN3A+ (blue) territories. The insert is oriented with the tectal ventricular surface marked by an asterisk. 1 and 2, arcs 1 and 2; IS, isthmus; OMC, oculomotor complex; rFP, rostral, or midbrain, floor plate; RN, red nucleus; tec, tectum; Tr, trochlear nucleus.