Preparation of the embryo. (A,B) Injection of ChABC into the fourth ventricle in the embryos was visualized by fast green included in the enzyme solution: (A) side view and (B) dorsal view of the embryos. The asterisk marks the position of the fourth ventricle. (C) The dashed red line outlines the hindbrain region to be dissected out from the embryo. (D) Schematic diagram showing cuts made in the dorsal neural tube to expose the ventricular surface of the hindbrain. (E) Flat-mounted hindbrain showing the DiI (red) and NeuroVue Maroon (blue) tracked fibers. Overlay of longitudinal gridlines (white dashed lines) assist fiber counting. The center of the dye injection (indicated by the asterisk) was designated as 0 μm. Gridlines of 100 μm were drawn parallel to the floor plate. (F) Schematic diagram showing a flat-mounted hindbrain with the ventricular surface facing up. The dashed outline in green indicates the extent of the VN complex (VNC) in the hindbrain. The dotted lines in black indicate the rhombomere boundaries. Targeting of DiI (red) to the VN neurons was confirmed by afferents that were labeled by NeuroVue Maroon (blue) introduced into the VIIIth cranial nerve root and their termination on the DiI-labeled VN neurons. Abbreviations: OV, otic vesicle; r, rhombomere; VIII, VIIIth cranial nerve.