Fig. 2. Axonal projections distinguish red nucleus and oculomotor complex territories within the medial arc. (A,B) Tracer deposits delivered to the third cranial nerve establish that OMC neurons lie within the PHOX2A-rich territory of the medial arc. Retrogradely labeled neurons identified by anti-fluorescein alkaline phosphatase histochemistry appear as dark purple cells in E5 midbrain whole-mount (A) and cross-section (B) preparations. (C,D) Whole-mount preparations of E6 explants injected with fluoresceinated dextrans and cultured for 4 hours. (C) Anterograde labeling from a deposit in ventromedial midbrain (asterisk) identifies a crossed projection from the medial arc to the contralateral hindbrain (arrow). (D) Retrograde labeling from a hindbrain tracer deposit (asterisk) identifies red nucleus (RN) neurons in contralateral midbrain. Arrowheads in C,D indicate the midbrain-hindbrain junction. Control deposits placed in more caudal and lateral hindbrain (n=54) failed to label contralateral ventral midbrain, suggesting that at E6 RN axons have not reached the spinal cord. (E,F) Cross-sections of E6 explants where PHOX2A (E) and BRN3A (F) gene expression were combined with retrograde labeling from tracer deposits in contralateral (left) hindbrain. The retrogradely labeled RN neurons lie pial to the PHOX2A+ OMC (E) and are contained within the BRN3A+ territory of the medial arc (F). III, third ventricle; FP, floor plate of hindbrain; IS, isthmus; OMC, oculomotor complex; rFP, rostral, or midbrain, floor plate; RN, red nucleus.

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