Fig. 1. Expression of Lmx1b and specification of early-born neurons in the spinal cord of Lmx1b^-/- mutants. (A-C) Expression of Lmx1b in the dorsal horn detected by in situ hybridization. (A) Lmx1b is expressed in dI5 interneurons at E10.5 (arrow). (B) At E11.5, in addition to dI5 neurons (small arrow), Lmx1b is expressed in neurons emerging from the dorsal VZ (large arrow). (C) Lmx1b expression is concentrated in laminae I-II neurons at E17.5. (D) Detection of BrdU⁺ (blue)/LMX1B⁺ (brown) in the spinal cord of wild-type E12.5 embryos. Arrows in D show nascent neurons co-labeled with anti-LMX1B and anti-BrdU antibodies. (E,F) Detection of LBX1 (dI4-6 marker, red) and BRN3A (dI1-3 and dI5 marker, green) in wild-type (E) and Lmx1b mutant (F) embryos at E11. (G,H) Detection of LBX1 (red) and PAX2 (dI4 and dI6 marker, green) in wild-type (G) and Lmx1b mutant (H) spinal cord at E11. (I,J) Detection of LMX1B (red) and PHOX2A (green) in dI5 neurons at E11.5; arrow in I and arrowheads in J (higher magnification of I) indicate double-stained cells. (K,L) Detection of LBX1 (red) and PHOX2A (green) in wild-type (K) and Lmx1b mutant embryos (L) at E11.5. Arrows indicate PHOX2A⁺ cells. (M) Statistical comparison of the number of PHOX2A⁺ cells in wild-type (white bar) and Lmx1b mutant (black bar) embryos. Asterisks indicate significant difference using Student's t-test P<0.001. Scale bars: 100 µm in A-C,E-I,K,L; 50 µm in D; 25 µm in J. MN, motoneurons.