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Fig. 4. Motoneurons in segments L1 and L2 assume identities of thoracic motoneurons in Hoxc10^-/-/Hoxd10^-/- double-mutant embryos. (A) Whole-mount ventral view of DiI-labeled axons from lumbar segments L1 and L2. In the control embryo, axons from segment L2 project to the limb (arrows), but in both double-mutant embryos these axons project to the body wall. Inset shows boxed area with axon terminations of L2 at higher magnification. (B) Cross-sections through ventral spinal cord segments T13-L2 of E13.5 control and Hoxc10^-/-/Hoxd10^-/- mutant embryos stained with anti-Lim3, which labels mMMC neurons, and with anti-Isl1, which labels the entire MMC and mLMC neurons. In control embryos, the pattern of labeled motoneurons differs significantly between thoracic and lumbar segments, whereas in Hoxc10^-/-/Hoxd10^-/- mutants the thoracic pattern extends into lumbar cord. Dashed lines encircle the MMC. Arrowhead, mLMC neurons. (C) Cross-sections through ventral spinal cord segments T13-L2 of E13.5 control and Hoxc10^-/-/Hoxd10^-/- mutant embryos stained with anti-nNOS (arrow) to label the PGC motor column in the intermediolateral spinal cord and with anti-Isl1 to label MMC and mLMC (arrowhead) motor columns in the ventral horn, as well as PGC motoneurons. In control embryos, nNOS is expressed in thoracic segments, with expression extending only to rostral L1. In Hoxc10^-/-/Hoxd10^-/- mutant embryos, however, nNOS expression extended beyond segment L2 (arrow). (D) Cross-section through lumbar segment L2 of Hoxc10^-/-/Hoxd10^-/- mutant embryo stained with anti-nNOS and anti-Isl1. Note that nNOS⁺ axons project to the sympathetic ganglia. Arrows point to axonal projection path. Arrowhead indicates sympathetic ganglion (SG). Scale bars: 100 µm for B-D.

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