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Fig. 2 LIM homeobox gene expression in identified primary motoneurons. Primary motoneurons were injected with fluorescent dyes, fixed at 22-24 hours, and hybridized with LIM homeobox RNA probes. The fluorescent signal was enhanced using an alkaline phosphatase-catalyzed colorimetric reaction (Materials and Methods). A, C, E, G, I, K show Nomarski optics images of doubly labeled neurons. B, D, F, H, J, L are fluorescent images of the same motoneurons showing axonal trajectories. Triangles indicate the ventral boundary of the spinal cord. Scale bar equals 25 mm. (A,B) Injected CaP showing hybridization with islet2 probe. (C,D) Injected CaP hybridized with lim3 probe. At this stage, lim3 probe labels more cells than the primary motoneurons in the ventral spinal cord. (E,F) Injected MiP hybridized with islet1 probe. (G,H) Injected MiP hybridized with lim3 probe. (I,J) Injected RoP showing hybridization with islet1 probe. A second cell, dorsal to RoP, was injected but does not show islet1 labeling. Also, RoP is bordered by islet1-labeled cells. At this stage, islet1 begins to label cells in addition to the primary motoneurons (see Fig. 5). (K,L) Injected RoP hybridized with lim3 probe. (M) Schematic representation of primary motoneuron positions within the spinal cord, their axonal projections into the adjacent somite and the combination of LIM homeobox genes each expresses at axogenesis. Yellow represents lim3 expression, blue represents islet1, and red represents islet2. M indicates the MiP axon, R the RoP axon and C the CaP axon. The primary motoneurons were drawn by tracing and superimposing images of cells from different 24 hour embryos from a video display.